Proceedings of the Indian Academy of Sciences
(Animal Sciences)
Volume 94, 1985
CONTENTS
Endocrine influence on protein synthesis in the fatbodies of female red cotton
bug, Dysdercus cingulatus Fabr Raji Raghavan and D Muraleedharan
Observations on the histology and histochemistry of Penetrocephalus plerocer-
coid (Pseudophyllidea: Cestoda)
K J Chandra, K Hanumantha Rao and K Shyamasundari
Effect of starvation on acid phosphatase activity in Gastrothylax crumenifer
M C Sathyanarayana
Four new species of trypanoplasms from the fresh water fishes of the genus
Mystus in Maharashtra M A Wahul
Mechanism of resistance in rice varieties showing differential reaction to brown
planthopper K Venugopala Reddy and M B Kalode
Larval and post-larvdl development of Spodoptera litura (Fabricius) on some
host plants G P Garad, P R Shivpuje and G G Bilapate
Activity-time budget in blackbuck N L N S Prasad
Some biochemical changes in the reproductive cycle of a hill stream teleost
Puntius chilinoides (McClelland) B P Nauriyal and H R Singh
Effect of carbaryl on esterases in the air-breathing fish Channa punctatus (Bloch)
5 Anmachalam, S Palanichamy and M P Balasubramanian
Irradiation effects on the adrenal gland of rats undergoing inanition stress
5 S Hasan and P K Chaturvedi
Laboratory culture of Diaphanosoma Senegal Gauthier, (Crustacea, Cladocera)
from South India K Venkataraman and S Krishnaswamy
Laboratory evaluation of anticoagulant treated baits against Indian field mouse,
Mus booduga Gray
M Balasubramanyam, M J Christopher and K R Purushotham
Effects of starvation on respiration and major nutrient stores of the prosobranch
snail Bellamya bengalensis (Lamarck)
D Satya Reddy and M Balaparameswara Rao
ii Contents
Influence of distillery effluent on growth and metamorphosis ofRana malabarica
(Bibron) M A Haniffa, Stephen T De Souza S J,
A G Murugesan and Barnabas Xavier 1 1 1
Bait preferences of rodents in their natural habitat
Nafis Ahmad and V R Parshad 1 1 7
Biochemical correlates of agonistic behaviour in Bandicota bengalensis: Hepatic
cholesterol and ascorbic acid Shakunthala Sridhara, T Somasekhar,
Elizabeth John, M V V Subramanyam and A Sundarabai 123
Topography of nervous system in two pouched Paramphistomes
Neerja Mishra and Veena Tandon 129
Effect of hypoxia on tissue metabolism of midgut gland of the scorpion
Heterometrus fulvipes G Ramesh Babu, G N Jyothirmayi and
P Venkateswara Rao 139
Male reproductive system of some digenetic trematodes
K Satya Gopal, C Vijayalakshmi and K Hanumantha Rao 145
Extraretinal photoreception involved in photoperiodic effects on gonadal
activity in the Indian murrel, Channa (Ophiocephalus) punctatus (Bloch)
S K Garg and S K Jain 1 53
Influence of food plants on the food utilization and chemical composition of
Henosepilachna septima (Coleoptera: coccinellidae)
G Ganga, J Sulochana Chetty, R Senthamil Selvi and T Manoharan 161
Foreword 169
Recent advances in animal behaviour K M Alexander 173
The Drosophila circadian clock M K Chandrashekaran 187
Hormones in insect behaviour V K K Prabhu 197
Hormonal rhythm and behavioural trends in insects D Muraleedharan 207
Behavioural energetics of some insects T J Pandian 219
Behavioural analysis of feeding and reproduction in haematophagous insects
RSPrasad 225
Feeding and ovipositional behaviour in some reduviids (Insecta-Heteroptera)
ETHaridass 239
A behavioural assessment of the impact of some environmental and physiolog-
ical factors on the reproductive potential of Corcyra cephalonica (Stainton)
(Lepidoptera: Pyralidae) S S Krishna and S N Mishra 249
Behavioural analysis of feeding and breeding in Orthopteran insects
S Y Paranjape 265
Physico-chemical factors in the acridid feeding behaviour (Orthoptera:
Acrididae) M C Muralirangan and Meera Muralirangan 283
Contents iii
Behavioural response (feeding preference and dispersal posture) of Aphis
gossypii Glover on brinjal crop T K Banerjee and D Raychaudhuri 295
Behavioural analysis of feeding and breeding in Lamellicorn beetles
G K Veeresh and K Veena Kumari 303
Evolution of insect sociality — A review of some attempts to test modern theories
Raghavendra Gadagkar 309
An analysis of the superparasitic behaviour and host discrimination of chalcid
wasps (Hymenoptera: Chalcidoidea) T C Narendran 325
Application of sex pheromones in sugarcane pest management
H David, B F Nesbitt, S Easwammoorthy and V Nandagopal 333
Role of behavioural studies in the development of management strategies for
forest insect pests K S S Nair 341
Behavioural strategies of emergence, swarming, mating and oviposition in
mayflies K G Sivaramakrishnan and K Venkataraman 351
Functional morphology of air-breathing fishes: A review
Hiran M Dutta and J S Datta Munshi 359
Bionomics of hill-stream cyprinids. I. Food, parasites and length-weight
relationship of Labeo dyocheilus (McClell) Sandeep K Malhotra 377
pH and dissolution of crystalline style in some bivalve molluscs of Porto Novo
coastal waters P Shahul Hameed 383
Studies on the induced spawning and larval rearing of a freshwater catfish,
Mystus punctatus (Jerdon) N Ramanathan, P Natarajan and N Sukumaran 389
Wing microsculpturing in the Brazilian termite family Serritermitidae
(Serritermes serrifer, Isoptera), and its bearing on phylogeny
M L Roonwal and N S Rathore 399
Electrical stimulation — Effects on the protein in the ventral nerve cord of
cockroach, Periplaneta americana
S L Maheswari, K Balasundaram and V R Selvarajan 407
Studies on the silk gland of Bombyx mori: A comparative analysis during fifth
instar development S K Sarangi 413
Studies on mating, spawning and development of egg in Macrobrachium nobilii
(Henderson and Mathai) C Balasundaram and A K Kumar aguru 421
Effect of castration and androgen treatment on the androgen dependent
parameters in the accessory glands of the slender loris, Loris tardigradus
lydekkerianus (Cabra) A Manjula and K M Kadam 427
Community and succession of the round-head borers (Coleoptera:
Cerambycidae) infesting the felled logs of White Dhup, Canarium euphyllum
Kurz TNKhan 435
iv Contents
Behavioural responses in terms of feeding and reproduction in some grass-
hoppers (Orthoptera: Insecta) T N Ananthakrishnan, K Dhileepan and
B Padmanaban 443
Investigations on Heliothis armigera (Hubner) in Marathwada — XXVIII. Key
mortality factors in regular and overlapping generations on pigeonpea
G G Bilapate 463
Transpiration rates and acclimation to water and temperature of the tropical
woodlice, Porcellionides pruinosus Brandt and Porcellio laevis Latreille
G Achuthan Nair and N Balakrishnan Nair 469
Changes in some biochemical constituents of the fiddler crabs Uca annulipes
Latreille and U. triangularis (Milne Edwards) in response to eyestalk removal
G V Krishna Rao, Y Prabhakara Rao and D G V Prasada Rao 475
Fecundity of the allochthonous feeder, Rasbora daniconius (Ham.) and of the
autochthonous feeder, Puntius amphibius (Val.)
K Prem Kumar, P A John and N K Balasubramanian 481
Development, endocrine organs and moulting in the embryos of Dysdercus
cingulatus Fabr (Heteroptera: Pyrrhocoridae)
Mariamma Jacob and V K K Prabhu 489
Seasonal variations and the role of neurosecretory hormones on the androgenic
gland of the prawn Macrobrachium lamerrii R Sarojini and G Gyananath 503
Mermithid nematodes as parasites of Heliothis spp. and other crop pests in
Andhra Pradesh, India
V S Bhatnagar, C S Pawar, D R Jadhav and J C Davies 509
Wing microsculpturing in the termite genus Amitermes (Termitidae,
Amitermitinae) M L Roonwal and N S Rathore 517
Effect of two graded doses of x-irradiation on the rat adrenal gland and its
protection by S-phenetyl formamidino 4(N-ethyl isothioamide) morpholine
dihydrochloride P K Chaturvedi and S S Hasan 523
Effects of DDT and malathion on tissue succinic dehydrogenase activity (SDH) and
lactic dehydrogenase isoenzymes (LDH) of Sarotherodon mossambicus (Peters)
K Ramalingam 521
Ultrastructure of the eggs of Reduviidae: I. Eggs of Piratinae (Insecta —
Heteroptera) E T Haridass 533
Host selection and food utilization of the red pumpkin beetle, Raphidopalpa
foveicollis (Lucas) (Chrysomelidae: Coleoptera) K Raman and R S Annadurai 547
Distribution of earthworms in Madras Sultan A Ismail and V A Murthy 557
Alkaline protease in the midgut of the silkworm Bombyx mori L.r changes during
metamorphosis S K Sarangi 567
Contents v
roceedings of the Symposium on 'Animal Communication9 February 1985,
ianga!ore
breword 573
to the communication of well-being
Madhav Gadgil, Malati Hegde, N V Joshi and Sulochana Gadgil 575
in recognition in social insects and other animals — A review of recent findings
id a consideration of their relevance for the theory of kin selection
Raghavendra Gadagkar 587
radeoffs in the evolution of frog calls A Stanley Rand 623
he evolution of communication and social behaviour in Dictyostelium dis-
rideum Vidyanand Nanjundiah 639
'ommunication and synchronization of circadian rhythms in insectivorous bats
G Marimuthu and M K Chandrashekaran 655
ubject Index 667
juthor Index 678
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 1-9.
© Printed in India.
Endocrine influence on protein synthesis in the fatbodies of female
red cotton bug, Dysdercus cingulatus Fabr
RAJI RAGHAVAN* and D MURALEEDHARAN
Department of Zoology, University of Kerala, Kariavattom 695 581, India
* Department of Zoology, Sree Narayana College, Punaiur, Kerala, India
MS received 24 August 1984; revised 29 November 1984
Abstract. Extirpation of pars intercerebralis neurosecretory cells (PINSC) of female
D. cingulatus significantly brought down the level of protein synthesis in the fatbodies 48, 72,
and 96 hr after the operation, while implantation of active PINSC into both normal and PINSC-
ablated females elevated substantially the protein content in the fatbodies. Additional supply
of JHa (FME) by topical application activated protein synthesis in the fatbodies both in the
ailatectomised and normal females. Histochemical studies to demonstrate the protein content
in the fatbodies of the above experimental insects also corroborated these findings. Probable
regulatory mechanism of protein synthesis in the fatbodies of female D. cingulatus by the
hormonal principles from PINSC and corpus allatum are discussed in the light of the above
findings.
Keywords. Dysdercus cingulatus; protein; fatbody; neurosecretory cells; juvenile hormone.
1. Introduction
Fatbodies are the site where most of the haemolymph proteins and vitellogenin are
synthesised and released in adult insects and thus it fulfils a variety of functions similar
to the hepatopancreas of molluscs and crustaceans or the liver in mammals (Telfer
1965; Chen 1978; Keeley 1978). So neurohormonally dependent changes in the protein
should reflect changes in the protein synthetic capacity of the fatbodies as well. As
Engelmann (1979) suggests, one of the most exciting aspects and one which attracted
increasing attention during the last few years, is the mechanism of control of
vitellogenin biosynthesis. The synthesis of haemolymph proteins seems to be con-
trolled, at least in part, by hormones of corpus allatum (Coles 1965a; Engelmann and
Penney 1966; Liischer 1968) and factors from the neurosecretory system (Hill 1962,
1965; Wyss-Huber and Liischer 1966). The Ca-hormones are intimately involved in
various phases of protein metabolism (Gilbert and Schneiderman 1961; Thomas and
Nation 1966). Juvenile hormone is demonstrated to influence the synthesis of storage-
proteins in the fatbody of Bombyx mori (Tojo et al 1981). In the American cockroach
the RNA content of the fatbodies is cyclic in nature and the protein level is contributed by
the fatbody (Mills et al 1966). In Leucophaea maderae fatbodies seem to react with an
increased release of proteins to the changed hormonal environment during oocyte
maturation (Wyss-Huber and Liischer 1972). The brain hormone could be acting
directly on the fatbody or through some other organ as the ovary itself (Hagedorn and
Fallon 1973).
In the present paper, results of our experiments performed to elucidate the influence
of hormonal principles from the pars intercerebralis neurosecretory cells (PINSC) and
Raji Raghavan and D Muraleedharan
hat from corpus allatum (JH) on the synthesis of fatbody proteins in the female red
otton bug, Dysdercus cingulatus are presented.
L Material and methods
LI The animal
rhe red cotton bug, Dysdercus cingulatus (Heteroptera: Pyrrhocoridae), was reared in
the laboratory at 29+3cC, r.h. 90 + 3% and 12:12iJD regime. The insects were fed
ad libitum on soaked cotton seeds. The newly emerged adults of both sexes were
separated within an hour after emergence from the stock colony and fed as described
earlier by Muraleedharan and Prabhu (1979) and adult females of appropriate age
groups were selected from among them for experimentation.
2.2 Surgical techniques
All the instruments used for microsurgery were washed well in distilled water and
sterilised in 70 % ethyl alcohol. Surgical procedures for extirpation and implantation of
PINSC and allatectomy were followed after Muraleedharan and Prabhu (1979, 1981).
Adult donor females within 3 hr after emergence were used for extirpation of PINSC and
newly emerged adults served as hosts. Sham-operated insects of corresponding age
groups served as controls for each category. Pieces of gut tissue were implanted into the
control instead of PINSC. Operated insects were disengaged from plasticine ribbons and
after mopping off the Ringer solution sticking to them, a thin film of anti-septic powder
consisting of penicillin, streptomycin and phenylthiourea in the ratio 1:1:2 was applied
on the wound. Adult females, 24 hr after their emergence, were used for allatectomy.
Twenty four hr after the operation such females were allowed to mix with young adult
males for free mating.
2.3 Protein estimation
Fatbodies from different experimental insects were dissected out 48, 72 and 96 hr after
each experimental manipulation. Pre-weighed specimen tubes containing 0-5 ml of
isotonic potassium chloride solution were again weighed along with the fatbodies and
the weight of fatbodies used for protein estimations were determined from the weight
difference. Protein extract of fatbodies was prepared in isotonic KC1 solution after
homogenisation, precipitation with 10 % TCA solution and subsequent centrifugation at
5000 g for 20 min. The residue dissolved in 1 ml of 0-1 N NaOH served as the protein
extract. Total proteins in the fatbodies were estimated according to the method of
Lowry et al (1951), using phenol reagent of Folin-Ciocalteau. Bovine serum albumin
(Sigma chemical Company, USA) was used as standard. Concentration of protein was
expressed in ug protein/mg tissue. Mean values of 8 different determinations were
adopted as the protein concentration in each group. Significance of the data were
analysed employing student's t test.
Endocrine control offatbody proteins 3
2.4 Histochemistry
For histochemical demonstration of proteins, the mercury bromophenol blue method
(Pearse 1968) was followed using formalin-fixed fatbodies from different categories of
experimental insects along with their respective controls.
2.5 JHa treatment
Farnesyl methyl ether (FME) (Econ. Control Inc., USA) was the juvenile hormone
analogue (JHa) used. FME was dissolved in acetone for topical application; the
concentration being 0-5 /ig/jd and 4 /zl (containing 2 /xg) was applied to each animal
(effective dose was determined in the preliminary experiments) with the aid of a
calibrated microcapillary. FME dissolved in acetone was topically applied underneath
the wings to mildly anaesthetised females. Controls were treated similarly with the same
quantity of acetone.
3. Results
Protein concentration in the fatbodies was significantly lower in the piNsoablated
females 48, 72 and 96 hr respectively after the operation than in the respective stages of
the sham-operated control groups (figure 1). Implantation of active PINSC into normal
females elevated the protein concentration to a significant level when compared with
that of the operated controls (P ^ 0-01). Substantial increase in the fatbody protein
concentration was noticed in piNSC-ablated insects when they were implanted with
active PINSC (figure 1). Histochemical observations corroborate these findings (figure 3;
1 to 4). Significant reduction in fatbody protein concentration was noticed in the
allatectomised females as well when these were estimated 48, 72 and 96 hr after the
operation ( P ^ 0-05). Topical application of FME (JHa) on the normal as well as
allatectomised females (figure 2) enhanced protein concentrations to a significant level
(P ^ 0-05). However, the rise in protein concentration in allatectomised females was
less than that in normal females supplied with additional JHa (figure 2). Histochemical
investigations also corroborate these findings (figure 3; 5 to 8).
4. Discussion
Present studies show that in the female D. cingulatus, synthesis of protein is inhibited in
the absence of PINSC while implantation of active PINSC into PiNsc-ablated insects
restores the level significantly; implantation into normal females enhances the protein
level well above the normal level. The protocerebral neurosecretory cells have been
reported to be indispensable for oogenesis in many insect species like Calliphora
zrythrocephala (Thomsen 1948, 1952; Possompes 1956), Schistocerca gregaria
(Highnam 1962a, b, c; Hill 1962), Schistocerca pararensis (Strong 1965a, b), Locusta
nigratoria (Girardie 1966), Anacardium aegyptium (Geldiay 1967), Dysdercus cingu-
latus (Jalaja et al 1973). However, this does not necessarily mean that PINSC control egg
maturation directly. In many insects, MNSC ablation from the brain results in a lowered
Raji Raghavan and D Muraleedharan
fH PI NSC ABLATED
EEE3 CONTRQL
| 1 p/A/SC IMPLANTED
p^r, -PINSC-f- PINSC
72 96
HOURS AFTER OPERATION
Figure 1. Histogram showing the effect of extirpation and implantation of PINSC on the
protein content in the fatbodies of female D. cingulatus. Each column represents mean of 8
values and the bars denote qrsEM.
food intake as in Calliphora erythrocephala (Thomsen and Moller 1963), D. cingulatus
(Muraleedharan and Prabhu 1979), Hyblaea puera (Muraleedharan and Prabhu 1981)
resulting in a low protein concentration and the subsequent cessation of oocyte
maturation. Therefore, the relationship between MNSC and oogenesis can only be
studied properly if MNSC cauterisation has been carried out in such a way that it does not
affect food intake. In the experiments performed on Schistocerca gregaria (Hill 1965)
and Leptinotarsa decemlineata (de Loof and de Wilde 1970; de Loof and Lagasse 1970)
this condition was fulfilled and the effect of MNSC on oogenesis was established. In
Schistocerca this is explained by a direct effect on the synthesis of vitellogenic proteins
and in Leptinotarsa by an effect partly on the fatbody in conjunction with the corpus
allatum (vitellogenic protein synthesis) and partly via the corpus allatum on the
terminal oocyte. Thomsen (1952) has shown that in C. erythrocephala females the
extirpation of MNSC causes total exhaustion of the cells of fatbodies. The rate of protein
synthesis in the fatbodies as well as the concentration of haemolymph proteins are
reduced in pars intercerebralis-cauterised female locusts (Hill 1962, 1965). These
findings by Hill corroborated a suggestion made earlier by Thomsen (1952) that a
principle liberated from pars intercerebralis regulates protein metabolism. Supporting
data for this may be found in the observation of low protein concentration in the
haemolymph of brain operated females of Gomphocerus rufus (Loher 1965), L. maderae
(Engelmann 1966; Engelmann and Penney 1966) and in young females of T. molitor
Endocrine control offatbody proteins
260
ALLATECTOMJSED
§3 CONTROL
Q FME TREATED
ALLATECTOMISED +
FME
HOURS AFTER ALL A TEC TOM Y AND JHA TREATMENT
Figure 2. Histogram showing the effect of allatectomy and topical application of FME (jna)
on the protein content in the fatbodies of female D. cingulatus. Each column represents mean
of 8 values and the bars denote +SEM.
(Mordue 1965). MNSC are known to stimulate protein synthesis in the fatbodies in many
species of insects such as S. gregaria (Highnam et al 1963; Hill 1963), M. sanguinipes
(Elliot and Gillot 1979). The content of the neurosecretory material in the MNSC of D.
cingulatus increases steadily during the early days of the first gonotrophic cycle when
active vitellogenesis is taking place and the protein build up in the haemolymph is under
the control of neurosecretion (Jalaja and Prabhu 1977). A sudden decline in the
haemolymph proteins is reported in the female D. cingulatus 72 hr; after emergence
which was suggested to be related to heavy yolk protein deposition in the ovaries (Jalaja
and Prabhu 1971). The present studies also demonstrate a sudden decline in the protein
concentration of fatbodies after 72 hr in the sham-operated controls of the PINSC-
ablated insects. This indicates that the decline found in the control is due to
vitellogenesis. So it is suggested that in D. cingulatus females hormones from PINSC
stimulate protein synthesis in the fatbodies during vitellogenesis.
A highly reduced level of protein synthesis as noticed in the allatectomised females
and its substantial increase when these insects are supplied with additional jna and also
the increase noticed in protein synthesis when normal females are supplied with extra
titre of JH in the form of JHa, demonstrate that JH stimulates protein synthesis in the
Raji Raghavan and D Muraleedharan
;/**;*v .
Figure 3. Sections of fatbodies fixed in formalin and stained for protein using mercury
bromophenol blue technique (Pearse 1968). I. and 3. 48 and 72 hr after PINSC ablation, 2. and
4. their respective controls. 5. and 7. 48 and 72 hr after allatectomy, 6. and 8. their respective
controls. (Nu-Nucleus, Pr-protein) Magnifications of all figures are x400.
Endocrine control offatbody proteins 7
fatbodies of female D. cingulatus. The rate of incorporation of radioactive amino acids
into fatbody proteins is reported to be significantly slow in allatectomised P. americana
females (Thomas and Nation 1966). Implantation of active corpus allatum into
decapitated N. cinerea is also reported to elevate the rate of protein synthesis in the
fatbody (Liischer 1968). However, allatectomised queens of Apis mellifica contain
vitellogenin at a high titre (eventhough some what lower than in operated controls) and
did even lay eggs when treated with CO2 (Engels and Ramamurthy 1976). Repeated
application of JH restored vitellogenin titres to those observed in normal queens
(Ramamurthy and Engels 1977). JH stimulates the protein content in the fatbodies of a
number of insects such as Musca domestica, Locust a migratoria, Melanoplus sangunipes
and Diatraea grandiosella (Adams and Nelson 1969; Lauwerjat 1977; Elliot and Gillot
1978; Turnen and Chippendale 1980). Enlargement of nuclei and abundance of rough
endoplasmic reticulum and golgi complexes were noticed in the fatbodies in connection
with the progress of vitellogenesis during which proteins were synthesised in
abundance in L. migratoria (Couble et al 1979). JH is involved in the synthesis of
vitellogenins in Rhodnius prolixus (Coles 1964, 1965 a, b) and in Sarcophaga bullata
(Wilkens 1969).
Muraleedharan and Prabhu (1981) have shown that in D. cingulatus aliatectomy does
not affect food consumption. So the decrease in the fatbody as noticed in the
allatectomised insects cannot be attributed to deficiency of food. It was demonstrated
by Jalaja and Prabhu (1977) that in D. cingulatus both MNC-hormone and JH are
involved in vitellogenesis and MNohormone stimulates the process by influencing the
production of JH by the corpus allatum. A neurosecretory influence is observed on the
protein synthesis while a direct gonotrophic effect is with corpus allatum and a
reciprocal relationship between the neurosecretory system and corpus allatum in which
interference with one component of the neuroendocrine system results in interference
with the other (Hill 1962; Highnam et al 1963).
In the light of the present findings and the pertinent available literature, it may be
suggested that in adult females of D. cingulatus hormonal principles both from PINSC
and corpus allatum have a stimulatory effect on the synthesis of proteins in the fatbody.
The influence imparted by PINSC seems to be either through a trophic mechanism on the
corpus allatum or by its direct effect on the fatbody while hormones from CA seem to
stimulate the process directly.
Acknowledgements
The authors are thankful to Prof. V K K Prabhu for critically going through the
manuscript and to Prof. K M Alexander for facilities. One of the authors (RR)
acknowledges the financial assistance from the University of Kerala in the form of a
Fellowship.
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Jalaja M and Prabhu V K K 1977 Endocrine control of vitellogenesis in the red cotton bug, Dysdercus
. cingulatus Fabr; Entomon 2 17-29
Jalaja M, Muraleedharan D and Prabhu V K K 1973 Effect of extirpation of median neurosecretroy cells in
the female red cotton bug, Dysdercus cingulatusi J. Insect Physiol. 19 29 — 36
Keeley L L 1978 Endocrine regulation of fatbody development and function; Ann. Rev. Entomol 23 329-352
Lauverjat S 1977 Le evolution post imaginale due tissue adipeux femelle de Locusta migratoria et son
controle endocrine; Gen. Comp. Endocrinol. 33 13-34
Loher W 1965 Hormonale Kontrolle der Oocytentwicklung bei der Heuschreeke Gomphocerus rufus L.; Zool.
Jahrb. Abt. PhysioL 71 677-684
Lowry D H, Rosenbaum N J, Farr A L and Randall R J 195 1 Protein measurement with Folin phenol reagent
J. Biol. Chem. 193 265-268
Endocrine control offatbody proteins 9
Liischer M 1968 Hormonal control of respiration and protein synthesis in the fatbody of the cockroach
Nauphoeta cinerea during oocyte growth; J. Insect Physiol 14 499-511
Mills R R, Greenslade F C and Cough E F 1966 Studies on vitellogenesis in the American cockroach; J. Insect
Physiol. 12 767-779
Mordue W 1965 The endocrine control of oocyte development in Tenebrio molitor L.; J. Insect Physiol. II
505-511
Muraleedharan D and Prabhu V K K 1979 Role of the median neurosecretory cells in secretion of protease
and invertase in the red cotton bug Dysdercus cingulatus; J. Insect Physiol 25 237-240
Muraleedharan D and Prabhu V K K 1981 Hormonal influence on feeding and digestion in a plant bug,
Dysdercus cingulatus and a caterpillar, Hyblaea puera\ Physiol. Entomol 6 183-189
Pearse AGE 1968 Histochemistry: Theoretical and applied 3rd edn (London: Churchill) 1
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313-314
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Schistocerca-$p. II. The innervation of the CA, the lateral neurosecretory complex and oocyte growth; J.
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blow fly Calliphora erythrocephala Meig; J. Exp. Biol 29 137-172
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 11-19.
© Printed in India.
Observations on the histology and histochemistry of
Penetrocephalus plerocercoid (Psendophyllidea: Cestoda)
K J CHANDRA, K HANUMANTHA RAO* and
K SHYAMASUNDARI*
Department of Fisheries Biology and Limnology, Bangladesh Agricultural University,
Mymensingh, Bangladesh
* Department of Zoology, Andhra University, Waltair 530003, India
MS received 17 November 1983; revised 8 October 1984
Abstract. The histology of plerocercoid of Penetrocephalus sp. reveal that the body surface
consists of tegument, basement membrane, epidermal longitudinal musculature, parenchy-
matic longitudinal musculature, transverse and dorsoventral muscle fibres. Three types of
glands could be recognized from the scolex of the plerocercoid. The musculature of the
plerocercoid consists of glycogen, acid, sulfated, neutral and carboxylated mucopolysac-
charides: basic proteins containing tyrosine, S-H and S-S groups, protein bound amino groups,
sulfhydril groups, glycoprotein and lipid.
The frontal glands contain carbohydrates (1,2 glycols, both acid and neutral mucopolysac-
charides), basic protein (tyrosine, S-H group, protein bound amino group) and phospholipids.
The structure, organization and histochemistry of the plerocercoid is discussed.
Keywords. Histology; histochemistry; Penetrocephalus', plerocercoid; cestoda.
1. Introduction
Hanumantha Rao (1960a) erected the genus Penetrocephalus for the form described by
him earlier as Bothriocephalus ganapatii (Hanumantha Rao 1954), recovered from a
teleost fish Saurida tumbil (Bloch). In his later paper (Hanumantha Rao 1960b)
observations on histochemistry and egg formation were furnished. Rama Devi (1970)
studied the histology and some aspects of histochemistry. But histochfemical work on
the plerocercoid of Penetrocephalus has not been carried out so far.
Histochemical investigation on pseudophyllidean cestodes probably started with the
work of Takahashi (1959) on Diphyllobothrium (Spirometrd) mansoni and then Arme
(1966) on Ligula intestinalis. But little attention has been paid to larval stages. Ohman
(1968) was the first to investigate the histochemistry of the larva of D. detriticum and
concluded that one cannot rely solely on morphological descriptions to solve the
taxonomic problems in Diphyllobothrium.
The present work deals with the histology and histochemistry of plerocercoid of
Penetrocephalus collected from a number of teleost fishes.
2. Material and methods
Plerocercoids of Penetrocephalus were recovered from various locations in the body
cavity of Saurida tumbil and 13 other species of teleosts, collected from the off-shore
12 K J Chandra, K Hanumantha Rao and K Shyamasundari
tishing station, Visakhapatnam and from local fish markets. The larvae were fixed ii
alcoholic Benin's, Susa, Carney and formal calcium, passed through grades of alcohol
cleared, embedded in paraffin wax (m.p. 58°C) and sections cut at 10-12 n thickness
Heidenhain's azan, and Mallory's triple stains were used for histological studies. Th<
histochemical tests employed were periodic acid schiff (PAS) technique for carbohydrate
containing groups, PAS saliva for glycogen, PAS after acetylation and deacetylation fo
1 : 2 glycol groups. Alcian blue (AB) 1 pH and AB 2-5 pH for acid mucins, AB (1 pH)/pAS t<
detect sulphate free sialic acid containing mucins, AB (2-5 pH)/PAS to distinguish neutra
from acid mucopolysaccharide and toluidine blue for the demonstration of ack
mucopolysaccharides. Aldehyde fuchsin (AF)/AB was employed to distinguish betweei
sulphated and non-sulphatcd mucosubstances and, to confirm the presence o
sulphated mticins, AB/safranin was performed. For basic proteins, mercury bromo
phenol blue was used and, potassium perrnanganate/alcian blue (KMnO4/AB) fo
disulphidc, ferric ferricyanide for sulfhydril group and Congo red for glycoprotein. T<
demonstrate lipids-Sudan black B and for phospholipids copper phthalocyanii
techniques were employed. Most of the procedures for histochemical tests wer
adopted from Pearse (1968).
3. Observations
Live specimens were obtained from the stomach wall, liver, muscles and other region
of the alimentary canal of the hosts. The worms appeared flat, elongated and milky
white in colour. The scolex was invaginated.
3.1 Histology
The outer layer of the larval worms of Penetrocephalus sp. is a thick tegument. It i
smooth, but not uniform throughout the body. Beneath the tegument is the basemen
membrane. This layer separates the inner muscular layer. It is single layered with n<
vacuoles, reticulations etc. Below the basement membrane, lies the epiderma
longitudinal musculature. The cells of this region are not widely spaced nor dense!
packed. A deeper parenchymal longitudinal musculature is also present. Transvers
muscles are situated between two rows of longitudinal muscles (figure 1). They are als<
found in the central region running towards periphery, but are more distinct in th
scolex.
In the invaginated anterior region of the larva, numerous frontal glands occur in th
medular parenchyma. They are irregular and of various shapes. Three types of gland
could be distinguished, though one type occupies the major part of the scolex. In th
central region there is a frontal pit filled with secretions. The entire part is covered wit!
thin fibrous parenchymal musculature (figure 2).
3.2 Histochemistry
The staining and histochemical reactions of the various anatomical regions especiall
the tegument and the frontal glands are summarized in table 1 and the reactions on th
various regions are shown in figures 3-8.
Histology and histochemistry of Penetrocephalus
13
*,**,
FP
FG
Figures 1-2. 1. Cross-section of the body showing general musculature (Azan). 2. Cross-
section through the scolex showing frontal glands (Azan).
From the ensemble of histochemical tests it could be stated that the tegument is
charged with acid, sulfated, carboxylated mucopolysaccharides with hyaluronic acid,
basic proteins containing tyrosine, S-S group, S-H group and lipids especially
phospholipids.
The muscles display carbohydrate containing glycogen, proteins containing tyrosine,
protein bound S-H and NH2 groups and lipids.
The frontal glands contain carbohydrates (1, 2 glycol groups and both acid and
neutral mucopolysaccharides), proteins (S-H, NH2 groups and tyrosine) and
phospholipids.
4. Discussion
The results of the study on the histology and histochemistry of the plerocercoid of
Penetrocephalus sp. agree to some extent with those obtained for the adult of
Penetrocephalus ganapatii.
The histochemical composition of the tegument has been studied in detail, more in
cyclophyllidean cestodes than in pseudophyllideans. Bogitsh (1963) found PAS positive
material in the tegument of Hymenolepis microstoma and he stated that it could
probably be a mucoprotein. Lumsden (1975) stated that the external limiting
14
K J Chandra, K Hanumantha Rao and K Shyamasundari
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Histology and histochemistry of Penetrocephalus
15
Figures 3-4. 3. Tegument and the deeper longitudinal musculature showing positivity
towards PAS. 4. Frontal glands showing positivity towards PAS.
membrane is coated with a layer of carbohydrate-rich polyelectrolyte, namely, a
glycocalyx which serves as a binding surface for inorganic ions and higher molecular
weight organic compounds including host enzymes. Hanumantha Rao (1960a) also
mentioned that the tegument of P. ganapatii is PAS positive and fast to saline. Trimble
and Lumsden (1975) found the tegument of larval Taenia crassiceps to possess a surface
coat rich in both neutral and acidic carbohydrates. Rama Devi (1970) worked on six
species of pseudophyllidean tapeworms and reported similar findings. She also detected
the presence of lipids. In Ptychobothrium cypseluri, Bothriocephalus manubriformis,
Oncodiscusfimbriatus and Bothriocephalus indicus the tegument was found to possess
acid mucopolysaccharides and glycogen (Rama Devi 1970). The present observations
reveal the tegument to consist of basic proteins containing tyrosine, glycoprotein, S-H,
S-S etc., in addition to mucopolysaccharides.
The basement membrane is composed of basic proteins containing tyrosine, S-H
groups, NH2 groups, protein bound amino acid groups and glycoproteins and lipids
bearing phospholipids. The epidermal and the parenchymal musculature contain basic
16
K J Chandra, K Hanumantha Rao and K Shyamasundari
4"
*£L.i **„ < **•' *. \j
: - -•; '• -• '
Figures 5-6. 5. Frontal glands showing metachromasia with toluidine blue. 6. Basement
membrane and the deeper longitudinal muscles showing the presence of basic proteins
(bromophenol blue).
Histology and histochemistry of Penetrocephaius
Figures 7—8. 7. Frontal glands indicating the presence of basic proteins (bromophenol
blue). 8. Frontal glands showing glycoproteins (Congo red).
Abbreviations. B- Basement membrane; Dl- Deeper longitudinal musculature;
EP ~ Epidermal longitudinal musculature; FG- Frontal glands; FP- Frontal pit;
T- Tegument; TM- Transverse muscle.
proteins with tyrosine, S-H groups, very little quantities of protein bound amino
groups and lipids.
The presence of glycogen in the parenchyma in various pseudophyllids was
demonstrated by many workers (Smyth 1946, 1947; Hanumantha Rao 1960a; Rama
18 K J Chandra, K Hanumantha Rao and K Shyamasundari
Devi 1970). In the present study also glycogen was detected in the parenchyma and
muscles.
In addition to glycogen, both epidermal and parenchymal musculature of the
plerocercoid contain lipids especially phospholipids. Smyth (1946) reported the
presence of lipid droplets of various sizes scattered throughout the parenchyma of
Ligula intestinalis. Rama Devi (1970) also reported the presence of lipid droplets in the
parenchyma of pseudophyllidean cestodes. Lipids may also be considered as break
down products of metabolism (Rama Devi 1970).
Baer (1956) reported that the scolex of Manor ygma perfectum displays a 'deep
staining granular mass' which was considered to evoke a pronounced host tissue
reaction. In Lecanicephalidae, a similar 'glandular complex' was described in
Polypocephalus rhinobatides and in P. radiatus by Subhapradha (1951). Smyth (1964)
reported the occurrence of glands in the restollum of Echinococcus granulosus. He
stated that the secretion of the gland is PAS negative and concluded that it is probably an
extremely labile lipoprotein or lipid-protein coacervate. In the present investigation on
the plerocercoid of Penetrocephalus sp. three types of gland cells could be recognized.
The glands are PAS positive. Rama Devi (1970) also recognized three types of glands in
the scolex of adult Penetrocephalus ganapatii. Wolffhugel (1938) reported the
occurrence of three types of gland cells in the scolex of Nematoparataenia southwelli.
Many speculations have been made as to the function of these glands. However,
Smyth (1964) mentioned that the glands cause contraction of the villi which can assist
the orientation of the scolex. Wolffhugel (1938) suggested that the enzymic secretion
of the gland assists the cellular digestion. He also assumed that the secretion was
hormonal in nature, related to the regulation of growth and maturation of the strobila.
According to Rama Devi (1970) the glands of P. ganapatii assist in penetration
activities.
Rawson and Rigby (1960) observed that in the cysticercoid of Choanotaenia
crassiscolex these glands secrete a lubricant into the rostellar sac to help movement of
the rostellum. Therefore, the gland cells in different cestodes may vary in structure,
anatomy, chemical nature and function.
It is believed that in Penetrocephalus also the scolex glands aid in the penetration
activities.
Acknowledgements
One of the authors (KJC) is grateful to the Ministry of Education and Culture,
Government of India, New Delhi, for financial assistance, the authorities of Andhra
University, Waltair for facilities and also to the authorities of Bangladesh Agricultural
University, Mymensingh for granting him study leave.
References
Arme C 1966 Histochemical and biochemical studies on some enzymes of Ligula intestinalis (Cestoda:
Pseudophyllidea); J. Parasitol. 52 63-68
Baer J G 1956 Parasitic helminths collected in West Greenland; Medeleser our Gronland ungirne of
Kominiss; F. Vidensk. Unders. Gronl. Bd. 124 55-58
Bogitsh B J 1963 Histochemical studies on Hymenolepis microstoma (Cestoda: Hymenolepididae);
J. Parasitol 49 989-997
Histology and histochemistry of Penetrocephalus 19
Hanumantha Rao K 1954 A new bothriocephalid parasite (Cestoda) from the gut of the fish Saurida tumbil
(Bloch); Curr. ScL 23 333-334
Hanumantha Rao K 1960a Studies on Penetrocephalus ganapatii, a new genus (Cestoda: Pseudophyllidea)
from the marine teleost Saurida tumbil (Bloch); Parasitology 50 155-163
Hanumantha Rao K 1960b The problems of Mehlis' gland in helminths with special reference to
Penetrocephalus ganapatii (Cestoda: Pseudophyllidea); Parasitology 50 349-350
Lumsden R D 1975 The tapeworm tegument: a model system for studies on membrane structure and function
in host-parasite relationship; Trans. Am. Microscop. Soc. 94 501-507
Ohman J C 1968 Histochemical studies of the cestode Diphyllobothrium dentritriticum Nitzsch, 1824;
Z. Parasitenkd. 30 40-56
Pearse AGE 1968 Histochemistry: Theoretical and applied 3rd edn (London: J A Churchill)
Rama Devi P 1970 Studies on pseudophyllidean cestodes of fishes of Visakhapatnam district, Andhra Pradesh
with special reference to Senga visakhapatnamensis sp. n. Ph. D. Thesis, Andhra University, Waltair, India
Rawson D and Rigby J E 1960 The functional anatomy of the cysticercoid of Choanotaenia crassiscolex
(Linstow 1890) (Dilepididae) from the digestive gland ofOxychilus cellarius (Mull.) (Stylommatophora)
with some observations on developmental stages; Parasitology 50 453-463
Smyth J D 1946 Studies on tapeworm physiology. I. The cultivation of Schistocephalus solidus in vitro; J. Exp.
Biol 23 47-70
Smyth J D 1947 Studies on tapeworm physiology. II. Cultivation and development of Ligula intestinalis
in vitro; Parasitology 38 173-181
Smyth J D 1964 Observations on the scolex of Echinococcus granulosus, with special reference to the
occurrence and chemistry of secretory cells in the rostellum; Parasitology 54 515-526
Subhapradha C K 1951 On the genus Polypocephalus Braun, 1878 (Cestoda) together with the description of
six species from Madras; Proc. Zool Soc. Lond. 121 205-235
Takahashi T 1959 Studies on Diphyllobothrium mansoni, histochemical studies on plerocercoid; Jpn.
J. Parasitoi 8 669-676
Trimble III J J and Lumsden R D 1975 Cytochemical characterization of tegument membrane associated
carbohydrates in Taenia crassiceps larvae; J. Parasitoi. 61 665-676
Wolffhugel K 1938 Nematoparataeniidae; Z. Infektionskr. V. Hyg. Haustere 53 9-42
A— 7
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 21-23.
© Printed in India.
Effect of starvation on acid phosphatase activity in
Gastrothylax crumenifer
M C SATHYANARAYANA
Zoology Department, A.V.C. College, Mannampandal 609 305, India.
Abstract. Effect of starvation on acid phosphatase activity in Gastrothylax crumenifer
showed that activity was greater in starved individuals than in well fed ones.
Keywords. Starvation; acid phosphatase; Gastrothylax crumenifer.
1. Introduction
Although considerable work has been done on the subject of stress very little is known
on stress and its effect on parasites or parasitism. Starvation in flukes produces increase
in membraneous autophagic vacuoles exhibiting hydrolytic activity as elicited in
Megalodiscus temper atus (Bogitsh 1973), Schistosoma mansoni (Bogitsh 1975),
Haematoloechus medioplexus (Davis et al 1969). A study was undertaken with a view to
elucidating the effects of starvation on the activity of phosphatases in Gastrothylax
crumenifer.
2. Material and methods
To detect the effects of starvation on phosphatase activity in G. crumenifer, live parasites
were obtained from the rumen wall. They were transported to the laboratory in
Hedon-Fleig medium and were washed thoroughly to clean off debris. One batch of
worms was taken separately and assayed for phosphatase activity and considered for
normal activity at 0 hr. The second batch was incubated in Hedon-Fleig medium with
0-5 % glucose (fed), one million units of penicillin and 2-5 g of streptomycin per litre as
described by Thorpe (1967). To determine the effect of starvation, another batch of
worms was incubated in Hedon-Fleig medium without glucose (starved). Ten to fifteen
worms were placed in 200 ml experimental liquid in finger bowls and the temperature
maintained at 37°C Solutions were changed after 12 hr and thereafter at 24 hr
intervals; the worms were observed twice daily. Dead worms were removed promptly.
A worm was considered to be "fully alive" when moving spontaneously, "moribund" or
"half alive" when sluggishly moving or responding only to mechanical stimulation and
"dead" when no movement was evident or no response was obtained. Usually active
worms were attached firmly to the sides of the glass jars.
Observations of the parasite's enzyme activity were made at 0 hr and at the end of
12 hr, and then at intervals of 24 hr, up to 120 hr (5 days), for both control (fed) and
experimental (starved) batches. At the commencement of the 6th day, parasites in the
non-nutrient medium were transferred to the nutrient (glucose) medium for measure-
ment of any difference in activity. As no parasite survived after the 6th day, even in the
21
22
M C Sathyanarayana
nutrient medium, the experiment had to be concluded at that stage. Phosphatase
activity was assayed by the method of King and Armstrong as described by Varley
(1967), using disodium phenyl phosphate (0-01 M) as substrate. The protein content of
the supernatant was determined following the method of Lowry et al (1951), using
human serum albumin as a standard.
3. Results
The phosphatase activity in G. crumenifer was estimated at 0 hr i.e., prior to subjecting
them to starvation for varying periods up to 120 hr. The phosphatase activity was
greater in starved individuals than in the fed ones at 12, 24, 48 and 120 hr (figure 1). The
phosphatase activity was greater in refed ones than in fed ones, at 144 hr.
4. Discussion
The results indicated that starvation/stress enhanced the acid phosphatase activity in
G. crumenifer. No biochemical work exists describing the effects of starvation on
0.107
0.024 -,'
0.016 '
0.004
144
Figure 1. Phosphatase activity in G. crumenifer with glucose (fed) and without glucose
(starved) at different hours in veronal buffer at pH 3 ( ( )-fed, ( ) starved).
Effect of starvation on phosphatase 23
phosphatase activity in digenetic trematodes. A few studies have been made histochemi-
cally on the effects of starvation on phosphatase activity in digenetic trematodes. The
stimulus for the increased synthesis of phosphatases remains unknown. It is possible
that the diminishing pressure of material in the lumen of the digestive tract is the
triggering mechanism (Bogitsh 1975). As the amount of food in the lumen is reduced,
the pressure is likewise lessened and an impulse is probably generated.
Bogtish (1973) suggested that starvation is a stimulus to which the gastrodermis of
M. temperatus reacts by sequestered areas with the enclosed material subsequently
being degraded. It has been reported that the golgi complexes become increasingly
active in their relationship with the lysosome system in other types of organisms
subjected to stress factors, such as starvation (Ericson 1969). Under conditions of stress
(e.g., starvation) organelle complexes are often found in increased numbers (Bogitsh
1973; Threadgold and Arme 1974). A marked increase in the number of acid
phosphatase positive, membrane-bound vacuoles was reported in starved M. tem-
peratus as compared to well fed worms (Bogitsh 1973).
The functional significance of this process lies in the possibility that it may represent
a survival mechanism for the tissue following stress. The metabolism of the
gastrodermis of M. temperatus may become reoriented so that the lytic rates become
significantly greater than the synthetic rates (Bogitsh 1973).
It is desirable that a larger number of trematodes be investigated to determine how
the stress would affect the various tissues, thus enabling a better understanding of this
aspect of trematode physiology.
Acknowledgement
The author is indebted to Prof. D W Halton, for his valuable suggestions and for
critically going through the manuscript. Thanks are due to late Dr Sita Anantaraman
and Prof. M Anantaraman, Madras for their help in preparation of the manuscript.
References
Bogitsh B J 1973 Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes:
IX Megalodiscus temperatus; Exp. Parasitol. 32 244-266
Bogitsh B J 1975 Cytochemistry of gastrodermal autophagy following starvation in Schistosoma mansoni;
J. Parasitol 61 237-248
Davis D A, Bogitsh B J and Nunnally D A 1969 Cytochemical and biochemical observations on the digestive
tracts of digenetic trematodes. Ill Non-specific esterase in Haematoloechus medioplexus; Exp. Parasitol.
24 121-129
Ericson J L E 1969 Mechanism of cellular autophagy in Lysosomes in biology and pathology (eds) J T Dingle
and H B Fall (Amsterdam: North Holland) Vol 2
Lowry O H, Rosenbrough N J, Farr A L and Rundall R F 1951 Protein measurement with the folin phenol
reagent; J. Biol. Chem. 193 265-275
Thorpe E 1967 Histochemical study with Fasciola hepatica; Res. yet. ScL 8 27-36
Threadgold L T and Arme C 1974 Electron microscope studies of Fasciola hepatica XI Autophagy and
parenchymal cell function; Exp. Parasitol. 35 389-405
Varley H 1967 Practical clinical biochemistry 4th edn (New York: William Heinmann-Medical Books Ltd
and Interscience Books Inc) p. 802
A-J
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 25-35.
© Printed in India.
Four new species of trypanoplasms from the fresh water fishes
of the genus Mystus in Maharashtra
M A WAHUL
Department of Zoology, Marathwada University, Aurangabad 431004, India
Present address: Department of Zoology, Vidarbha Mahavidyalaya, Amravati 444 604, India
MS received 25 July 1984; revised 27 October 1984
Abstract. Four new species of haemoflagellates of the genus Trypanoplasma, Laveran and
Mesnil, 1901 viz T. krishnamurthyi, T. cavacii, T. vidyai and T. seenghali are described from the
fresh water fishes of the genus Mystus in Maharashtra.
Keywords. Haematozoa; haemoflagella'.ss; Trypanoplasma.
1. Introduction
Biflagellate organisms of the genus Trypanoplasma Laveran and Mesnil 1 90 1 from fresh
water fishes have been recorded from various parts of the world. The major reviews of
4his group are those of Pavlovaskii (1964), Becker (1970, 1977) and Lorn (1979).
However, this group received little attention in India. Mandal (1979) and Joshi (1982)
are the only workers who have described one and two species respectively. The present
contribution is the third of a series on this group in Maharashtra.
%
2. Material and methods
The material for this investigation was obtained from two rivers in two different
localities, namely Purna and Aurangabad in Maharashtra. The fishes were brought
alive to the laboratory for examination or were examined on the spot itself using a field
microscope. Smears were made from the blood obtained from the heart and no anti-
coagulent was used. The smears were air-dried, fixed in acetone-free methyl alcohol for
8-10 min and stained with Giemsa's stain diluted with phosphate buffer. The drawings
were using a Leitz camera lucida at a magnification of about 2000 x . The photo-
micrographs were taken with Leica M-3 camera. The identification of the fish hosts was
based on Day (1875), Shrivastava (1968) and Jhingran (1982). The slides of the type
material are deposited in the Protozoology Section, Department of Zoology,
Marathwada University, Aurangabad, India.
3. Observations and discussion
3.1 Trypanoplasma krishnamurthyi sp. nov. figures 1-4
Host: Mystus cavacius Hamilton.
Locality: River Puma, Parbhani Dist, Maharashtra, India.
Site of infection: Blood.
25
26
M A Wahul
Figures 1-4. Trypanoplasma krishnamurthyi sp. nov. from M . cavacius.l~3. Camera lucida
drawings. 4. Photomicrograph.
This trypanoplasm was present in two out of the four fishes (Mystus cavacius)
examined. The infection was moderate in both the cases.
Morphology
Cell body: The body of the trypanoplasm is short, broad and often irregular in shape.
Cytoplasm: The cytoplasm is vacuolated and granular, but does not show homo-
geneous appearance because of varying patterns of granulation in different regions.
Nucleus: The nucleus is dorsal and very characteristic in having a distinctly ovoidal
shape (length: width = 2:1) consistently. This is the only species found during the
present study having a consistent nuclear shape.
Kinetoplast: The kinetoplast is absent which is extremely characteristic, as no dis-
kinetoplastic trypanoplasm has ever been described so far, from fresh water fishes.
Flagella and undulating membrane: The two flagella arise from the kinetosomes which
Trypanoplasms from the fresh water fishes of the genus Mystus 27
are distinct and rod-like. The anterior flagellum is about as long as the body. The
posterior flagellum forms an *S* configuration as it runs over the body. The free trailing
portion of the posterior flagellum is long, being slightly less than the body length.
There is no clear evidence of the presence of an undulating membrane.
The details of dimensions of the trypanoplasm are given in table 1.
This is the first trypanoplasm to be described from this host species and the third
from a fish of the genus Mystus in India, the earlier records being those of T. indica
from M . vittatus and T. mysti from M. aor.
A comparison of this species with T. indica (Mandal 1979) shows that it is distinctly
smaller in size, measuring 15-53 -28-24 x 5-17 - 16 /im (19-89 x 10-13 /an) as against
25— 30-5 x 6 — 10-5 ^m (28-5 x 8 ^m) in the present form. This form is unique in
lacking a kinetoplast, while T. indica has a conspicuous one. It also has a much longer
trailing flagellum than T. indica.
It is marked off from T. mysti Joshi, 1982, by its smaller size, (19-89 x 10-13 /-im as
against 28*2 x 10-9 /-on) larger nucleus and distinctly longer flagella. The monomorphic
nature of this trypanoplasm distinguishes it from T. atti Joshi, 1982 which exists in two
forms. The two species are also marked off by differences in morphology and
morphometrics.
The only other record of trypanoplasm from fishes of the family Bagridae is
T. pseudobagri Chang, 1964 from Pseudobagrus fulvidraco in China. Unfortunately
neither its description nor any information about this species could be procured by the
author and hence no comparison could be made.
The only trypanoplasm without a kinetoplast recorded earlier is T. beckeri Burreson,
1979. That species was described from a marine fish Scorpaenichthyes marmoratus of
family Cottidae in the United States, while the present species is from a fresh water fish
of the family Bagridae in India. The body of the present form is relatively short and
broad (19-89 x 10-13 //m) as contrasted with the extremely long and twisted body of
T. beckeri (109 x 6-5 |im).
In view of its distinctness, this species is considered new to Science and designated
Trypanoplasma krishnamurthyi sp. nov. after Dr R Krishnamurthy of the Marathwada
University, Aurangabad, in grateful appreciation of his active guidance throughout the
course of this work.
Table 1. Body dimensions of T. krishnamurthyi sp. nov. from
M. cavacius (based on 50 forms).
Particulars (/im) Minimum Maximum Average
Length of ceil body
15-53
28-24
19-89
Width of cell body
5-17
16-00
1W3
Length of nucleus
3-28
11-29
6-91
Width of nucleus
141
6-12
3-08
Length of kinetoplast
—
__
—
Width of kinetopiast
—
—
—
Length of anterior
free flagellum
10-35
30-13
22-52
Length of posterior
free flagellum
11-29
27-30
18-38
28
M A Wahul
3.2 Trypanoplasma cavacii sp. nov. figures 5-10
Host: Mystus cavadus Hamilton.
Locality: Kham river, Waluj, Aurangabad, Maharashtra, India.
Site of infection: Blood.
This trypanoplasm was found as the only species in 10 out of the 51 fishes
(M. cavadus), examined. The infection was light in all the cases and a few dividing
forms were also observed (figure 10).
Morphology
Cell body: The body of the trypanoplasm is variable in shape. The elongated forms
have a T shaped configuration (figures 6, 7) with the anterior part being broad and
forming the longer arm and the posterior third being relatively narrower and forming
the shorter arm. In other cases, the body is short, broad and almost straight (figure 5).
- fr°m
p ' - ' • • an*« uc
9. Photomicrograph of 'Trophozoite'. 10. Photomicrograph of dividing form.
Trypanoplasms from the fresh water fishes of the genus Mystus
29
Cytoplasm: The cytoplasm is vacuolated and shows distinctly coarse chromophillic
granules, often abundant near the posterior end (figures 6, 7).
Nucleus: The nucleus is ovoid (figures 6, 7) to elongated (figure 8) and placed dorsally,
close to the anterior end.
Kinetoplast: The kinetoplast is relatively short (L:W = 2-4:1) distinctly triangular
(figure 6) or fusiform (figure 7) in the elongated specimens and slightly curved and
somewhat crescentic in the short forms (figure 5). The kinetoplast is ventral in position.
Flagella and undulating membrane: The two flagella arise from the kinetosomes, which
are placed just anterior to the kinetoplast. The anterior flagellum becomes free from the
body soon after the origin, and it is about as long as body or slightly more. The
posterior flagellum forms 2-3 distinct folds along the length of the body, before
becoming free. The free trailing part is about as long as the body.
There is no clear evidence of an undulating membrane.
The details of the dimensions of the trypanoplasm are given in table 2.
This trypanoplasm is described from the same host (i.e. M. cavacius) as the preceding
species i.e. T. krishnamurthyi sp. nov. It is easily distinguished from the earlier species by
the presence of a distinct kinetoplast, its slightly smaller size (17-63 x 9-58 urn as against
19-89 x 10-13 um) and by the presence of distinctly coarse chromophillic granules in the
cytoplasm. The present species is much smaller in size than T. indica (17-63 x 9*58 um as
against 28-5 x 8 jum), but has a nucleus and kinetoplast which are only slightly smaller.
It also has a conspicuously longer trailing flagellum (16-94 /mi) compared with T. indica
(10-5 Jim).
Compared with four other species of trypanoplasms described from fresh water
fishes in Maharashtra, i.e. T. saranae, T. lomi and T. solapurensis, Wahul (under
publication) and T. quadrii, Krishnamurthy and Wahul (under publication) it is marked
off by differences in the shape and size of the body and kinetoplast, the pattern of
distribution of chromophillic granules in the cytoplasm and by a much longer trailing
flagellum. Further it is distinguished from T. qadrii by its monomorphic nature.
This species has distinctly smaller body dimensions than T. mysti but has a larger
nucleus, a better developed kinetoplast and distinctly longer flagella. This species is also
much smaller than T. atii and is typically monomorphic besides having distinctly longer
flagella.
Table 2. Body dimensions of T. cavacii sp. nov. from M. cavacius
(based on 25 forms).
Particulars (/im)
Minimum Maximum Average
Length of cell body
15-06
20-24
17-63
Width of cell body
6-59
14-59
9-58
Length of nucleus
4-23
847
6-25
Width of nucleus
141
4-23
2-65
Length of kinetoplast
2-11
6-12
4-33
Width of kinetoplast
047
3-29
1-43
Length of anterior
free flagellum
13-65
23-06
20-41
Length of posterior
free flagellum
12-17
21-18
16-94
30
M A Wahul
It has an overlapping range in body length with T. bore Hi, Laveran and Mesnil ( 1 90 1 ),
T. cataractae Putz (1972), and T. cyprini, Plehn (1903) but is clearly much broader. It has
a larger nucleus than T. borelli and T. cataractae and a smaller kinetoplast than T.
cyprinL It has an overlapping but narrower length range with T. varium, Leger (1904),
but is contrasted from it by its definite shape, smaller kinetoplast and longer trailing
flagellum. It is marked off from all the other species by its size, being smaller than T.
abramidis Brumpt (1906), T. barbi, Brumpt (1906), T. acipenseris, loffet al (1926) and T.
guernei, Brumpt (1906) and larger than T. makeevi, Achemerov (1959), T. salmositica,
Katz (1951), T. markewitschi, Schapowal (1953) and T. pseudocaphirhynchi, Ostroumov
(1949).
In light of the above discussion, it is considered new and named Trypanoplasma
cavacii sp. nov. after the specific name of the host in which it was found.
13
10 urn
Trypanoplasms from the fresh water fishes of the genus Mystus 31
3.3 Trypanoplasma vidyai sp. nov. figures 11-15
o
Host: Mystus seenghala Sykes.
Locality: River Purna, Parbhani Dist., Maharashtra, India.
Site of infection: Blood.
This trypanoplasm was found in 8 (Mystus seenghala) out of the 28 fishes examined.
Out of the 8 infected fishes it occurred alongwith another species of Trypanoplasm
(T. seenghali sp. nov.) and a species of Trypanosoma in one while in the other seven it
occurred with another species of Trypanosoma. The infection was moderate in all cases.
Morphology
Cell body: The body of the trypanoplasm is short, broad and stumpy (L : W 2 : 1 ) with a
distinctly convex dorsal margin and a straight (figures 13-14) or curved (figures 11,12)
concave ventral margin. The anterior end is broad and rounded while the posterior is
bluntly conical (figure 14).
Cytoplasm: The cytoplasm is highly vacuolated (figure 14) and stains homogeneously
and intensely.
Nucleus: The nucleus is spherical (figure 11) to ovoidal (figure 14) and lies along the
dorsal margin in the anterior third of the body.
Kinetoplast: The kinetoplast is relatively short, broad and variable in shape, the L : W
ratio varying from 2:1 to 4: 1. The width of the kinetoplast is not uniform and the
posterior end is generally pointed. In most cases it runs partly along the ventral margin
and turns away from the margin into the cytoplasm (figures 11, 14).
Flagella and undulating membrane: The two very delicate flagella arise from the
kinetosomes which are placed just anterior to the kinetoplast. The anterior flagellum is
relatively long, being one and one-third times the body length. The posterior flagellum
is extremely characteristic, running along or very close to the dorsal surface of the body,
almost up to the posterior tip. In most cases there are hardly any undulations visible
(figure 11) and where they are present, the undulations are numerous, very small and
shallow attaching the flagellum to the body surface at several points (figures 12-14). It is
also characteristic by its free trailing part, which in almost all cases, recurves and
extends forward along the dorsal surface of the body and ends in a loop. The free part is
about as long as the body.
There is no clear evidence of the existence of a distinct undulating membrane.
The details of the dimensions of the trypanoplasm are given in table 3.
Table 3. Body dimensions of T. vidyai sp. nov. from M. seenghala
(based on 50 forms).
Particulars (^m) Minimum Maximum Average
Length of cell body
15-53
30-13
19-16
Width of cell body
- 7-53
15-53
11-84 .
Length of nucleus
4-70
9-41
6-76
Width of nucleus
1-88
5-64
3-15
Length of kinetoplast
2-82
6-82
4-78
Width of kinetoplast
0-70
3-29
1-43
Length of anterior
free flagellum
20-24
30-60
25-05
Length of posterior
free flagellum
11-77
26-36
18-06
32 MA Wahul
This is the first trypanoplasm to be described from M . seenghala and the fifth from
fishes of the genus Mystus. A comparison of this species with the other four as well as
the other species described from various other fresh water fishes show it to be distinct.
It has a relatively short, broad and stumpy body as contrasted with T. indica (19-16
x 11-84 /an as against 28-5 x 8-0 /on). The kinetoplast is shorter and broader than in
7. indica and the flagella distinctly longer. The presence of a distinct kinetoplast and a
definite body shape distinguish this species from T. krishnamurthyi sp. nov. Compared
with T. cavacii sp. nov. it is distinctly larger with a more stumpy appearance because of
its broad body. It also has a larger nucleus which is more spherical or ovoid and clearly
longer flagella than the latter. Its smaller size, typical and almost constant body shape,
much larger kinetoplast and distinctly longer and delicate flagella, distinguishes this
species from T. mysti.
Its monomorphic nature, longer flagella and the shape and size of the kinetoplast
differentiate it from T. aiti and T. qadrii Krishnamurthy and Wahul (under publication).
The shape and size of its body, the short and broad nature of its kinetoplast and long
flagella demarcate this species from the others described from this area, namely T.
saranae, T. lomi and T. solapurensis Wahul (under publication).
The broad and stumpy body of this species and the nature of its kinetoplast
distinguish this species from all the others described so far.
The species under discussion is unique in having a posterior flagellum running very
close to the dorsal surface throughout the length of the body and in having the free
trailing part recurving forwards and often forming a loop.
In view of these differences it is considered new and designated Trypanoplasma vidyai
sp. nov.
3.4 Trypanoplasma seenghali sp. nov. figures 16-20
Host: Mystus seenghala Sykes.
Locality: River Purna, Parbhani Dist, Maharashtra, India.
Site of infection: Blood.
This trypanoplasm was found in only one fish (Mystus seenghala) out of the 28
examined. This fish also harboured another species of Trypanoplasma (T. vidyai sp.
nov.) and a species of Trypanosoma.
The infection was light in the case of the present species.
Morphology
Cell body: The body of the trypanoplasm is elongated (L:W = 3: 1) with a typical 'C
(figure 19) or 1U' (figure 18) shaped configuration. The anterior third is almost twice as
broad as the posterior third. The anterior end is bluntly conical and the posterior end
tapering to a rounded tip.
Cytoplasm: The cytoplasm is vacuolated, granular and stains homogeneously.
Nucleus: The nucleus is oval (figure 19) to elongated (figure 16) and situated dorsally in
the anterior third.
Kinetoplast: The kinetoplast is elongated (L:W = 6:1) apparently straight, stiff and
rod like (figures 17, 18). The anterior half is broader than the posterior. The posterior
end tapers to a point and runs along or close to the ventral margin.
Trypanoplasms from the fresh water fishes of the genus Mystus
33
19
• . •
Figures 16-20. Trypanoplasma seenghali sp. nov. from M. seenghala. 16-19. Camera lucida
drawings. 20. Photomicrograph.
Flagella and undulating membrane: The two delicate flagella arise from the kineto-
somes, which are placed just anterior to the kinetoplast. The anterior flagellum is
almost as long as the body. The posterior flagellum runs along the dorsal surface and
becomes free posteriorly. The free trailing part of the posterior flagellum is about three
fourths of the body length. During its course the posterior flagellum is thrown into two
conspicuous folds, one near the junction of the anterior and middle thirds of the body
and the other near the junction of the middle and posterior thirds (figures 17, 18) and in
some cases two or three very small folds in the posterior third (figures 17, 19).
There is no clear evidence of a distinct undulating membrane.
The details of dimensions of the trypanoplasm are given in table 4.
This is the second species of trypanoplasm to be described from this host species (i.e.
M. seenghala). Though it is described from the same host as T. vidyai sp. nov., it is easily
marked off from that species by conspicuous differences in the shape and dimensions of
A— 10
34 MA Wahul
Table 4. Body dimensions of T. seenghali sp. nov. from
M. seenghala (based on 25 forms).
Particulars (/mi) Minimum Maximum Average
Length of cell body
15-53
32-95
25-27
Width of cell body
5-17
10-82
8-00
Length of nucleus
3-76
9-88
6-93
Width of nucleus
1-41
4-23
2-80
Length of kinetoplast
3-76
8-00
5-70
Width of kinetoplast
0-47
1-88
0-98
Length of anterior
free flagellum
20-71
37-30
23-69
Length of posterior
free flagellum 12-24 25-42 17-84
the body, the different nature of the kinetoplast and the pattern of undulations of the
posterior flagellum. Its characteristic shape, typically elongated kinetoplast and longer
flagella contrast it from T. indica. Its distinctly larger size and presence of a kinetoplast
differentiate it from T. krishnamurthyi sp. nov. which is smaller and lacks a kinetoplast.
It differs from T. cavacii sp. nov. being larger in size and having a relatively elongated
and narrow kinetoplast.
It is marked off from T. mysti by its distinctly longer kinetoplast, larger nucleus and
conspicuously longer flagella.
Its monomorphic nature marks it off from T. atti and T. qadrii Krishnamurthy and
Wahul (under publication), while its characteristic shape, kinetoplast and body
dimensions distinguish it from all the species described so far from this area. This is the
largest of the trypanoplasms found during the present study and the only species which
is close to this in body dimensions is T. solapurensis Wahul (under publication) which
has a length range of 16-94 x 28-24 /an as compared with 15-53 x 32-95 /an here.
However, the present species is much broader and differentiated from that species by
the shape and size of the nucleus and kinetoplast and distinctly longer flagella.
Compared with the various other monomorphic trypanoplasms described so far, it
has an overlapping length range with T. abramidis and T. barbi. However, its body is
broader than that of T. barbi and narrower than that of T. abramidis. It also has a
kinetoplast which is shorter than in the two species and a trailing flagellum which is
distinctly much longer, besides slight differences in the size, shape and position of the
nucleus.
Its body dimensions mark it off from the rest of the species.
In view of the above discussion, this species is considered new and designated
Trypanoplasma seenghali sp. nov. after the specific name of the host.
Acknowledgements
The author is thankful to Dr R Nagabhushanam for laboratory facilities and
Dr R Krishnamurthy for constant encouragement and guidance. Thanks are also due
to the UGC, New Delhi, for the award of a Teacher-fellowship.
Trypanoplasms from the fresh water fishes of the genus Mystus 35
References
Becker C D 1970 Haematozoa of fishes, with emphasis on north American records. Special publication No. 5
82-100 American fisheries Society of Washington
Becker C D 1977 Flagellate parasites of fish in Parasitic protozoa (ed) Kreier (New York: Academic Press)
Vol. I 357-416
Brumpt E 1906a Sur quelques especes nouvelles de-trypanosomes parasites des poissons deaes douce: leur
mode devolution; C.R. Soc. Biol Paris 60 160-162
Brumpt E 1906b Mode de transmission et evolution des trypanosomes des poissons. Description de quelques
especes de trypanoplasmes des poissons d'eau douce. Trypanosome d un Crapaud africain; C.R. Soc. Biol.
Paris 60 162-164
Burreson E M 1979 Structure and life cycle of Trypanoplasma beckeri sp. n. (Kinetoplastidae) a parasite of the
cabezon, Scorpaenichthys marmoratus in Oregon coastal waters; J. Protozool 26 343-347
Day F 1875-78 The fishes of India, being a natural history of fishes known to inhabit the seas and fresh water
of India, Burma and Ceylon. Text and Atlas in 4 Pts. London,
loffl G, Bozhenko V B and Levashev M M 1926 Trypanoplasma acipenseris sp. n. a new parasite of sterlet
blood. (In Russian.) Russkhyi Gidrobiologhicheskyi Zhurnal 5 103-110
Jhingran V G 1982 Fish and fisheries of India. II Ed. (Delhi: Hindustan Publishing Corporation).
Joshi B D 1982 Three new species of haematozoans from fresh water teleosts (pisces); Proc. Indian Acad. Sci.
(Anim. Sci.) 91 397-406
Katz M 1951 Two new haemoflagellates (genus Cryptobia) from some western Washington teleosts;
J. Parasitol 37 245-250
Krishnamurthy R and Wahul M A 1984 Studies on the morphology of some trypanoplasms from the blood
of fresh water fishes in Maharashtra. II. From the fishes of the genus Labeo\ Arch. Protistenk. (in press)
Laveran A and Mesnil F 1901 Sur les flagelles a membrane andulante des poissons (generes Trypanosoma
Gruby et Trypanoplasma n. gen.). C.R. Acad. Sci. Paris 133 670-675
Leger L 1904 Sur les haemoflagelles du Cobitisbarbatula L. Comptes Rendus de la Societe de Biologic 57
344-345
Lorn J 1979 Biology of trypanosomes and trypanoplasma offish in "Biology of kinetoplastida Vol. II (ed)
W H R Lumsden and D A Evans, (London: Academic Press) 269-337
Mandal A K 1979 Studies on the haematozoa of some cat fishes, belonging to the genus Mystus scapoli from
India; Bull. Zool Surv. India 2 17-23
Pavlovaskii E N 1964 Key to parasites of fresh water fish of USSR Academy of sciences of the USSR.
Zoological Institute, Isreal Program for scientific Translations, Jerusalem
Plehn M 1903 Trypanoplasma cyprini sp. n.; Arch. Protistenk 3 175-180
Putz R E 1972 Cryptobia cataractae sp. n. (Kinetoplastida, Cryptobiidae) a haemofiagellate of some cyprinid
fishes of West Virginia. Proc. Helminth. Soc. (Washington) 39 18-22
Srivastava G 1968 Fishes of Eastern Uttar Pradesh. Vishwavidyalaya Prakashan, Varanasi
Wahul M A 1984 Studies on the morphology of some trypanoplasms from the blood of fresh water fishes in
Maharashtra, I. From fishes of the genus Puntius; Arch. Protistenk. (in press)
A—
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 37-48.
© Printed in India.
Mechanism of resistance in rice varieties showing differential
reaction to brown planthopper
K VENUGOPALA REDDY and M B KALODE
Directorate of Rice Research (Formerly AICRIP), Rajendranagar, Hyderabad 500030, India
Abstract. A total of 1070 rice varieties, mainly from Assam Rice Collection, were evaluated
to identify better sources of resistance to brown planthopper, Nilaparvata lugens (Stal). In
mass screening replicated tests 17 varieties were identified as resistant. Moderate resistance was
observed in 73 varieties.
All the resistant and moderately resistant varieties were relatively less preferred by nymphs
and there was a positive correlation between the number of nymphs settled and the damage
score. Test varieties non-preferred by adult insects for feeding and shelter were also less
suitable for oviposition with the exception of ARC 13854, and ARC 14766A. On resistant
varieties the nymphal survival was much lower (18-5-28-4%) and nymphal duration was
prolonged by 5-7 days as against those on the susceptible check. Results of probing behaviour
tests indicated that resistant varieties received more number of probing punctures (80-121)
than the susceptible check (31). Further, insects caged on resistant varieties quickly lost their
body weight while those on the susceptible check registered gain in weight. Honey dew
excretion by brown planthopper adults on resistant varieties was 6-6 to 1 1-9 times less than that
on susceptible T(N)1. Selected varieties showing moderate damage reaction (ARC 5918, ARC
10443, ARC 13984, ARC 14529 and ARC 14864) exhibited more feeding marks, greater amounts of
excretion, and higher gain in body weight of the insects, thus confirming a moderate degree of
resistance. Based on various parameters, ARC 5780, ARC 5988 and ARC 14394 were comparable
to resistant check, Ptb 33 in level of resistance. No association of Lemma and Palea colour with
brown planthopper resistance was observed in the rice varieties tested.
Keywords. Varietal resistance; rice varieties; Nilaparvata lugens.
1. Introduction
In India the brown planthopper (Nilaparvata lugens (Stal)) has assumed greater
importance since its outbreak in Kerala during 1973-74 and subsequently in many
other parts of the country. Host plant resistance as a component of pest management
programme is being successfully utilized in Philippines and Indonesia in controlling
this pest. In view of the more virulent biotype in India, screening for resistance to brown
planthopper was carried out at various research institutes and the number of resistant
donors identified have been reported (Kalode and Krishna 1979; Kalode et al 1983;
Kalode 1983). Though there are reports from India about nymphal non-preference for
certain rice varieties, no detailed information is available on adult preference.
Investigations were, therefore, undertaken at the AICRIP, Hyderabad to study the
reaction of selected varieties/cultures to both nymphs and adults of brown planthopper
(BPH) as well as to understand the mechanism manifesting different degrees of
resistance, so that better varieties (donors) with desirable characters could be utilized
effectively in resistance breeding programme.
37
38 K Venugopala Reddy and MB Kalode
2. Material and methods
2.1 Mass rearing and varietal screening v
BPH was reared on 30 day old T(N)1 plants inside the green house provided with
coolers to maintain the temperature at 30 ± 5°C to ensure uniform and steady supply of
insects. The rearing cages (70 x 62 x 75 cm) were provided with glass panels with a
small window on one side and fine nylon wire mesh on the other sides. Pre-mated gravid
females were allowed to oviposit on plants for two days and the emerging nymphs were
further maintained to get age specific insects for different experiments.
ARC cultivars (1000), 20 varieties from IRRI and another 50 cultures were screened by
adopting the modified mass screening layout (Kalode et al 1975). Pre-germinated seeds
were sown in rows in wooden flats along with susceptible and resistant check, which
were then transferred to galvanized iron trays filled with water to maintain adequate
humidity and to prevent ants. Seven day old seedlings were infested with a large number
of 1-2 instar nymphs so as to get 5 to 10 insects/seedling and were scored for damage
reaction on a 0-5 scale when more than 90 % of T(N)1 seedlings were killed. Test
varieties showing damage score up to 2-5 in a preliminary test were retested, replicated 3
times to confirm their reaction.
2.2 Studies on preference/non-preference mechanism on selected rice varieties
2.2a Response of nymphs: During the retest, the number of nymphs settled on each
seedling was counted at different intervals viz., I day, 3 days, 5 days and 7 days after
infestation to assess the nymphal preference for different varieties.
2.2b Response of adults for settling and oviposition: About 30 selected varieties
including resistant and susceptible checks were grown randomly (8cm apart) in
polythene sheet lined wooden flats. Each variety was replicated four times with seven
seedlings per replication. Thirty days after sowing, each wooden flat was transferred to
a suitable cage and a large number of adults were released. The counts of adult insects
settled on each seedling were taken at 12, 24 and 48 hr after release. The plants were
then cut as close to the base as possible and the number of eggs laid per seedling was
recorded by staining in 1 % erythrocin dye in an aqueous solution as suggested by Naito
(1964) under a binocular microscope.
2.3 Studies on antibiosis mechanism
2.3a Survival and development of nymphs: Thirty six resistant and moderately
resistant varieties were included along with resistant and susceptible checks Ptb 33 and
T(N)1, respectively. Seeds of each variety were sown in earthen pots and each variety
was replicated 6 times. Thirty days after sowing each plant was caged with 10 freshly
hatched nymphs in mylar film cages (5 x 45 cm) the open end of which was closed with
fine muslin cloth. The counts of surviving nymphs were taken, 24 hr after infestation
and thereafter once in five days till 20 days.
Mechanism of resistance in rice varieties
39
2.3b Feeding response of adult brown planthopper: Thirty varieties which were
identified as resistant and moderately resistant in mass screening test were included for
various investigations. The varieties were grown in wooden flats along with the resistant
and susceptible checks.
2.3b(i) Attempts of feeding — Probing marks: Seven days after germination, the
seedlings of each variety were removed from the flats and washed and then transferred
individually into test tubes (2x17 cm) containing water. Two gravid females were
released in each tube. Twelve hours later, the seedlings were transferred into 70 % ethyl
alcohol. These were then stained and the probing marks counted as described earlier.
2.3b(ii) Amount of feeding — change in body weight: Five adult insects per replication
were first weighed in a small vial and then starved for 3 hr. The insects were then
allowed to feed on 15-day old seedlings in a test tube for double the time of starvation
i.e. 6 hr. The insects were again weighed to assess gain or loss in body weight. Each
variety was replicated 5 times.
2.3b(iii) Amount of feeding — honey dew excretion: Each variety was replicated five
times with single thirty day old plant per pot. The plant was drawn through a wooden
plank which rested on the rim of the pot. Whatman No. 1 filter paper was placed on the
plank by drawing the plant through a slit made in the centre. Then each plant was caged
with an inverted glass funnel along with ten pre-starved adult insects (figure 1). The
Figure 1. A set-up for honey dew collection.
40 K Venugopala Reddy and M B Kalode
adults were allowed to feed for 24 hr. The filter papers were removed, dried and sprayed
with 0-2 % ninhydrin solution, which turned the areas of honey dew excretion to
pinkish violet. The coloured portions were dissolved in 80 % ethanol and the amount of
honey dew excreted was determined by reading the concentration in the spectrophoto-
meter at 540 m^u.
2.4 Association of Lemma and Palea colour with resistance
Lemma and Palea colour of the grains of all the varieties (1070) was noted and
statistically analysed by the %2 test to know the association of Lemma and Palea colour
with BPH resistance.
3. Results and discussion
3.1 Mass screening
Of the 1070 varieties evaluated in the preliminary tests, 152 varieties showed damage
grade up to 2-5 on a 0-5 scale. These were retested in replicated test for confirmation of
reaction. ARC 5754, 5757, 5764, 5780, 5838, 5973, 5981, 5500, 5988, 13507, 12864, 13854,
13966, 14394, 14539, 14766(A) and 14903 were resistant (damage score up to 1-5) and 73
varieties indicated moderate degree of resistance recording damage score of 1-6 to 3.
The rest of the entries recorded higher damage reaction indicating that these varieties
might have escaped the damage in the preliminary test. About 24-36 varieties showing
different degrees of resistance were selected for various tests.
3.2 Studies on preference /non-preference mechanisms
3.2a Response of nymphs: It is evident from table 1 that all the resistant and
moderately resistant varieties were relatively less preferred as compared to susceptible
check T(N)1. The nymphs could locate the feeding site within 24 hr and no distinct
variation was observed between the varieties after 24 hr of release. However, on
majority of the resistant varieties there was a decreasing trend in the number of
nymphal population settled between 1 day and subsequent observations while on the
susceptible variety T(N)1 more number of nymphs were noted. On an average, 4-2-6-5
and 4-5-7-2 nymphs were recorded on resistant and moderately resistant varieties
respectively as against 12-7 nymphs on the susceptible check T(N)1. Non-preference
mechanism was reported to be a factor of resistance in BPH as early as 1969 (IRRI, 1969).
Kalode and Krishna (1979) and Kalode et al (1978) reported that Ptb 33, Ptb 21, Leb
Mue Nahng, ARC 6650 and CR 57-MR 1523 had less number of BPH nymphs as compared
to T(N)1 and suggested the possibility of some attractants in the susceptible variety.
Absence of feeding stimulants or presence of feeding deterrants/repellents could be
other possible reasons for non-preference. In the present investigation, a positive
correlation was observed with regard to the number of nymphs and damage grade.
Higher the number of nymphs settled greater was the damage and vice-versa. It appears
that non-preference has a definite role in the manifestation of resistance in some of the
varieties tested.
Mechanism of resistance in rice varieties
41
Table 1. Preferential response of N. lugens (Stal) nymphs and adults on selected rice
varieties.
Reaction to nymphs
Reaction to adults
Damage
ARC No. score
Average of nymphs
settled after days*
No. of adults/seed-
ling after hours
Av. no. of
eggs per
seedling
1
7
12
36
Resistant (R)
5780
1-1
4-2
4-2
1-3
1-0
17-7
5973
1-1
5-7
4-7
2-2
2-2
33-2
14539
1-4
5-1
4.9
2-1
1-3
32-7
13854
1-4
5-1
4-8
3-0
4-3
66-3
5838
1-5
6-7
6-1
1-7
1-2
20-1
5981
1-5
6-6
5-7
1-7
1-8
28-0
5754
1-5
7-3
6-5
1-0
1-6
22-1
14394
1-5
4-4
4-1
1-3
1-0
34-1
14766A
1-5
5-1
4-9
1-7
2-9
41-7
13507
1-5
5-1
5-1
3-3
1-7
36-3
5988
1-5
5-3
4-7
1-4
1-6
23-7
Moderately resistant
(MR)
5913
1-6
7-3
6-3
2-2
2-2
26-8
14426
1-7
4-5
4-5
2-9
3-8
63-2
15381
1-7
5-3
5-3
2-9
3-9
38-2
5916
1-7
5-7
5-3
1-6
2-1
30-7
5912
1-8
6-2
6-0
2-5
3-0
44-7
13522
1-9
5-4
5-2
1-8
1-0
29-5
5906
2-0
8-1
7-2
1-6
2-0
39-8
5924
2-0
6-0
5-7
1-6
2-5
40-2
5918
2-0
6-1
5-6
1-8
2-8
42-3
Ptb21
2-1
5-5
5-2
2-3
1-3
28-0
14864
2-1
5-8
5-6
1-9
2-5
38-0
13984
2-2
5-7
5-0
2-0
1-0
25-0
10443
2-2
6-0
5-7
2-7
3-3
44-1
Ptb33
1-2
5-0
4-2
1-3 to
0-9 to
16.5 to
(Resistant check)
1-5
1-1
25-9
T(N)1
5-0
10-4
12-7
6-0 to
8-5 to
186-9 to
(Susceptible check)
8-9
9-1
210-3
* Average of 4 observations.
3.2b Response of adults: Marked differences were observed in the preference of
adult BPH after 36 hr, although some differences were apparent even after 12 hr of their
release (table 1). ARC 5780 was least preferred by adults followed by resistant check Ptb
33 and ARC 5838, ARC 5754, ARC 5988 for settling and oviposition. Even on moderately
resistant varieties the number of adults settled (1-3-9) and the number of eggs laid
.(25-63-2) were comparatively lower as compared to the susceptible check T(N)1 on
which 8-5-9 adults settled and 186-9-2 103 eggs were laid. However, on some of the
resistant varieties (ARC 13854 and ARC 14766A) comparatively more number of eggs
42
K Venugopala Reddy and M B Kalode
were deposited as compared to other resistant varieties. Choi et al (1979) reported that
resistant varieties which were non-preferred for feeding did not exhibit the same trend
towards opposition also. The reasons could be inconsistent feeding on resistant
varieties. In the present investigation varieties which were non-preferred for feeding
and shelter were also non-preferred for egg laying with the exception of ARC 13854 and
ARC 14766A. The ovipositional preference for these two resistant varieties, may be due
to the presence of ovipository stimulants which needs further investigation.
3.3 Studies on antibiosis mechanism
3.3a Survival and development of nymphs: Antibiosis studies carried out with thirty
six varieties indicated that some resistant varieties had adverse effects on BPH nymphs
resulting in low survival of the insects (figure 2) as evident with resistant check Ptb 33
(16-5 %), ARC 5780 (18-5 %), ARC 5988 (22-5 %), ARC 5838 (25-6 %), ARC 5981 (26-5 %), ARC
5973 (27-5 %), ARC 5782 (28-5 %), ARC 14766A (26-7 %) and ARC 14394 (28-4 %) as against
90-93-4% on T(N)1. These varieties also adversely affected the development where the
nymphal period was delayed by 3-7 days as compared to tJiat on T(N)1. BPH nymphs
took 23 days on resistant check Ptb 33 and ARC 14394; 22 days on ARC 5780, ARC 14766A
and ARC 13854.
It was observed that from the 6th to the 21st day there was a gradual decrease in the
survival of nymphs. This might be due to nutritional deficiency in the test varieties.
However, some varieties viz., ARC 5780, ARC 5988, ARC 5838 had shown relatively higher
antibiosis effects. Karim (1975), reported that the survival of the nymphs on resistant
varieties *B5, Ptb 20 and Mudgo ranged from 8-17 % just 3 days after caging and only
1 and 2 % of the caged nymphs on Ptb 20 and xB5 reached the adult stage.
In the present study, a high level of nymphal mortality was not observed as evident by
only 19-5-30 % mortality up to the sixth day. This may be either due to the ability of the
, V. nymphal survival at 21 days afttr caging
15
10-
/-
-SO
to
25-
20 -
IS -
10 -
5-
(O 2
0. _.
Figure 2.
varieties.
Rate of development and percentage survival of N. lugens nymphs on selected rice
Mechanism of resistance in rice varieties
43
biotype involved in the present study to tolerate antibiosis effects in earlier stages of
development or the varieties involved might not be having such high concentrations of
toxic components required to induce a high mortality in a short period. However, the
resistant varieties could confirm their resistance by virtual low survival of BPH on them,
while some moderately resistant varieties had relatively higher survival of BPH nymphs
and had relatively low antibiosis effects on them. This indirectly suggests that these
varieties might be tolerant to the BPH.
3.3b Feeding response of adult BPH
3.3b(i) Attempts of feeding — Probing marks: The results of the probing behaviour
indicated that the resistant varieties received more number of probing punctures than
the susceptible ones. T(N)1 the susceptible check recorded the least number of probing
punctures (31-2) whereas a greater number of probing punctures was observed on
resistant varieties (figure 3). Resistant varieties viz., ARC 14394 (121), ARC 14766A (1 19-4),
ARC 13507 (115-6), ARC 5780 (100-2), ARC 5838 (94-6), ARC 5754 (83-6), ARC 5988 (83), ARC
5973 (80) and moderately resistant variety ARC 14803 (100-2) received the maximum
number of punctures. On the other hand, varieties viz., Mudgo and ASD 7 reported to be
resistant to biotype 2 in Philippines, received less number of probing marks compared
to T(N)1 in the present investigation indicating that these varieties were suitable for
feeding by the test insect.
It was also observed that the percentage of probing marks on the leaf blade was more
on the resistant varieties than on the susceptible varieties. The results indirectly revealed
that non-preference of BPH to certain varieties may be gustatory rather than olfactory or
visual.
120 r
I 80
D
on leaf sh«ath
on leaf blade
uo
100
60
20
^ in Jr _.
3 « 0- -
Figure 3. Probing punctures made by N. lugens adults on selected rice varieties.
44
K Venugopala Reddy and M B Kalode
Karim (1975) reported that the varieties xB5 and HR 12 received significantly higher
number of punctures (50-9 and 48-2 respectively) which were about 2-10 times more
than that received by other resistant varieties.
3.3b(ii) Amount of feeding — change in body weight: In earlier experiments it was
observed that the insects suffered high mortality and made more feeding marks when
caged on resistant varieties. In order to know whether the insects had actually fed on the
test varieties or not, experiments were carried out on the amount of feeding done by the
insects. The results showed that the insects caged on the resistant varieties lost their
body weight; while on some of the moderately resistant varieties and susceptible check
they gained weight (figure 4). Insects lost their body weight up to a maximum of over
33-3 % on ARC 5780 followed by 30 % on ARC 13854 and ARC 13507. However, insects fed
on ARC 10443, ARC 6601, ARC 5918, ARC 14864, ARC 13984 and ARC 14529 gained some
body weight (5-7 to 13-9%) as compared to 27-6% on the susceptible check.
Sogawa (1982) reported that the reduced concentration of phagostimulant amino
acids might be the reason for less intake of sap and loss in body weight of the insects
which fed on resistant varieties.
3.3b(iii) Amount of feeding — honey dew excretion: Amount of feeding by the insects
was judged by the honey dew deposited when they were allowed to feed for specified
period of time on selected varieties. The results indicated that the BPH adults fed very
little on resistant varieties and in turn excreted honey dew in traces (table 2). On
resistant varieties ARC 5780, ARC 5973, ARC 5858, ARC 5754, ARC 14539, ARC 13854, ARC
14394, ARC 14766A and ARC 13507, the hoppers excreted as little as 6-6-11-9 times less
than that on susceptible T(N)1. As observed in the previous experiments, there was a
higher nymphal mortality and loss in body weight on these varieties. These varieties had
also received higher number of probing punctures. The less honey dew excretion while
I20
j£ -20
Ftfl
I
f K
i °
-20
-40
Figure 4, Gain or loss in body weight of N. lugens adults on selected rice varieties.
Mechanism of resistance in rice varieties 45
Table 2. Amount of honey dew excreted by brown planthopper adults
caged on selected rice varieties for 24 hr.*
Variety Average Ratio
Resistant (R)
ARC 5780 0-20 10-10
ARC 5973 0-29 6-52
ARC 5838 0-17 11-88
ARC 5981 0-23 7-83
ARC 5754 0-28 6-75
ARC 5988 0-22 9-18
ARC 14539 0-34 6-47
ARC 13854 0-28 7-88
ARC 14394 0-20 9-45
ARC 14766A 0-18 10-80
ARC 13507 0-13 6-66
Moderately resistant (MR)
ARC 5913 0-66 2-86
ARC 5916 0-29 6-52
ARC 10840 0-56 3-38
ARC 5912 0-33 6-12
ARC 5924 0-65 2-91
ARC 5906 0-83 2-43
ARC 5918 0-87 2-32
ARC 5927 0-58 3-30
ARC 10443 0-73 2-60
ARC 14803 0-56 3-93
ARC 14426 0-49 4-49
ARC 15381 0-73 3-01
ARC 13522 0-81 2-72
Ptb 21 0-78 2-82
ARC 14864 0-84 2-62
ARC 13984 1-03 2-13
ARC 14529 1-05 2-10
Ptb' 33 (Resistant check) 0- 14- 1 1 -78-
0-16 14-42
T(N)1 (Susceptible check) 1-89- 1-00
2-20
Ten insects per plant; five replications per variety.
feeding on resistant variety may be due to the presence of feeding repellants and/or
feeding deterrants or lack of feeding stimulants. It was also observed that the amount of
honey dew excreted was in accordance with the degree of resistance, i.e., the insects
excreted comparatively little honey dew on resistant varieties than on moderately
resistant varieties and susceptible check T(N)1.
Honey dew excretion was considered to be directly proportional to food intake by
the insects (Maxwell and Painter 1959; Sogawa and Pathak 1970). Sogawa (1982)
opined that reduced concentration of phagostimulant ammo acids might be one of the
reasons for the less amount of excretion of honey dew on resistant varieties. Kalode and
46
JiT Venugopala Reddy and M B Kalode
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Mechanism of resistance in rice varieties 47
Krishna (1979) reported that resistant cultivars (Ptb 33, Ptb 21, MR 1523 and ARC 6650)
restricted insect feeding and only a little amount of honey dew was excreted by the
insects during feeding on these varieties. They further stated that on Leb Mue Nahng
and T(N)1 the insects had excreted heavily.
3.4 Association of Lemma and Palea colour with brown planthopper resistance
Lemma and Palea colour of 1070 rice varieties noted during the preliminary mass
screening test was statistically analysed. The results were non-significant indicating that
there was no association of Lemma and Palea colour with BPH resistance.
The overall performance of selected rice varieties based on various parameters to
understand the mechanism of resistance as summarised in table 3 indicated that insects
fed little on the resistant varieties viz., ARC 5780, ARC 5988, ARC 14394, ARC 5838, ARC
14539, ARC 14766A, ARC 13507, ARC 5981, ARC 5754, ARC 5973, ARC 13854, ARC 14803 and
lost their body weight. The nymphs also exhibited high mortality on these varieties, and
their development period was also prolonged. The mechanism in manifestation of
resistance to BPH appeared to be due to non-preference and antibiosis. Most of the
varieties which were less preferred by nymphs were also less preferred by adults for
settling and oviposition.
However, in some of the moderately resistant varieties like ARC 5918, ARC 10443, ARC
13984, ARC 14529 and ARC 14864 relatively less number of feeding marks, higher amount
of honey dew excretion and gain in body weight were observed as compared to the
resistant varieties indicating intermediate reaction to the pest. It is evident that the
number of probing punctures, amount of honey dew excretion and gain or loss in body
weight are all inter-related to the degree of resistance. Based on these parameters ARC
5780, ARC 5988 and ARC 14394 were comparable to resistant check, Ptb 33 in level of
resistance.
This paper is part of the M.Sc. thesis submitted by the senior author to the Andhra
Pradesh Agricultural University, AP.
References
Choi S Y, Heu M H and Lee J O 1979 Varietal resistance to brown planthopper in Korea, in Brown
planthopper: Threat to rice production in Asia (Los Banos: International Rice Research Institute)
pp. 171-186
International Rice Research Institute (IRRI) 1969 Annual Report (Los Banos: International Rice Research
Institute)
Kalode M B 1983 Leaftiopper and planthopper pests of rice in India; Proc. of First Int. Workshop on
Lea/hoppers and Planthoppers of Economic Importance (London: Commonwealth Institute of
Entomology) pp. 225-245
Kalode M B and Krishna T S 1979 Varietal resistance to brown planthopper in India, in Brown planthopper:
Threat to rice production in Asia (Los Banos: International Rice Research Institute) pp. 187-199
Kalode M B, Kasi Viswanathan P R and Seshu D V 1975 Standard test to characterise host plant resistance to
brown planthopper in rice; Indian J. Plant Prot. 3 204-206
Kalode M B, Krishna T S and Gour T B 1978 Studies on pattern of resistance to brown planthopper
Nilaparvata lugens in some rice varieties; Proc. Indian Natl. Sci, Acad. 44 43-48
Kalode M B, Mangal Sain, Bentur J S and Kondal Rao Y 1983 Investigations on host resistance in rice to
insect pests; Proc of Rice Pest Management Seminar (Coimbatore: Tamil Nadu Agricultural University)
pp. 182-191
48 K Venugopala Reddy and M B Kalode
Karim A N M R 1975 Resistance to the brown planthopper Nilaparvata lugens (Stal) in rice varieties M.S.
Thesis, University of Philippines p. 131
Maxwell F G and Painter R H 1959 Factors effecting the rate of honey dew deposition byThesiaphis maculata
(Buck) and Toxoptera graminum (Rond.); J. Econ. Entomol. 52 368-373
Naito A 1964 Methods for examination of the feeding of leafhoppers and planthoppers and its application;
Jap. Plant. Prot. 18 482-484
Sogawa K 1982 The rice brown planthopper: Feeding physiology and host plant interaction; Ann. Rev.
Entomol. 27 49-73
Sogawa K and Pathak M D 1970 Mechanism of brown planthopper resistance in Mudgo variety of rice
(Homoptera: Delphacidae); Appl. Entomol. Zool 5 145-158
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 49-56.
© Printed in India.
Larva! and post-larval development of Spodoptera litura
(Fabricius) on some host plants
G P GARAD, P R SHIVPUJE and G G BILAPATE
Marathwada Agricultural University, Parbhani 431401, India
MS received 19 January 1984; revised 20 November 1984
Abstract. The tobacco caterpillar, Spodoptera litura (Fabricius) was reared on different host
plants under laboratory conditions. Th's article reports the growth and development of
S. litura on different host plants.
Keywords. Spodoptera liturai host plants; larval development; post-larval development.
1. Introduction
The tobacco caterpillar, Spodoptera litura is a serious and polyphagous pest of many
economically important crops (Basil 1943; Thobbi 1961; Bhattacharya and Rathore
1977). It is a well-documented fact that food plants play a vital role in development,
survival and reproductive potential of insects. In the present article we report the
growth and development of S. litura on different host plants.
2. Material and methods
The eggs obtained from the moths reared as larvae on the leaves of castor, okra,
groundnut and sunflower were kept in petridishes for hatching. The neonate larvae
(100) were reared individually in plastic boxes (5x5 cm) on the respective host plants.
Food was changed when required. Larvae pupated in the sieved moist soil provided in
the plastic boxes. Observations on larval duration, per cent larvae pupated and larval
weight on the llth and 14th day after hatching were recorded. Sexing was made in the
pupal stage. The pupal length, width and weight were recorded. The adults emerging
from the respective host plants on the same day were paired and released for egg laying
in plastic jars (12 x 15 cm) covered with muslin cloth held in position by rubberband.
Cotton swabs soaked in 10 % honey was provided daily for adult feeding. Paper strips
(7-5 x 5 cm) folded in a zig-zag fashion was provided as oviposition sites. The egg
clusters laid were separated from the muslin cloth and paper strips and thus the
fecundity was worked out.
3. Results and discussion
The host plants exhibited differential response with respect to the percentage pupation,
larval duration and growth index of S. litura (table 1). The percentage pupation ranged
from 44-33-92-21 % on groundnut and okra. The order of suitability of host plants for
AQ
50
G P Garad, P R Shivpuje and G G Bilapate
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Larval and post-larval development of S. litura 51
pupation was okra > sunflower > castor > groundnut. The larval duration also varied
significantly and ranged from 11-50 days on castor to 19-52 days on groundnut. The
suitability of host plants for larvae was in the order: castor > sun-
flower > okra > groundnut. The mean larval weight on the ilth day of age was high
(941-26 mg)on castor followed by sunflower, okra and groundnut
A perusal of table 2 indicates that the host plants influenced the prepupal, pupal
duration and mean per cent adult emergence. The mean prepupal and pupal period was
longest on sunflower but pupal period on okra was similar to that of sunflower. The
mean percentage adult emergence ranged from 41-11 on groundnut to 82-22 on okra. In
general, females emerged earlier than males in all the host plants tested.
The results on the pupal measurements are dipicted in table 3. Significant differences
were recorded in pupal length, width and weight of S. litura when the larvae were reared
on different hosts. There was no relation between the pupal length, width or weight and
the pupal duration.
The mean preoviposition and oviposition periods did not differ significantly except
that on sunflower the preoviposition period was shorter (table 4). The highest fecundity
was noted on sunflower (3649-4 eggs) while the lowest was on groundnut (3121-8 eggs).
Reproductive index was calculated by dividing mean fecundity by average female pupal
weight (Hough and Pimental 1977). On the basis of the reproductive index the host
plants could be arranged in a descending order as: groundnut (10-68), okra (9-80),
Sunflower (9-75) and castor (8-46). The life cycle from egg to adult emergence was
shorter on castor (25-07 days) followed by sunflower (26-66 days), okra (30-03 days) and
groundnut (32-80 days). In general females had a shorter life cycle than males. The
females lived longer than males.
The mortality in the early instars of the larvae was 55-67 % on groundnut. Tiwari et al
(1980) observed mortality in the early instars up to 12 days of the larval period due to
. less intake of leaf tissues of groundnut. The work of Thobbi (1961) and Singh and Byas
(1975) also indicated heavy mortality on cotton. The mortality of larvae on okra,
sunflower and castor was 7-79, 11-01 and 15-66 %, respectively. However, Singh and Hoi
(1972) observed. 22% larval mortality when reared on castor. Bhattacharya and
Rathore (1977) recorded 84-70, 80, 73-50 and 77-10 % pupation and growth index values
of 5-39, 4*57, 2-93 and 3-97 on castor, cabbage, cotton and soybean, respectively.
Bilapate and Thombre (1979) recorded 89-47% pupation on sunflower at 26 ± 1°C
temperature. A similar trend was observed in the present studies also. The differences in
the larval period of S. litura feeding on many host plants have been reported by many
researchers. Basu (1945) reported larval duration of 18-20 days on cauliflower, 14 days
on castor and 14-16 days on okra (Thobbi 1961), 18 days on castor (Rattan Lai and
Nayak 1963), 18-7 days on castor (Patel et al 1965), 15-4 days on castor (Singh and Hoi
1972), 14-18 days on castor (Aleernuddin 1979). The results obtained in the present
investigations on larval period on okra and sunflower are in agreement with those of
earlier workers (Thobbi 1961; Bilapate and Thombre 1979). Host plants which
supported poor larval development gave smaller growth index values as in the case of
groundnut. Thobbi and Srihari (1967) obtained high larval weight after 1 1 days on HC-
6 irrigated castor variety^ The differences in pupal durations of S. litura feeding on
different host plants have been similarly demonstrated by different workers (Basu 1943;
Singh and Byas 1975; Bhattacharya and Rathore 1977). Basu (1943) established a direct
relationship between host plant and pupal duration. In the present investigation
however, the larvae with higher growth index values do not necessarily yield pupae of
52
G P Garad, P R Shivpuje and G G Bilapate
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Larval and post-larval development of S. litura
53
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G P Garad, P R Shivpuje and G G Bilapate
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shOTBr ««*. and as such •
s
-
(1979) reported 1721-23 onca..
Aleemuddin (1979) observed 26 06 eggs ^pe workers These variations may be due
the fecundity is at vanance wtth ,thos ot P16^ care durm rearing. Aleemuddm
to the substrate used, ^n^ch^^in order to understand the suitably of
(1979) reported a 25-69 days hfe cycle <» ^^^fc were considered. The host
host plants for S. litura, in all thirt ™***£"£^&** four points for the first
plants were arranged for each *^$^*^Tte total score for each
position and reducing one pomt for each subsequp suitabilit The host plants
Sort Plant was considered by «r^^S"^^^^^dunt?n>
e arranged in an ^D^°^^^^gAt larval weight, growth index
c^pe^P^tKmpe^I^1^!^ ^riod, fecundity, longev.ty of
cent adult emergence, pupal weigM, ov^p ^ of host plants with the
groundnut (22).
Ackeowledgement
The
References
than, Prof. P . O,op*Q*r f<"
^
Res. 1 14-23.
56 G P Garad, P R Shivpuje and G G Bilapate
Singh H N and Byas 1975 Larval development of Prodonia litura (F) in relation to certain cultivated food
plants; Indian J. Entomol 37 1-6
Thobbi V V 1961 Growth potential ofProdenia litura F in relation to certain food plants at Surat; Indian J.
Entomol 23 262-264
Thobbi V V and Srihari T 1967 Growth response ofProdenia litura F in relation to the castor leaves obtained
from irradiated plants; Indian J. Entomol. 29 154-156
Tiwari S N, Rathore Y S and Bhattacharya A K 1980 Note on the survival and change in the weight of the
larvae ofSpodoptera litura F feeding on some promising groundnut varieties; Indian J. Entomol. 42 283
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 57-65.
© Printed in India.
Activity-time budget in blackbtick
N L N S PRASAD*
Department of Zoology, Osmania University, Hyderabad 500007, India
*Caesar Kleberg Wildlife Research Institute, Campus Box 218, Texas A&I Univ. Kingsyille,
Texas 78363, USA
MS received 28 July 1983; revised 12 November 1984
Abstract. The activity patterns of blackbuck observed at Mudmal showed that feeding
accounted for the maximum frequency (75 %) with an average duration of 77 sec followed by
standing with a frequency of 62 % and an average duration of 19-9 sec. Lying although had only
4 % frequency, showed an average duration of over 30 min.
The hourly time budgets for basic activity patterns during the day in a season varied greatly
for both females and territorial males. Rhythms of feeding and lying peaks occurred alternately
during the day in all seasons. The time budgets for the activity patterns showed seasonal
variation. Lying time per day was more than the average time allotted to any activity. In the
case of females, the average time spent for feeding per day during summer was 25 % which was
more than that of monsoon and winter. The time spent in lying was 39 % which increased to
48 % in monsoon and winter. The average time spent in walking and standing did not show any
significant seasonal variation. The time budgets for the territorial males also showed the same
tendency as that of the females in all seasons. During winter, however, the feeding time per day
was 1 1 % while the lying time was 57 %, the former being significantly less and the latter
significantly greater than the females.
Keywords. Activity-time budget; blackbuck.
1. Introduction
Few quantitative studies have been published on the daily activity patterns of
blackbuck. These include the work of Schaller (1967) and Nair (1976) in India and
Mungall (1978) in Texas which describe the distribution of percentage of animals
engaged in basic activities in hourly classes from dawn to dusk. Based on a month's
study Mungall et al (1981) have compared the quantitative time budgets of males and
females of different age-classes in different social categories.
The activity patterns are determined by a wide range of factors, both biotic and
abiotic, and information is lacking as to how blackbuck adapt and maintain themselves
to seasonal changes. In the present paper, the time budgets for basic activities of
blackbuck are described for both sexes based on the studies at Mudmal (16 24'N and 77
27'E) during 1979-80 and the seasonal differences discussed.
2. Methods
Data were recorded on all activity categorised as feeding, walking, running, standing,
lying, and 'other activities' from dawn to dusk. The recordings made were as follows:
The activity of 11 identified territorial males was separately recorded at different times
of the day with the help of a stop watch. Recording of activity of females, however, was
58 NLNSPrasad
difficult due to difficulty to distinguish them individually. One female was randomly
selected from a herd and its activities recorded. On occasions when such an individual
under observation was confused or was out of sight, another adult female was
randomly selected from among the members visible. As a large number of observations
thus made showed synchrony among female members, the data collected were fairly
accurate. The activity patterns of the territorial males and females obtained thus
represent their activities from all social categories of different herd sizes although
majority of them were from mixed herds of atieast 10 head.
An activity was considered when the time spent in that activity exceeded 30 sec before
changing to the next activity. The average time spent by the territorial males in each
hour of daylight was calculated separately for summer, monsoon, and winter seasons
and expressed in percentage. Thus the activity curves composed of a fraction of the
12 hr rhythms of the individuals recorded on different days in a season.
Most of the continuous observations lasted for 4-6 hr, although on a few occasions
activity records were obtained for lOhr continuously without external disturbance.
Observations without continuous record between two consecutive daylight hours were
not included in calculations.
The seasonal differences in the activity patterns of the territorial adult males and
females were separately calculated for all daylight hours by employing the following
formula:
d_
where k1 = ajn^ k2 = a2/n2 and
a = total time spent in a given activity in a daylight hour over the period of
observation in a given season,
n = total time spent for all activities in that hour for the period of
observation in that season.
The mean time spent in an activity category in a day was calculated and comparisons
made between territorial males and females and the seasonal differences within them.
Apart from the preparation of time budget, nearly 5 hr of continuous record of
observation was utilized for obtaining the frequency of occurrence of the activity
categories and the association between them through hierarchical cluster analysis (De
Ghet 1978). Thus, v
Jaccard's association coefficient s= A/(A)+ (B)+ (C)
where, A = number of times both activities (say feeding and walking) occurred,
B=first activity (feeding) occurred and second (walking) not occurred '
C = second activity (walking) occurred and first (feeding) not occurred'
The transition probabilities of one activity following the other, as for example
feeding followed by walking and so on, was calculated using Markov models (Fagen
and Young 1978). Thus, transition probability of an activity Pr = (Number of times
activity t followed i by activity ;)/(Total number of times of'occurrence X^™"
The expected probability of an activity / was calculated by the following equation:
Sum of occurrence of an activityj^su^^ j
Total number of times of occurrence of all activities "
Activity-time budget in blackbuck 59
3. Results
3.1 Activity frequency
The frequency distribution of the activity categories is shown in table 1. Feeding
showed highest frequency (37-69%) followed by standing (31-16%). The average
duration of feeding, however, was just over a minute. Feeding was disrupted frequently
either by standing or by walking. Lying which showed only 4 %, on the other hand, was
continuous for a longer spell with an average duration of over 30 min. The maximum
duration of an individual continuously in lying activity was 2-5 hr.
3.2 Activity sequence
The association between the activity categories is shown in figure 1. Feeding and
standing were in maximum association with each other with a value of 0-79. The
association index of walking and feeding with standing was 0-71 followed by running
(0-36). The association index of lying with the rest was 0-03.
Table 2 gives the transition probability of one activity following the other along with
the expected values. The probability of standing followed by feeding was higher than
feeding followed by standing. The probability of feeding followed by walking is below
the expected value. On the other hand, walking followed by feeding showed a
probability higher than that expected. Running had an equal probability of being
followed by standing and walking. Similarly lying was either followed immediately by
Table 1. Transition probabilities of one activity following the other.
Preceding
acts
Following acts
F
S
W
R
L
0
Feeding (F)
0-0
0-0
0-55
051
0-42
0-43
0-0
0-02
0-03
0-03
0-0
0-02
Standing (S)
0-69
0^7
0-0
0-0
0-29
0-18
0-02
0-007
0-0
0-01
0-02
0-007
Walking (W)
0-63
£52
0-31
0-43
0-0
0-0
0-02
0-015
0-02
0-02
0-02
0-015
Running (R)
0-0
0-385
0-5
0-315
0-5
0-265
0-0
0-0
0-0
0-015
0-0
0-02
Lying (L)
0-0
0-39
0-67
0-32
0-33
0-27
0-0
0-01
0-0
0-0
0-0
0-01
Other (0)
activities
0-0
0-385
0-5
0-315
0-5
021
0-0
0-02
0-0
0-01
0-0
0-0
ft nnriftrlinftd mimhftrs indicate thft ftTfTV*rtftH nrnhahilitifts while th*» nthers are
60
NLNS Prasad
S w
i.o -
as -
as
c
o
0-4
0.2
F = Feeding
S ^ Standing
W = Walking
R s Running
L = Lying
Activities
Figure 1. Dendrogram showing association between basic activity patterns.
Table 2. Frequency and duration of activity categories.
Activity
Total Average
% duration % duration
Frequency Frequency (sec) Duration (sec)
Feeding
75
37-69
5809
34-29
77-45
Walking
54
27-14
2461
14-53
45-57
Running
2
1-01
8
0-05
4-00
Standing
62
31-16
1237
7-30
19-95
Lying
4
2-01
7375
43-53
1843-75
Other activities
2
1-01
53
0-31
26.50
standing or walking before the start of any other activity, although the former h
higher probability than the latter.
The 'other activities' usually were followed either by standing or by walking
equal chance of occurrence.
3.3 Activity-time budget of females
The time spent by females in the activity categories from 7-8 hr during the three sea
is shown in figure 2. The activity records between 13-17 hr were not represente<
summer as the observations were discontinuous. This data, however, showed
animals spending most of the time lying down. Due to this reason it became too difl
to locate them.
In summer bouts of feeding were noticed in all daylight hours observed with at
20 % of time. In winter there were distinct peaks of feeding with a maximum of
Activity-time budget in blackbuck
61
FE E 01 NG
WAL KING
STANDING
Summer ;
§ Monsoon ;
Figure 2. Seasonal activity patterns of females.
between 7-8 hr and 12-13 hr. During the rest of the daylight hours feeding time ranged
between 8% and 15%. The time spent in feeding between 7-8 hr in winter was
significantly greater than that of summer (d = 7-02, p < 0-001).
During monsoon, in the early hours feeding was very less (8 % between 8-10 hr).
However, it increased to 22% during 10-11 hr. Between 11-12 hr feeding time was
32 %, the maximum noticed in the season. During the rest of the daylight hours the time
apportioned for feeding ranged from 12-26 %. There were no significant differences in
the feeding time spent in 10-11 hr, 11-12 hr and 17-18 hr of monsoon and summer;
15-16 hr and 17-18 hr of monsoon and winter; and 8-9 hr and 17-18 hr of winter and
summer. During the rest of the day hours the differences in feeding time between
seasons was significant at p < 0-001.
During summer and monsoon, the time for walking showed a gradual increase from
7-13 hr. Contrary to this, winter walking time decreased from 26% (9-10 hr) to 5%
(15-16 hr) and again showed an increase after 17 hr.
Standing showed an increase from morning with an afternoon peak in all seasons.
The maximum time spent in an hour was 44 % (12-13 hr) in summer, 26 % (13-14 hr) in
monsoon and 48% (12-13 hr) in winter.
Lying showed definite peaks in monsoon and winter. In monsoon, when data was
recorded from 8-18 hr, the maximum time spent was 80 % between 8-9 hr. The rest of
62 N L N S Prasad
Table 3. Average proportion of time spent per day by females and territorial males
in the activity categories.
Category
Season
Feeding
% Time
Walking
Standing
Lying
Other
activities
Terri-
Summer
25
11
23
37
4
torial
Monsoon
17
13
17
47
6
males
Winter
11
11
16
57*
5
Females
Summer
25
11
24
39
1
Monsoon
19
10
23*
48
0
Winter
16*
10
21*
48
5
* Comparison between females and territorial males and allotment of significantly
more time than the other at p < 0*001.
the daylight hours showed 30% (ll-12hr) to 64% (9-10 hr). During winter the
maximum time allotted was 94 % (9-10 hr) in the morning and 64 % (15-16 hr) in the
afternoon. During the remaining hours of the day the amount of time spent ranged
between 10% (8-9 hr) and 72% (16-17 hr).
During summer, over 50 % time was spent on lying between 7-8 hr which decreased
to 34 % between 9-10 hr. It again increased to 40 % between 10-1 1 hr and fell to 28 % in
the succeeding hour. During 17-18 hr again 45 % of the time was spent in this activity.
Females spent an average of 25 % of the day time for feeding, 39 % for lying while
walking and standing shared 11% and 14% respectively during summer season
(table 3). The average feeding time decreased to 19 % in monsoon and 16 % in winter
and the time spent in lying increased to 48 % in these two seasons. Walking and
standing showed a slight decrease.
3.4 Activity-time budget of territorial males
The activity-time budget of the territorial males in the three seasons is shown in figure 3.
As in females, feeding and lying were the main activities to be focussed upon among all
activities in all seasons. During summer two peaks of feeding were clear between
13-14 hr (58 %) and 16-17 hr (61 %). Between 7-8 hr and 17-18 hr about 38 % of the
time was spent in this activity. The minimum time spent in this activity was 14 %
between 8-9 hr. The rest of the daylight hours have feeding time ranging between 20
and 26 %. Between 14-15 hr the data were not represented as the number of
observations were very few.
During monsoon and winter there were no significant peaks in feeding activity. The
maximum and minimum amount of time spent in feeding was 25 % (12-13 hr) and 7 %
(17-18 hr) respectively. The time allotment for feeding between 7-8 hr, 13-14 hr and
15-16 hr during summer is significantly greater (p < 0-001) than monsoon and winter
in the corresponding hours. The total time spent in feeding per day was also higher in
summer (25 %) than in monsoon (17 %) and (11 %) which was statistically significant
(d = 8*78 for monsoon and summer; d = 15-84 for winter and summer; p < 0*001). The
total time spent in feeding per day during monsoon was significantly more than that
during winter (d = 9-17; p < 0-001).
Activity-time budget in blackbuck
63
FEEDING
78 10 12 14
HOURS OF DAY
HI Summer^ 1=} Monsoon j O Winter
Figure 3. Seasonal activity patterns of territorial males.
There were marked differences in lying in the three seasons. In summer lying was
more between 7-8 hr, 11-12 hr and 15-16 hr and the time spent ranged between
54-62 %. Between 12-13 hr there was practically no lying. During monsoon, as in the
case of summer, morning and evening hours showed more lying activity and there was
an afternoon dip. Fifty to sixty per cent of the time was spent in this activity between
7-10 hr while between 10-14hr only 34-42% time was spent. The subsequent day
hours till 18 hr had 46-68 % of time in this activity. During winter, there were periodical
oscillations in lying. Thirtyeight per cent of time was spent between 7-8 hr which
significantly increased to 78 % between 9-10 hr. Lying time decreased to 21 % during
11-12 hr and again increased to 42 % between 12-13 hr. In the subsequent hours there
was once again a decrease in this activity. But between 15-17 hr it touched a maximum
of 80 %. Compared to summer and monsoon seasons, winter had significantly more
time in lying between 9-10 hr 11-12 hr; 13-14 hr and 15-17 hr (p < 0-001). The total
lying time per day was also maximum during winter (57 %) followed by monsoon (47 %)
and summer (37 %) and the differences were statistically significant (d = 10-37 for
summer and monsoon, and for winter and monsoon; d = 17-45 for winter and summer;
p < 0-001).
64
N L N S Prasad
4. Discussion
The maintenance of life in a mammal requires a variety of activities associated with
procurement of food, shelter and protection. Each of these activities had a certain
benefit and cost attached to it (Sharatchandra and Gadgil 1980). An obvious principle is
that the extent of activity must be adequate to maintain the kind of life permitted by the
animal's anatomic and physiological adaptations (Davis and Golley 1963). Through the
time budget for basic activities of blackbuck, the following explanation emerges to
show as to how they maintain themselves in different seasons.
During summer forage material in general and fresh foliage in particular was very
scarce. Blackbuck being mainly grazers and preferring the tender leaf were rather
forced to spend much of their time in search of food. This is the reason for their
allotting a significantly large proportion of time for this purpose as compared to other
seasons (table 3). During monsoon sprouting of grasses was in abundance and they
could easily obtain their nourishment. During winter blackbuck take to crops, which
are more readily available. The crops have more crude protein content than grasses and
hence a lesser quantity may suffice their energy requirements. This probably is the cause
for a further drop in feeding time during winter. This enabled them to spare more time
for lying and ruminating in monsoon and winter.
Lying time was significantly more while the feeding and standing time was less in
40
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SUMMER
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:— ~ ^.-£- <s«^:iir:^*^^i-.Tjr.
.-•^. c
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8 10 12 14 16 18 8 10 12 14 16 18
,HOIRS OF DAY
Figure 4. Comparison of activity patterns between females and territorial males. A to E:
Feeding, Walking, Standing, Lying and other activities, respectively. Broken line indicates the
activity of females while continuous line refers to the activity of the territorial males.
Activity-time budget in blackbuck 65
territorial males than in females during winter and monsoon. A major portion of lying
time was spent by territorial males within or adjacent to their territories which were
located near the cultivated fields. They thus had a better access for a quick bite and
hence could spend the remaining time for lying and ruminating. They could also watch
the activities of females and keep the bachelor associations away from their territories,
which move from one area to the other.
The females showed a significantly more time for standing than the territorial males.
A plausible explanation for this could be, the females in general were more cautious and
watchful, both while feeding and while at rest, than the territorial males. This may be
because the females mostly were seen in groups and could easily be spotted and would
probably need to doubly ensure whether there would be any sort of danger before they
resort to bedding.
If we look at the hourly activity-time budgets, in most cases the general pattern of
curves in both territorial males and females showed a more or less similar tendency of
increase and decrease (figure 4). This indicates that their response to the changing
conditions in different seasons is more or less the same. The differences in some daylight
hours may be due to the environmental parameters such as temperature, relative
humidity, presence of shade in the area, wind speed etc. which, however, were not
measured during the present study.
Acknowledgements
The author is indebted to Prof. J V Ramana Rao, Prof. Madhav Gadgil, Dr N V Joshi,
Dr S N Prasad, Mr P V K Nair for helping him in many ways. The financial help by the
Ministry of Agriculture, Govt. of India through the President, FRI and Colleges, Dehra
Dun, for the work is gratefully acknowledged.
References
Davis D E and Golley F B 1963 Principles of mammalogy (New York: Reinhold) 335
De Ghet V J 1978 Hierarchical cluster analysis in Quantitative ethology, (ed) P W Colgan (New York: John
Wiley) 116-144
Fagen R M and Young D Y 1978 Temporal patterns of behaviours: Durations, intervals, latencies, and
sequences in Quantitative ethology, (ed) P W Colgan (New York: John Wiley) 80-114
Mungall E C 1 978 The Indian blackbuck antelope: A Texas view. College Station, Texas, The Texas Agric. Exp>
Station, Texas A and M Univ. System. 184
Mungall E C, Patel B H, Prasad N L N S, and Dougherty S E 1981 Conservation and management of blackbuck
antelope (Antilope cervicapra), (Lewisville, Texas: Old Orchard Printers) 111
Nair S S 1976 A population survey and observations on the behaviour of blackbuck in the Point Calimere
Sanctury, Tamil Nadu; J. Bombay Nat. Hist. Soc. 73 304-310
Sharatchandra H C and Gadgil M 1980 On the time-budget of different life-history stages of chital (Axis
axis). J. Bombay Nat. Hist. Soc. 75 949-960
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 67-72.
© Printed in India.
Some biochemical changes in the reproductive cycle of a
hill stream teleost Puntius chilinoides (McClelland)
B P NAURIYAL and H R SINGH
Department of Zoology, Garhwal University, Srinagar Garhwal 246174, India
MS received 16 April 1984; revised 29 October 1984
Abstract. The protein content was highest in the ovaries of Puntius chilinoides during the
maturing stage and in the testes during the mature stage. The activity of the acid phosphatase
and the number of isozymes decreased in the testes during maturation, whereas in the ovaries
the activity increased during the maturation and spent stages. The alkaline phosphatase
activity in the testes increased during maturation phase, while in the ovaries the highest activity
of the enzyme was recorded at the maturing stage and the lowest during the mature stage.
Cholesterol level in the ovaries was highest during the maturing stage, while in the testes it was
noticed during the immature stage. The sugar contents in the gonads were highest at the
mature stage. The results are discussed in relation to the reproductive cycle in P. chilinoides.
Keywords. Biochemical changes; protein; acid phosphatase; alkaline phosphatase; choles-
terol; sugars.
1. Introduction
It is well known that several metabolic changes occur during the development of
gonads and in fact all the metabolic activities inside a developing tissue are ultimately
under some biochemical control. The metabolic activities are controlled by the
enzymes. Now it is clear that lysosomes are the main organelles where the acid
hydrolases like the acid phosphatase are localised. The alkaline phosphatase is also
much important in animal tissues. Phosphatases in general play a very important role in
phosphate (Pf) availability in the tissues. Inorganic phosphate (Pt) is required in the
synthesis of several metabolites during developmental stages. On the other hand,
carbohydrates, fats and cholesterol also play a significant physiological role during the
developmental stages in gonads.
There are several reports on the biochemical changes that occur during growth and
development in fish, Lai (1963) reported decline in protein contents in the ovaries in
Cirrhina mrigala during maturation. Contrary to this, Ehlebracht (1973) reported an
increase in the protein content during maturation. Wegmann and Goetting (1971)
studied a distribution of protein, polysaccharides, nucleic acids and fats in
Xiphophorous helleri. Shaffi et al (1974) have reported higher alkaline phosphatase
activity in the ovaries of Clarias batrachus during maturation. Siddiqui (1966) in
Channa punctatus, Singh and Singh (1979) in Heteropneustes fossilis and Sen and
Bhattacharya (1981) in Anabas testudineus reported the cholesterol level in different
stages of maturity in the gonads.
Studies on the biochemical changes during the development of the gonads in
hillstream fishes are scanty. Therefore, in addition to the seasonal morphohistological
68 B P Nauriyal and H R Singh
studies of the gonads and pituitary gland, the biochemical changes in protein, acid
phosphatase, alkaline phosphatase, cholesterol and sugar contents of the gonads of a
hillstream minor carp Puntius chilinoides of Garhwal Himalaya during different stages
of development were studied.
2. Material and methods
On the basis of detailed seasonal morphohistological changes in the gonads and
pituitary gland of P. chilinoides, the following stages of development viz the immature,
maturation, mature, spent and resting stage have been studied. The important
morphohistological changes have been observed during immature, maturation, mature
and spent stages, therefore, the present biochemical study has been conducted during
these stages of maturity. Sexually mature P. chilinoides of each sex were collected
regularly from the Khandagaad, a tributary of Alaknanda. The soluble protein contents
of the gonads were determined by the method of Lowry et al (1951). The soluble sugar
contents were estimated by the Anthrone method (Mac Cready et al 1950). The total
cholesterol was determined by the modified method of Zlatkis et al (1953). For the
determination of phosphatases, the homogenates were prepared in cold grinding
medium consisting of 0-1 M Tris-HCl buffer (pH 7-5) and centrifuged at 2000 x g. The
acid phosphatase activity was determined by the method of Baijal et al (1972). The assay
system comprised 1 ml of 0-2 M acetate buffer (pH 5-5) 0-1 ml 0-2 M MgSO4 and
enzyme preparation and water, making the volume 1-9 ml. The alkaline phosphatase
activity was determined following the method described by Bodansky (1932), using
1ml 0-2 M barbitone buffer (pH 9). For both the phosphatases 0-1 ml of 0-1 M
jS-glycerophosphate was added as a substrate. Phosphate was determined following the
method described by Fiske and Subbarow (1925). The unit of the enzyme activity was
expressed as the amount which librates one \JL mole of pi per minute at 37°C.
Change in protein profile and isozymes of acid phosphatase in the gonads of
P. chilinoides were analysed for qualitative changes in their protein and acid
phosphatase composition by means of disc electrophoresis. A 20 % (W/V) homogenate
was prepared by grinding the tissue in pre-chilled tris-HCL buffer (pH 7-5) at 0-5°C
and centrifuged at 2000 x g. The supernatant was used for electrophoritic separation of
protein and acid phosphatase using three gels for each sample. The method of disc
electrophoresis in polyacrylamide gel as described by Davis (1964) was followed.
0*15 ml extract with 0-05 ml of 1 M sucrose was layered on 7-5 % acrylamide gel using
bromophenol blue as a tracking dye. The samples were run in cold at pH 8-3 using Tris-
glycine buffer with a current of 3 mA per tube. The process was carried out till the
tracking dye reached the lower end of the gel. The gels were then taken out. For
proteins, the gels were stained in 0-25% Coomassie brilliant blue for 15hr and
destained in 7% acetic acid at 5 mA current per tube.
For isozymes of acid phosphatase the gels were incubated in proper incubation
mixture as described by Brewbaker et al (1 968). The zymograms of the stained gels were
prepared and the transmittance of bands was measured with the help of a densitometer
(Toshniwal, type CM 11).
Changes in the reproductive cycle of P. chilinoides 69
3. Observations
3.1 Protein
The protein contents were higher in the ovaries in comparison with the testes during all
the four stages (immature, maturing, mature and spent) (figure 1A). In the testes, the
protein contents increased during the maturing stage and the highest value of protein
was observed at the mature stage; and a sharp decline in the spent stage was observed. In
the ovaries, the protein contents increased only during maturing stage (stage II). The
protein contents then showed a slight decline in the mature period and finally a sharp
decline at the spent stage.
3.2 Acid phosphatase
The activity of the acid phosphatase showed a marked decrease during maturation in
the testes. The activity was highest at the immature stage and a significant decrease in
250
f 200
•g 150
Cl
J 10°
? 50
o .2-5
,5 ° w.Q3
30 -J--02
5 5 '-6 -01
IB)
in iv
i ii in
^
(C)
C '
&
"o
D.
1"
.02
/
>-
/
'.H
.01
<K //
o
0
, V\*=^2r ,
i n in iv
n m iv
m iv
Figure 1. Seasonal biochemical changes in the testes (•-•) and ovary (•-•) of
P chilinoides. A. Protein; B. Acid phosphatase; C. Alkaline phosphatase; D. Cholesterol;
E. Sugar. (I, II, III and IV represent immature, maturation, mature and spent phases
respectively).
70
B P Nauriyal and H R Singh
Protein
Testes
B a a
Ovary
• B HE
• II I
TB^I a
in
II I I B
IV
Acid phosphatasc
. Testes
i n B a i
II H
i
n
\ 1
a i
in
1 81
i i i
IV
Ovary
1 H.B 0
I
I
i en
1
n
1 6B 1
in
III 1
S 1
IV
Figure 2. Zymograms of protein and acid phosphatase by polyacrylamide gel elec-
trophoresis of the gonads of P. chilinoides. (I, II, III and IV represent immature, maturation,
mature and spent phases respectively).
the activity was observed at maturing and mature stages. During the spent stage the
activity increased significantly. The activity of this enzyme showed a different pattern in
the ovaries. The acid phosphatase activity in the ovaries was found highest at the
immature stage. At the maturing stage a significant decrease was noticed after which
there was a continuous increase at the mature and spent stages (figure IB). The activity
of the acid phosphatase and the number of the isozymes decreased in the testes during
maturation, whereas in the ovaries the activity increased during maturation and spent
stages (figure 2).
3.3 Alkaline phosphatase
In the ovaries the highest alkaline phosphatase activity was recorded at the maturing
stage which decreased sharply up to the mature stage. At the spent stage the activity
again increased. In the testes it showed a different pattern. The activity was lowest at the
maturing stage and then continuously increased up to the spent stage (figure 1C).
3.4 Cholesterol
The cholesterol content in the testes was highest in the immature stage. The highest
cholesterol level in the ovaries was found during the maturing and the lowest during the
immature stage (figure ID).
Changes in the reproductive cycle of P. chilinoides 71
3.5 Sugars
The amount of soluble sugars in both the gonads was highest at the mature stage as
compared to the other three stages (figure IE).
3.6 Changes in protein profile and isozymes of acid phosphatase
Five protein bands were observed at all the four stages of development in the ovary.
However, in the mature stage the first band had a very low Rf value, whereas the band
having the highest Rf value in the immature, mature and spent stages was not detected
in the maturing stage. In the testes four protein bands were detected at all the stages
except at the mature stage where only three bands were detected and the fourth band
(the band of highest Rf value) was absent. The Rf value of each individual band was
different at different stages.
In the ovary four bands of acid phosphatase were detected at the spent stage, only
two bands at the mature stage and three bands at the immature and maturing stages.
The first and the second band (I and II from origin) had the same jR/values at all the four
stages. In the testes acid phosphatase activity appeared as four bands at the immature
and spent stages with the same Rf values, although the Rf values of the third and fourth
band in the spent stage was slightly less. The maturation and mature stages showed only
two bands, one band in common (figure 2).
4. Discussion
The protein contents in the ovaries was much higher than in the testes during the annual
cycle which is in consonance with previous studies. The low protein content at the
spent stage in P. chilinoides is indicative that rapid protein synthesis is necessary only
during maturation for the developing oocytes and sperms. The protein profile of the
ovaries and testes also indicated that the original bands disappear during the
developmental stages, indicating that the new proteins are synthesized.
The activity of the acid phosphatase and the number of isozymes decreased in the
testes during maturation and spent stages. These results indicate that in the ovaries the
acid phosphatase plays a significant role in the synthesis of essential metabolites by
liberating Pi. However, in the testes the decline in the acid phosphatase activity is
probably indicative of the fact that the enzyme apparently plays a less significant role
during maturation (stage II and III), but seems to play a significant role during the spent
stage as the level of the enzyme activity increases during the spent stage.
Shaffi et al (1974) have reported higher alkaline phosphatase activity in the ovaries of
Clarias batrachus during maturation. In P. chilinoides the alkaline phosphatase activity
increased in the ovaries during the maturing phase (stage II) indicating that during this
period the synthesis of new proteins takes place as alkaline phosphatase has been
reported to be involved in protein synthesis (Shaffi et al 1974). At the mature stage the
activity of alkaline phosphatase was lower, showing a decline during the process of
maturation. In the testes the alkaline phosphatase activity showed a different pattern.
The activity was lowest at the maturing stage (stage II) and then continuously increased
72 B P Nauriyal and H R Singh
up to the spent stage, suggesting that in the testes the alkaline phosphatase plays an
important role during the development of the sperms.
Siddiqui (1966) recorded maximum ovarian cholesterol level in the gonads ofChannc
punctatus at the end of the maturing phase, while in Heteropneustesfossilis Singh and
Singh (1979) observed a decline in the cholesterol level of the ovaries during the pre-
spawning phase, but an increase during the spawning phase. In Anabas testudineus Ser
and Bhattacharya (1981) reported low ovarian cholesterol level during the pre-
spawning phase and high cholesterol level during the post-spawning phase.
In P. chilinoides, the high cholesterol level in the ovaries was found during th<
maturing stage and the lowest during the immature stage, while in the testes the higt
level of cholesterol was noticed during the immature stage and lowest during the spen
stage. It is considered that the high cholesterol level in the gonads acts as a reservoir tc
meet the cholesterol demand of the maturing gonads and the decreased level might b<
due to the increase in the rate of steroidogenesis.
The sugar contents in the gonads of P. chilinoides were highest at the mature stage
suggesting that during maturation the accumulation of sugars takes place in the ovariei
and testc 5. The sharp decline in the sugar contents during the spent stage, confirms th<
previous studies (Lai 1963).
References
Baijal M, Singh S S, Shukla R N and Sanwal G G 1972 Enzyme of banana, optimum condition for extractior
Phyto. Chem. 11 929-930
Bodansky A 1932 Determination of serum phosphatase activity; J. Biol 99 197
Brewbaker J L, Upadhyay M D, Makinan Y and Mac Donald T 1968 Isozymes polymorphism in flowin;
plants III. Gelelectrophoretic methods and application; Physioi Plant. 21 930-940
Davis B J 1964 Disc electrophoresis. II Method and application to human serum proteins; Am. N. Y. Acad. Sc
121 404-427
Ehlebracht V J 1973 Stoffiche Verandrungen Wahrond des Reifezylus in ovarian von Herbat-Und Fruhjj
Chrsheringen des westlichen Ostec; Berolt Wils Kommn mearesforsch 23 47-83
Fiske C H and Subbarow Y 1925 The Colorimetric determination of phosphorus; J. Biol Chem. 66 375-40
Lai B 1963 Biochemical Studies of the ovaries of Cirrhina mrigala (Ham.) during different maturity stage
Sci. Cult. 29 305-306
Lowry O H, Kosenbrough N J, Farr A L and Randall R J 1951 Protein measurement with the folin phen<
reagent; J. Biol. Chem. 192 265-275
Mac Cready R M, Guggolz J, Silviera V and Dwen H S 1950 Determination of starch and amylase i
vegetables; Analyt. Chem. 22 1156-1158
Medford B A and Mackay W C 1978 Protein and lipid contents of gonads, liver and muscle of northern pil
Esox lucius in relation to gonad growth; J. Fish Res. Bd. Can. 35 213-219
Sen S and Bhattacharya S 1981 Role of thyroxine and gonadotropins on the mobilization of ovaria
cholesterol in a teleost, Anabas testudineus (Bloch); Indian J. Exp. Biol 19 408-412
Shaffi S A, Jafri A K and Khawaja D K 1974 Alkaline phosphatase activity in the ovary of the catfish Clark
batrachus (Linn.) during maturation; Curr. Sci. 43 51
Siddiqui M A 1966 Seasonal variations in total cholesterol content in different tissues of Ophiocephah
punctatus (Bl.); Indian J. Exp. Biol 4 122
Singh A K and Singh T P 1 979 Seasonal fluctuations in lipid and cholesterol content of ovary, liver and bloc
serum in relation to the annual sexual cycle in Heteropneustes fossilis (BL); Endocrinologie 73 47-5
Wegmann I and Goetting K J 197 1 Studies on yolk formation in oocytes of Xiphophorus helleri; Z. Zellforsc
microscop. anal. 119 405-433
Zlatkis A, Zak B and Boyle A J 1953 A new method for the direct determination of serum cholesterol; J. La
Clin. Med. 41 486-492
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 73-77.
© Printed in India.
Effect of carbaryl on esterases in the air-breathing fish
Channa punctatus (Blocfa)
S ARUNACHALAM, S PALANICHAMY
and M P BALASUBRAMANIAN
P.G. and Research Department of Zoology, A.P.A. College of Arts and Culture,
Palani 624 602, India
MS received 29 March 1984 #
Abstract. Electrophoretic analyses of liver and muscle of Channa punctatus revealed that
they contain atleast six and eight fractions of esterase, respectively. Characterization of
esterase was made on the basis of their responses towards certain inhibitors. Liver esterase
consists of acetylesterase and carboxyl esterases, whereas the muscle esterase has three types
namely acetylesterase, carboxylesterase and cholinesterase. The liver and muscle of
C. punctatus subjected to maximum sublethal concentration of carbaryl were electrophoreti-
cally analysed and it was found that both liver and muscle showed only three fractions of
esterase.
Keywords. Channa punctatus; esterases; characterization; liver; muscle.
1. Introduction
Although a number of workers have studied the effects of industrial pollutants and
agricultural pesticides on the activity of hepatopancreatic enzymes in many vertebrates
(Bhattacharya and Mukherjee 1976; Thomas and Murthy 1976), they have not reported
the findings of the electrophoretic analysis on these enzymes. The present study deals
with the electrophoretic characterization of liver and muscle esterases of a freshwater
air breathing fish Channa punctatus and the effect of carbaryl upon this enzyme.
2. Material and methods
Channa punctatus were collected from the local pond and acclimated to laboratory
condition and feeding schedule keeping them in a large glass aquarium.
For separation and characterization of the enzyme, two groups offish were reared:
one in pesticide-free water and another in a medium containing 5 ppm concentration
of carbaryl (N-methyl carbamate) (Union Carbide India Ltd) (sublethal level:
Arunachalam et al 1984) for 15 days. The liver and muscle were separated after killing
the fish in each group and homogenized in 40 % sucrose solution. The homogenate was
centrifuged at 5000 g for 10 min and the supernatant was used as the enzyme source.
Disc gel electrophoresis was carried out as previously described by Balasubramanian
et al (1982). Esterases were visualized by staining the solution containing 1 % 1-napthyl
acetate and 1 % fast blue RR in phosphate buffer (M/15, at pH 7) at 37°C for 15 min.
For characterizing the enzymes, gels were incubated in different inhibitors of varying
strength solutions for 30 min and then stained for esterase. By comparing with the
control gel, different types of esterases have been identified.
73
74 S Arunachalam, S Palanichamy and M P Balasubramanian
3. Results and discussion
Electrophoretic analyses of liver and muscle of C. punctatus revealed that they contain
atleast six and eight esterase fractions, respectively. Based on the mobility of the enzyme
fractions they have been designated as LEst-1 to LEst-6 and MEst-1 to MEst-8,
respectively (figure la,c), indicating that the esterase of both liver and muscle of
C. punctatus exists in multiple form. This is in accordance with the findings of Varma
and Frankel (1980).
Effects of certain inhibitors on esterase fractions of liver and muscle of C. punctatus
are presented in tables 1 and 2. Characterization of esterases was made on the basis of
its responses towards certain inhibitors. Among the liver esterase fractions of
C. punctatus, LEst-1 and LEst-2 were partially inhibited by silver nitrate and other
chemicals like p-CMB, EDTA, eserine sulphate and organophosphate had no effect on
these two fractions which are acetylesterases (Bergmann and Rimon 1958; Dickinson
and Johnson 1978; Balasubramanian et al 1982). LEst-3, 4, 5 and 6 were inhibited by
Table 1. Effect of inhibitors on various fractions of esterases in the liver of
C. punctatus.
Inhibitors LEst 1 LEst 2 LEst 3 LEst 4 LEst 5 LEst 6
Control + +
p-CMB 10'2M + +
EDTAlO~2M + +
Organophosphate
10"4M + +
Eserine sulphate
10'4M + +
AgNO310"2M +/- +/- + + +/- +/-
acetyl acetyl carboxyl carboxyl carboxyl carboxyl
— represents inhibition of enzyme activity; + 25 % activity; + + 50 % activity;
+ + + maximum activity or 100% activity.
Table 2. Effect of inhibitors on various fractions of esterases in the muscle of C. punctatus.
Inhibitors
MEst 1
MEst 2
MEst 3
MEst 4 MEst 5
MEst 6
MEst 7
MEst 8
Control
p-CMB 10 ~2M
EDTA 10~2M
Organophosphate
KT4M
+ + +
+ + +
+ + +
+ +
+ +
+ +
+ +
+ +
+ + + +
+ + + +
+ + + +
+ + +
+ + +
+ + +
+ + +
+ + +
-
Eserine
sulphate 10 ~4M
AgNO3
+ + +
+ +
+ +
+ +
+ + + +
+ + +
+ +
+ + +
++
acetyl
carboxyl
choline
choline choline
carboxyl
carboxyl
carboxyl
- represents inhibition of enzyme activity; + 25 % activity; + + 50% activity; + + + maximum activity or
100% activity.
Effect of carbaryl on Channa punctatus
75
organophosphate, but not by p-CMB, EDTA and eserine sulphate and these fractions are
the carboxylesterase (Ahmad 1976; Payne 1978; Varma and Frankel 1980).
Regarding the fish muscle esterases, MEst-1 is partially inhibited by silver nitrate and
not by other chemicals. This fraction is acetylesterase (Bergmann and Rimon 1958;
Dickinson and Johnson 1978; Balasubramanian et al 1982). MEst-3, 4 and 5 which were
inhibited by organophosphate and eserinesulphate are probably cholinesterases
(Augustinsson 1961; Holmes and Masters 1968) and MEst-2, 6, 7 and 8 which were
inhibited by organophosphate but not by p-CMB and EDTA are carboxylesterase
(Ahmad 1976; Payne 1978; Varma and Frankel 1980).
When fishes are exposed to pollutants whether industrial or agricultural, organs like
liver and kidney are affected much (Brown 1970), since most of the toxic substances
passing through these organs may cause histopathological and enzymatic changes.
The esterases of liver and muscle offish reared in sublethal concentration of carbaryl
are shown in figure 1. In liver, only three fractions i.e. LEst-3, 4 and 6 were identified,
while others (LEst-1, 2 and 5) were not exhibited (figure Ib). In the fish muscle also
there are only three fractions i.e. MEst-2, 6 and 8 (figure Id), and the other fractions
were not exhibited. Relative mobility of enzyme fractions, LEst-3, 4 and 6 and MEst 2,
6 and 8 in comparison with that offish reared in pesticide-free water showed that these
are carboxylesterases.
7a
Jb
ll.EST-6
5
7c
Jd
IM.EST-S
7
6
5
Figure 1. Zymogram pattern of esterases in Channa punctatus. (la — Liver; Ib — Liver of
treated animal; Ic — Muscle; Id — Muscle of treated animal).
76 S Arunachalam, S Palanichamy and M P Balasubramanian
Therefore it appears that acetylesterases of liver and acetylesterases and cholineste-
rases of muscle in C.punctatus were inhibited. Such inhibition on esterases in different
vertebrates due to certain pesticides has been reported (Mendoza and Hatina 1970).
Industrial effluents like sodium sulphide, phenol, ammonia and copper sulphate have
similar effects on liver esterase in C. punctatus and Glorias batrachus (Bhattacharya and
Mukherjee 1976). The intensity of the activity of the esterases of liver and muscle of the
treated fish was low when compared with that of fish reared in pesticide free-water.
Previous studies reported that carbaryl present in the medium decreased the growth
rate of fishes (Arunachalam and Palanichamy 1982; Arunachalam et al 1984).
Inhibition of acetylesterase and the consequent low activity observed in the present
study may be the reasons for the decreased growth. Cholinesterase is an important
enzyme in the excitable tissues of brain and muscle of teleost fishes (Nachamanson et al
1941; Weiss 1958; Lundin 1959). Inhibition of cholinesterase may lead to changes in the
normal behaviour. This may be the reason for the erratic movements and imbalance in
the fish exposed to pesticides (Arunachalam et al 1984).
Acknowledgements
The authors thank Prof. K Baskaran and Mr Ponnuraj for help.
References
Ahmad S 1976 Larval and adult housefly carboxylesterase: isozymic composition and tissue patterns; Insect
Biochem. 6 541-547
Arunachalam S and Palanichamy S 1982 Sublethal effects of carbaryl on surfacing behaviour and food
utilization in the air-breathing fish Macropodus cupanus; Physiol. Behav. 29 23-27
Arunachalam S, Palanichamy S and Balasubramanian M P 1984 Toxic and sublethal effect of carbaryl on
survival, food utilization and 02 consumption in air-breathing fish Channa punctatus; J. Environ. Biol. (in
press)
Augustinsson K B 1961 Multiple forms of esterase in vertebrate blood plasma; Ann. NY. Acad. Sci. 94
844-860
Balasubramanian M P, Nellaiappan K and Ramalingam K 1982 Characterization of esterase isozyme of
Raillietina tetragona (Molin, 1858) (Cestoda); Vet. Parasitoi 10 313-316
Bergmann P and Rimon S 1958 The effect of pH variations on the activities of C. esterase; Biochem. J 70
339-344
Bhattacharya S and Mukherjee S 1976 Activity of the hepatopancreatic protease and esterase in fish exposed
to industrial pollutants; Comp. Physiol Ecol I 45-50
Brown V M 1970 The prediction of the acute toxicity of river waters to fish; Proc. I V British Coare. Fish conb.
Liverpool University
Dickinson J P and Johnson A D 1978 Electrophoretic separation of esterases of Alaria mariianae (LaRue
1917) (Trematoda); Comp. Biochem. Physiol. B52 141-144
Holmes R S and Masters C J 1968 A comparative study of multiplicity of mammalian esterases; Biochem.
Biophys. Ada. 151 147-158
Lundin S J 1959 Acetylcholinesterase in goldfish muscle: studies on some substrate and inhibitors- Biochem J
12 210-214
Mendoza C E and Hatina G S 1970 Starch electrophoresis of liver esterases selectively inhibited by pesticides;
Bull. Environ. Contam. Toxicoi 5 181-190
Nachmanson D, Coats C W and Cox R T 1941 Electrical potential and cholinesterase in the electric organ of
Electrophorous electricus; J. Gen. Physiol 25 75-88
Effect of carbaryl on Channa punctatus 77
Payne D W 1978 The effect of activators and inhibitors on midgut esterase activity in the desert locus,
Schistocerca gregaria (Forsk); Acrida. 1 197-206
Thomas P C and Murthy T L 1976 Acid phosphatase activity in a freshwater air-breathing fish
Heteropneustesfossilis and the effect of certain organic pesticides on it; Indian J. Biochem. Biophys. 13
347-349
Varma A K and Frankel J S 1980 A comparison of tissue esterases in the genus Barbus by vertical gel
electrophoresis; Comp. Biochem. Physiol. B65 267-273
Weiss C M 1958 The determination of cholinesterase in the brain tissues of three species of fresh water fish
and its activation in vivo; Ecology 39 194-199
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 1, January 1985, pp. 79-86.
© Printed in India.
Irradiation effects on the adrenal gland of rats undergoing inanition
stress
S S HASAN and P K CHATURVEDI
Zoology Department, Garhwal University Campus, Pauri-Garhwal 246001, India
MS received 30 July 1984; revised 1 October 1984
Abstract. The effect of total body x-irradiation was studied on rats under inanition stress. In
response to irradiation an increase in the activity of cortex and medulla was noted in inanition
stress administered rats rather than in the normally fed animals. Similarly, rising levels of
urinary catecholamines and 5-hydroxytryptamine were observed in the starved animals after
irradiation.
Keywords. Inanition stress; irradiation; adrenal gland.
1. Introduction
Earlier studies suggest that ionizing radiation decreases the production of corticoids
from the adrenal cortex (Nabors et al 1974; Nabors 1962; Berliner et al 1962; Stevens
et al 1963). Similarly, earlier investigators (Nair 1965; Hasan et al 1977, 1978, 1979;
Veninga and Brinkman 1962; Renson and Fischer 1959; Varagic et al 1967) have
demonstrated that the radiation elicits release of 5-HT and catecholamine in rats.
Further, the response of adrenal activity is mostly dose dependent to radiation
(Dougherty and White 1946; French et al 1955). However, no result seems to have been
reported on the effect of x-irradiation on the adrenal gland of rats undergoing inanition
stress. In this investigation, urinary metabolites of catecholamine and 5-hydro-
xytryptamine viz VMA and 5-HiAA have been studied in relation to inanition stress after
total body irradiation.
2. Material and methods
Male Holtzman strain rats (1 10-120 g) were used in all control and experimental
groups. Water was allowed ad libitum to each group of rats.
Group I: rats were starved for 10 days.
Group II: rats were starved for 10 days and on the 6th day of starvation they were
exposed to x-rays.
Group III: rats were fed on a standard commercial diet (Hindustan Levers Ltd., India)
to serve as control for groups I and IV.
Group IV: rats were fed on a standard diet as mentioned in the group III and exposed
to x-rays, on the day when rats of group II were irradiated.
Total body of animals of groups II and IV were exposed to x-rays, 1000R (80 kV;
200 MAS; 1 sec; 80 cm distance).
79
80
S S Hasan and P K Chaturvedi
The control and experimental rats were killed by decapitation at intervals of 24, 48
and 96 hr after irradiation /on the 6th day after starvation. Adrenals were dissected out
and fixed in Bouin's fluid. Paraffin sections (5p) were cut and stained with haematoxylin
and eosin. Before the sacrifice, urine of each rat of the control and the experimental
groups was collected in a specially devised box for 24 hr for the biochemical
investigation of S-HIAA (Subramaniyam and Narayanan 1973) and VMA (Armstrong et al
1957). Rate of mortality was also recorded during the stretch of experiments.
3. Results
The group undergoing inanition stress showed 294 % mortality from the 5th to the
10th day of starvation. On the other hand the group receiving a combined treatment of
starvation and irradiation exhibited nearly 33-3% mortality during this period which
Table 1. Chart of mortality.
Groups
Starvation
Normal -f
control Radiation Starvation Radiation
A B C D
Total no. of
rats taken
25 25 34 36
1st day of
starvation
— — — —
2nd day of
starvation
— —
3rd day of
starvation
— — — —
4th day of
starvation
— — — —
5th day of
starvation
— — 2 2
6th day of
starvation
— — 4
7th day of
starvation/
— — 2 4
24 hr after
irradiation
8th day of
starvation/
— — — —
48 hr after
irradiation
9th day of
starvation/
— — 2 4
72 hr after
irradiation
10th day of
starvation/
____ <-\
96 hr after
irradiation
Percentage
mortality
— — 2941% 33-33%
Irradiation and adrenal gland 81
indicated an increase in the rate of mortality as compared to the non-irradiated starved
rats (table 1).
3. 1 a Adrenal cortex: Cells of the normal adrenal cortex were tightly packed and had
a dense staining cytoplasm. Throughout the cortex, there was a rich vascular bed of
sinusoids (figure 1). In rats undergoing inanition stress there was hypotrophy of cortical
cells and most of the cells appeared vacuolated at the end of the 7th day of starvation.
With increase in post-starvation period the columnar cells were widely separated by the
appearance of large sinusoids which were conspicuously present in the region of zona
reticularis (figure 3). Irradiation of the rats undergoing starvation brought about
gradual hypertrophy of cells accompanied by degranulation of the cytoplasm; besides
nuclei were markedly atrophied and had agranular nucleoplasm. By the end of 96 hr of
post-irradiation most of the cells in the cortex appeared hypertrophied and showed
increased depletion of granular contents of the cytoplasm indicating hyperactivity of
cortical cells in response to irradiation (figure 4) as compared to the non-irradiated
starved rats (figure 3). Contrary to this, normal diet fed irradiated rats showed gradual
hypertrophy and vacuolization of cortical cells (figure 2) but the extent of hypertrophy
and vacuolization was lesser than that observed in the starved irradiated rats (figure 4).
3.1b Adrenal medulla: Chromaffin cells of normal control animals contained
secretory granules and the nuclei were large and prominently stained and had granular
nucleoplasm (figure 5). In response to inanition stress the medullary cells got
hypotrophied and the cells appeared vacuolated. With increase in post-inanition period
the cells further became hypotrophied, the cytoplasm appeared almost degranulated,
sinusoids were found filled with erythrocytes (figure 7). Total body x-irradiation of
starved rats hastened the process of vacuolization of the chromaffin cells with nuclear
atrophy and dilation of blood vessels (figure 8). In addition, sinusoids were found filled
with erythrocytes (figure 8). Normal diet fed irradiated rats showed degranulation of
the cytoplasm of chromaffin cells and copious secretions were observed around the
nuclei indicating the secretory activity of the medullary cells (figure 6) in response to
whole body irradiation.
3.2 Biochemical studies
3.2a S-HIAA (table 2): In all the three experimental groups, i.e., the normal diet fed
group, the inanition stress administered rats and the irradiated starved rats there was
rising concentration of S-HIAA in their urine as compared to the normal control. But
the level of urinary excretion of S-HIAA was greater in the irradiated rats undergoing
inanition stress than in the normal diet fed irradiated group and the non-irradiated
inanition administered group.
3.2b VMA (table 3): As compared to the normal control the level of urinary
concentration of VMA was higher in the normal diet fed irradiated control rats and the
starved irradiated rats. However, there was an increase in the concentration of VMA in
the urine of rats receiving combined treatment of starvation plus irradiation compared
with the levels of the normal diet fed irradiated control and the non-irradiated inanition
administered rats.
82
S S Hasan and P K Chaturvedi
Irradiation and adrenal gland
83
Li^fA^^jMi^^srSffiSB j •
n*Si^**wMF
C^1RfJ9*V^
•im*^v,y r. f^^vj^
&£'¥«<&*
ssWlti.?.** M^
84 S S Hasan and P K Chaturvedi
Table 2. Urine- S-HIAA (mg/24 hr) (mean ± so).
Time of
Normal
Radiation +
collection
control
Radiation
Starvation
Starvation
of urine (hr)
A
B
C
D
24
0-916
1-644
1-282
1-894
±0-125
±0-106
±0-051
±0-245
A\B P<0-01
^:CP<0-01
A\D P <0-01
B:C P<0-01
5:/)P<0-02
C:D P<0-01
48
0-888
1-094
1-674
2-325
±0-082
±0-068
±0-086
±0-165
A:B P<Wl
^:CP<0-01
A-.D P<0-01
J5:CP<0-01
B.D P<0-01
C:/)P<0-01
96
0-799
1-087
1-826
3-539
±0-077
±0-211
±0-263
±0-122
A:B P>0-01
A\C P<0-01
A:D P<0-01
B:C P<0-01
J9:/)P<0-01
C:£>P<0-01
Table 3. Urine-
3-methoxy, 4-hydroxy
mandelic acid (mg/24 hr)
(mean ± SD).
Time of
Normal
Starvation -f
collection
control
Radiation
Starvation
Radiation
of urine (hr)
A
B
C
D
24
0-965
1-257
1-231
1-535
±0-195
±0-089
±0-134
±0-143
A:B P<0-01
A:C P>0-01
A:D P<0-01
B:C P>0-01
JB:/)P<0-01
C:DP<0-01
48
0-826
1-242
1-475
1-700
±0-072
±0-078
±0-127
±0-056
X:£P<0-01
A:C P<0-01
A:D P<0-01
B:CP<0-01
B:D P<0-01
C:/)P<0-01
96
0-832
1-098
1-769
1-956
±0-072
±0-081
±0-080
±0-101
A:B P<0-01
/4:CP<0-01
A:D P<0-01
5:CP<0-01
J5:DP<0-01
4. Discussion
From the results it appears that administration of inanition stress leads to atrophy anc
vacuolization of cortical cells; besides there occurs dilation of vascular beds of sinusoids
in the adrenal cortex. This indicates that the adrenal cortex continues to secrete £
minimum amount of cortical hormone in order to meet the salt, water and glucos<
metabolism of the body during the course of inanition stress. The rats undergoing
Irradiation and adrenal gland 85
inanition stress when exposed to total body irradiation showed increased hypertrophy
of the cortical cells accompanied by almost complete depletion of the cytoplasm
indicating enhanced hyperactivity of the adrenal cortex following total body
x-irradiation during the inanition stress. In response to irradiation the non-starved rats
also showed hypertrophy and vacuolization of cells in the cortex but the extent of
changes were much more striking than those observed in the starved rats. Similar
changes in the adrenal cortex of rats following irradiation have been reported in our
earlier studies (Hasan et al 1977, 1978, 1979). This investigation attributes to the fact
that x-irradiation induces hyperfunctioning and forced elimination of cortical hor-
mone from the adrenal cortex in the starved animals rather than in the normally fed
animals.
It is observed that under the influence of inanition stress there occurs regression in
the size of medullary cells with consequential nuclear atrophy. In addition, chromaffin
cells showed vacuolization and sinusoids appeared dilated suggesting increased
liberation of hormones from the chromaffin tissues in the circulation. Starved rats when
exposed to total body x-irradiation show an enhanced depletion of content of
chromaffin cells and dilation of blood vessels with increasing number of erythrocytes in
the sinusoids. Hyperactivity of the adrenal medulla is also demonstrated after 60Co
irradiation (Hasan et al 1977, 1978, 1979). These histological changes indicate increased
hyperactivity of the medullary cells in the starved rats compared with the normally fed
rats after total body irradiation.
Increased excretion of urinary catecholamine and S-HIAA was noted after total body
x-irradiation. The rise in concentration of S-HIAA and VMA may be associated with the
hyperactivity of 5-hydroxytryptamine and catecholamine in the body as the former
happens to be the metabolite of the latter. Similarly, increase in the contents of 5-HT and
catecholamine was reported by earlier workers after irradiation (Nair 1965; Hasan et al
1977, 1978; Veninga and Brinkman 1962; Renson and Fischer 1959; Varagic et al 1967).
Rats undergoing inanition stress showed an increase in the excretion of 5-HiAA and VMA
in the urine. Thus, it seems that during the period of inanition stress activity of 5-HT and
nor-adrenalin gets augmented and probably this increase leads to rising levels of 5-HiAA
and VMA in urine of starved rats. Further, the starved rats when exposed to x-rays
showed an increase in the excretion of 5-HiAA and VMA and the concentration of these
urinary metabolites were greater than in those of the non-irradiated starved rats and the
normally fed irradiated rats. Further rise in the levels of 5-HiAA and VMA may be
attributed to a stimulatory action of x-irradiation which possibly seems to have
accelerated the already enhanced activity of 5-HT and nor-adrenalin owing to inanition
stress. Thus, the biochemical studies in the present investigation further corroborate
our histological observations on the adrenal gland.
The fact that x-irradiation induces more severe changes in the adrenal gland and
brings about an increased excretion of urinary catecholamine and 5-HT in the starved
rats rather than in the normally fed rats, favours our data recorded on the rate of
mortality.
Acknowledgement
The authors are grateful to CSIR, New Delhi for financial assistance.
86 S S Hasan and P K Chaturvedi
References
Armstrong M D, Me Millan A and Shaw D M 1957 VMA in the body fluids; Acta Biochem. Biophys. 25 421
Berliner D L, Berliner M L and Dougherty T F 1962 The effects of chronic irradiation by internally deposite
radionucleides on corticosteroids biosynthesis, in Some aspects of internal radiation (Oxford: Pergamc
Press) pp. 179-186
Dougherty T F and White A 1946 Pituitary-adrenal cortical control of lymphocyte structure and functic
as revealed by experimental irradiation; Endocrinology 39 370-385
French A B, Migeon C J, Samuel L T and Bowers J Z 1955 Effect of whole body x-irradiation c
17-hydroxycorticosteroid levels, leukocytes and volume of packed red cells in the rhesus monkey; Am.
Physioi 182 469-476
Hasan S S, Sarkar F H, Sharma T R, Prasad G C, Pant G C and Udupa K N 1977 Response of adrenal glar
to whole body 60Co irradiation; Indian J. Exp. Biol 15 513-516
Hasan S S, Sarkar F H, Pandey S N and Udupa K N 1978 A note on the response of adrenal gland and i
catecholamine to chemical radioprotector; Indian J. Anim. Sci. 48 698-700
Hasan S S, Srivastava L K and Udupa K N 1979 Effect of radioactive iodine on adrenal gland and i
bioamines in normal and stressful conditions; Indian J. Exp. Biol 17 380-384
Nabors C J Jr 1962 A model system for the study of radiation effect on adrenal steroidogenesis; Radial. Rt
16557
Nabors C J Jr, Hinckley J S and Fowkes K L 1974 Steroid 11 jS-hydroxydehydrogenase activity in beagl
bearing 241 Am; Radial Res. 59 (abst.) 212
Nair V 1965 Regional changes in brain serotonin after head x-irradiation and its significance in tl
protentiation of barbiturates hypnosis; Nature (London) 28 1293-1294
Renson J and Fischer P 1959 Liberation de 5-hydroxytryptamine par le rayonnment x-Arch; Physu
Biochem. 67 142-144
Stevens W, Berliner D L and Dougherty T F 1963 The effect of x-irradiation on the conjugation of sterok
Radiat. Res. 20 510-518
Subramaniyam S and Narayanan S 1973 A quick colorimetric method of estimation of 5-HiAA in urine; Cui
Med. Proc. 46 17
Varagic V, Stepanovic S, Svecenski N and Haydukovic S 1967 The effect of x-irradiation on the amount
catecholamines in the heart atria and hypothalamus of the rabbit and in brain and heart of rat; Int.
Radial. Biol 12113-119
Veninga T S and Brinkman R 1962 Random liberation of biogenic amines as a cause of early irradiati<
effects; Int. J. Radial. Biol 5 283-289
Proc. Indian Acad. Sci. (Anim. Sci.)> Vol. 94, No. 2, April 1985, pp. 87-91.
© Printed in India.
Laboratory culture of Diaphanosoma Senegal Gauthier, (Crustacea,
Cladocera) from south India
K VENKATARAMAN and S KRISHNASWAMY
Department of Environmental Biology, School of Biological Sciences, Madurai Kamaraj
University, Madurai 625021, India
MS received 3 March 1984
Abstract. Laboratory studies on growth and reproduction of Diaphanosoma Senegal
Gauthier show that this species has a life span of 18-7 days. Three preadult and sixteen adult
instars were recorded at a temperature range of 28-30°C. Maximum body size is attained at the
end of its life-cycle and the growth increment is more during preadult instars. The present
observations are compared with other laboratory studies on tropical South Indian
cladocerans.
Keywords. Growth; reproduction; Diaphanosoma Senegal.
1. Introduction
The various members of the zooplankton, inspite of some convergent similarities, have
different types of life history (Hutchinson 1967). According to Edmondson (1955),
laboratory studies of the life span, instar duration, egg production and growth are
valuable sources of information for zooplankton and secondary productivity studies.
Earlier studies on some tropical species of Cladocera like Moina micrura Kurz
(Murugan 1975a), Ceriodaphnia cornuta Sars (Murugan 1975b), Scapholeberis kingi
Sars (Murugan and Sivaramakrishnan 1976) and Daphnia carinata King
(Venkataraman 1981) from the freshwater ponds have shown some important
intraspecific differences. Since differences in the life histories are likely to involve
different relationships, it was felt that it would be of interest to study the growth and
reproduction of Diaphanosoma Senegal Gauthier under laboratory conditions
(28~30°C), recorded for the first time in South India.
2. Material and Methods
Ovigerous fetnales of D, Senegal were collected from a seasonal pond near the
University campus (Lat.: 9° 53' N; Long.: 78° E) and were acclimated to the laboratory
temperature (28-30°C). Just born neonates were separated from the mothers and were
reared in petridishes (50 ml) with pond water. The method used by Venkataraman
(1981) was followed to study the life history. Table 1 shows the details of complete life-
cycle on ten individuals.
3. Result
The body length of newly hatched Diaphanosoma which is a miniature form of adult in
all respects measures about 0-59 mm. There are three preadult instars and sixteen adult
87
88
K Venkataraman and S Krfshnaswamy
Table 1. Mean growth, egg number per brood and duration of instar
of D. Senegal at 28-30;C
Instar
number
Mean
body size
(mm)
Mean
carapace
size (mm)
Mean
egg
number
Cumulative
frequency
of eggs
Mean
duration
of instar(hr)
Cumulative
duration
of instar(hr)
1
O59
0-20
13-2
13-2
2
0-88
0-26
—
18-8
31-0
3
1-22
0-33
—
—
23-8
54-8
4
1-37
0-45
3-2
3-2
37-3
92-1
5
1-47
0-49
2-6
5-8
25-6
117-7
6
1-53
052
2-9
8-7
23-7
141-4
7
1-56
0-52
2-8
11-5
23-5
164-9
8
1-63
0-55
3-0
14-5
24-0
188-9
9
1-70
0-55
3-5
18-0
23-2
212-1
10
1-73
0-55
2-6
206
21-6
233-7
11
1-73
0-59
2-7
23-3
23-4
257-2
12
1-76
0-59
2-8
26-1
23-6
280-7
13
1-76
0-59
2-6
28-7
24-8
305-5
14
1-79
0-59
3-0
31-7
24-1
329-6
15
1-83
0-59
2-2
33-9
24-3
353-9
16
1-86
0-62
1-3
35-2
23-2
377-1.
17
1-86
0-62
3-0
38-2
22-6
399-7
18
1-89
0-62
2-3
40-5
24-0
423-7
19
1-89
0-62
—
4O5
24-0
447-7
instars. The body length of the first adult instar (4th instar) is 1-37 mm. An average
maximum length of 1*89 mm is attained during the 19th instar and the mean life span is
about 18-7 days (table 1). A comparison between the percentage of the preadult growth
in relation to instar number of a few tropical and temperate cladocerans is shown in
figure 1. It was observed that D. Senegal has better preadult growth. The growth
increment in relation to percentage of initial length and total length is shown in figure 2.
Maximum number of eggs per brood of a single individual is 6 and the maximum
cumulative frequency of egg production in a single individual is 56. The mean number
of eggs produced and the cumulative frequency of egg production are shown in table 1.
A comparison of egg production of a few tropical cladocerans in relation to instar
number is shown in figure 3.
4. Discussion
The study shows that D. senega! has a total of nineteen instars (three preadult and
sixteen adult) with an average life span of 18-7 days (447-7 hr) (table 1). The instar
number of D. senega! is less than that of Simocephalus acutirostratus (22) and
Ceriodaphnia comma (20), but greater than that of Daphnia carinata (18) and Moina
micrura ( 1 3). These differences in the instar numbers may be due to hereditary factors as
well as differences in the culture medium (Anderson and Jenkins 1942; Venkataraman
1 983). The duration of instars in D, senega! varies throughout its life-cycle. Primiparous
instar (4th instar, 37-3) is longer than the longest preadult instar (table 1). In this respect
Laboratory cultures of D. Senegal
89
100-
i
cr
UJ
QL
,S.a.
0 1 2 3 A 5 6
PREADULT INSTARS
Figure I. Percentage of preadult growth increment in relation to instar number of a few
tropical and temperate cladocerans: S.a. — Simocephalus acutirostratus; D.t — Daphnia
thomsoni; D.m — Daphnia magna; D.s — Diaphanosoma Senegal', D.p — Daphnia pulex\ D.a —
Daphnia atkinsoni; D.c — Daphnia carinata; M.m — Moina micrura.
INSTAR NUMBER
Figure 2. Growth increment as percentage of total body length and initial body length of
Diaphanosoma Senegal
this species differs from S. kingi&nd M. micrura (Murugan and Sivaramakrishnan 1976;
Murugan 1975a) where the duration of preadult and adult instars is uniform (24 hr)
throughout their life cycle. D. Senegal is similar to 5. acutirostratus and D. carinata
in having the longest preadult instar (Murugan and Sivaramakrishnan 1973;
Venkataraman 1981).
Preadult growth increment as percentage of initial length of tropical and temperate
cladocerans is shown in figure 1. The greatest growth increment does not always occur
at the end of the adolescent instar or more rarely even earlier (Green 1955). In D. Senegal
the growth increment as percentage of initial length is 45-6 % initially and reaches
71 -6 % at the 3rd instar (figure 2). From the 4th instar (adult instar) onwards the growth
increment as percentage of initial length decreases suddenly. In the middle of its life
span (at 10th instar) it attains 90 % of growth (figure 2). The body length of D. Senegal at
the 4th instar is 1-37 mm. Comparison of preadult growth increment as percentage of
90
K Venkataraman and S Krishnaswamy
30-
cr
u
CD
o
o
UJ
18
12
So.
10
INSTAR NUMBER
15
20
Figure 3. Egg production in relation to instar number of some tropical cladocerans: S.a —
Simocephalus acutirostratus; S.k — Scapholeberis kingi; C.c — Ceriodaphnia cornuta; Mm —
Moina micrura; D.c — Daphnia carinata; D.s — Diaphanosoma Senegal.
initial length of a few temperate and tropical cladocera show that the temperate
daphnids like D. thomsoni, D. magna, D. atkinsoni and D. pulex (Green 1955) have the
greatest growth increment during the preadult instar. S. acutirostratus (Murugan and
Sivaramakrishnan 1973) attains maximum growth increment in the 3rd preadult instar
(84%) and this is the highest growth increment reported in any temperate or tropical
species. M. micrura on the other hand shows minimum growth increment during the
preadult instar and it recovers its growth only during the adult instars (Murugan
1975a). A study of D. Senegal reveals that the highest rate of growth increment is seen
during preadult instar 3 (45-6 %).
Progressive increase in body size is a measure of growth rate of the individual
(Edmondson 1 955). The mean growth increment of D, Senegal is rapid during the earlier
phase and is very slow during the reproductive phase. Rapid preadult growth increment
seems to be a common feature for Cladocera irrespective of physiological and
physicochemical factors (Venkataraman 1983). The growth pattern of D. Senegal shows
a s-shaped curve. According to Hutchinson (1967) the growth rate per instar is always
correlated with food supply, but growth pattern studies of D. Senegal in relation to food
or temperature has not been made.
The mean number of eggs per brood in relation to instar number of D. Senegal is
compared with a few tropical South Indian cladocerans (figure 3). The maximum
number of eggs per brood under laboratory conditions (28-30°C) of a single
individual is 6 and the total number of eggs produced in a life span is 56 (maximum
number). Comparative study of egg production of tropical South Indian cladocerans
under the same conditions reveals that S. acutirostratus (Murugan and
Sivaramakrishnan 1973) produces the maximum number of eggs per brood (27) when
compared to other tropical Cladocera. D. Senegal produces a lesser number of eggs per
brood, with a maximum of 3-5 eggs at the 9th instar (figure 3). The variation in egg
Laboratory cultures of D. Senegal 91
production in the species of Cladocera can be attributed to the amount of food
available for the organism during its life span (Dunham 1938; Anderson and Jenkins
1942), temperature of the culture medium (Me Arthur and Baillie 1929), and genetic
makeup of the animal (Banta and Wood 1939).
An interesting aspect of this study is the record that D. Senegal produces sexual eggs.
The parthenogenetic eggs of D. Senegal are oval but the sexual eggs are white in colour.
The egg is covered by an outer leathery coat. When the eggs are dried, the outer shell
shrinks and floats but when wet they sink to the bottom. The sexual eggs are entirely
different from those of D. carinata which have a black, thick chitinous outer coat.
Acknowledgement
One of the authors (KV) is indebted to the CSIR for the award of a fellowship.
References
Anderson D G and Jenkins J C 1942 A time study of events in the life span of Daphnia magna; Bull. Biol. 83
260-272
Banta A M and Wood T R 1939 General studies in sexual reproduction; (In Banta 1939) 131-181
Dunham H H 1938 Abundant feeding followed by restricted feeding and longevity in Daphnia; Physioi Zool
11 399-407
Edmondson W T 1955 Seasonal life history of Daphnia in an arctic lake; Ecologia 36 439-455
Green J 1955 Growth, size and reproduction in Daphnia (Crustacea: Cladocera); Proc. Zool. Soc. Lond. 126
173-204
Hutchinson G E 1967 A treatise on limnology (New York: John Wiley) Vol. II. 575-582
Me Arthur J W and Baillie W H T 1929 Metabolic activity and duration of life I. Influence of temperature
on longevity in Daphnia magna; J. Exp. Zool. 53 221-242
Murugan N 1975a Egg production, development and growth in Moina micrura Kurz (Cladocera, Moinidae);
Freshwater Bioi 5 245-250
Murugan N 1975b The biology of Ceriodaphnia cornuta Sars (Cladocera, Daphnidae); J. Inland Fish. Soc.
India 1 80-87
Murugan N and Sivaramakrishnan K G 1973 The biology of Simocephalus acutirostratus King (Crustacea,
Cladocera; Freshwater Biol 3 77-87
Murugan N and Sivaramakrishnan K G 1976 Laboratory studies on the longevity, instar duration, growth,
reproduction and embryonic development of Scapholeberis kingi Sars (1903); Hydrobiology 50 75-80
Venkataraman K 1981 Field and laboratory studies on Daphnia carinata King (Cladocera, Daphnidae) from
a seasonal tropical pond; Hydrobiology 78 221 — 225
Venkataraman K 1983 Taxonomy and ecology of Cladocera of southern Tamil Nadu Ph.D. thesis, Madurai
Kamaraj University, Madurai. 190 pp.
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 2, April 1985, pp. 93-97.
© Printed in India.
Laboratory evaluation of anticoagulant treated baits against Indian
field mouse, M us booduga Gray
M BALASUBRAMANYAM, M J CHRISTOPHER and
K R PURUSHOTHAM
Pesticide and Industrial Toxicology Centre, Department of Zoology, S. V. University, Tirupati
517502, India
Abstract. The toxicity of warfarin (0-025%), bromadiolone (0-005%) and brodifacoum
(0-005 %) to the Indian field mouse, Mus booduga Gray was determined. A single feeding of
bromadiolone or brodifacoum resulted in 83% mortality, while the same mortality was
obtained with warfarin only after 6 days of continuous feeding. A single day feeding of a single
dose chronic poison was effective against M. booduga than multiple dose chronic
rodenticide.
Keywords. M. booduga^ no-choice tests; mortality; susceptibility; warfarin; second gener-
ation anticoagulants; bromadiolone; brodifacoum.
1. Introduction
The Indian field mouse, Mus booduga Gray is a predominant rodent pest in irrigated
lands nesting in shallow burrows with single or branched tunnels, particularly in South
India and causes damage to paddy, ragi, sorghum, sugarcane and chillies at seedling,
growth and harvest stages (Chandrahas 1 974; Purushotham and Mohana Rao 1 979). In
Andhra Pradesh it breeds from August to February with a gap in summer months
(Mohana Rao 1980). The field mice can be controlled either by placing baits such as
cracked rice or bajra with 1 % groundnut oil treated with 1-2 % zinc phosphide or by
using 0-025 % warfarin or fumarin in the preferred bait. Of late the second generation
anticoagulants emerged in view of the bait shyness developed with zinc phosphide and
resistance with warfarin by a number of rodents. However, the use or value of
rodenticides against M. booduga can be assessed basing on data from laboratory and
field investigations. The present study aimed at evaluating the two second generation
anticoagulants along with the warfarin (rodafarin 6C), helps to make good the
deficiency.
2. Materials and methods
Mice captured around Tirupati (Andhra Pradesh) were acclimatised to laboratory
conditions for 2 weeks by maintaining them individually on food (Mohana Rao et al
1978) and water ad libitum. Experimental groups consisted of equal number of males
and females.
The mice were provided with anticoagulant treated baits for 1, 2 and 3 days in case of
second generation anticoagulants; 2, 4 and 6 days for warfarin separately. Daily intake
of poisoned bait was recorded by offering freshly prepared bait every day, besides
93
94 M Balasubramanyam, M J Christopher and K R Purushotham
noting down the day of mortality of the animal. The mortality due to poison was
confirmed by autopsy and signs of anticoagulant poisoning.
Technical grade bromadiolone (1%) and brodifacoum (025%) were mixed with
cracked bajra (Pennisetum typhoides) to give 0-005 % concentration. Warfarin (0*5 %
technical grade) was mixed with the bait to give 0-025 % concentration. The methods
provisionally recommended by WHO (1976) were followed for determining the
susceptibility of rodents to anticoagulant rodenticides.
3. Results
Data on the no choice tests with two anticoagulants viz, bromadiolone and
brodifacoum (tables 1 and 2) show that both these poisons caused complete mortality
within two weeks. A single feeding of bromadiolone and brodifacoum mixed baits
resulted in 83% mortality; while 100% mortality was obtained after two and three
feedings respectively. Warfarin is the least effective giving 83 % kill after 6 days of
continuous feeding (table 3). The mean days taken for causing 83 % mortality using
bromadiolone, brodifacoum (in single dose) and warfarin (in multiple dose) was 9*2, 9*4
and 8-6 days respectively. Mortality occurred after day 3, on consumption of bait
containing either bromadiolone or brodifacoum, whereas, death occurred only after
day 5 when fed on warfarin treated bait. Bait intake in no-choice tests was high up to
4-5 days which later declined possibly due to the development of symptoms of
anticoagulant poisoning. In case of all the three rodenticides, increase in the number of
feedings resulted in the lowering of the time taken to die.
4. Discussion
A single day feeding of second generation anticoagulants resulted in 83 % mortality (in
9 days) in M. booduga. Mathur and Prakash (1980) noted 66 % mortality (in 7-6 days) in
Funambulus pennanti with a single feeding of brodifaeoum mixed bait. The combined
sex mortality for brodifacoum (in 6-25 days) on Gerbillus gleadowi was 50 % (Soni and
Prakash 1981). A similar study by Renapurkar and Kamath (1982) revealed 75, 80 and
100 % mortality (in 10 days) for Rattus rattus, R. norvegicus and Bandicota bengalensis.
Fifty and 35 % mortality (in 74 and 5*6 days) were obtained from a single feeding of
0*002 % brodifacoum and 0*005 % bromadiolone respectively in cotton rat, Sigmodon
hispidus (Gill and Redfern 1980). Meehan (1978) reported 100% mortality (in 6-8 days)
in R. norvegicus. The mortality (83 % in a single dose) seen in M. booduga using
bromadiolone and brodifacoum indicate that these chemicals are effective in single
dose against test species. Since there is no appreciable attenuation, in the time taken for
death between 1 and 2 feeding periods and since the maximum mortality was evidenced
in single dose feeding itself, control of M. booduga with single day feeding of either oi
these two poisons may be expected in the crop fields. Although the average time to elicit
83 % mortality for all the three poisons was around 9 days, the use of single dose
anticoagulants saves a lot of bait material and the manual operations involved as
against the warfarin, a multiple dose poison.
The evaluation of warfarin against jR. rattus (Agarwal et al 1979); R. argentiventei
(Buckle et al 1980); B. bengalensis (Brooks et al 1980); and a variety of desert rodents
(Mathur and Prakash 1981) has been made. The range and mean days to death
Anticoagulant treated baits against M. booduga
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Anticoagulant treated baits against M. booduga 97
warfarin in M. booduga can be comparable to the other species. In 2, 4 and 6 days
feeding of warfarin, mortality started from day 6 and lasted up to day 16 and the
maximum kill occurred between 6 and 10 days. Similar mortality rates were noted for
bromadiolone and brodifacoum when used for 1 day only (tables 1 and 2). Comparing
susceptibility of warfarin to M. booduga with other species, it is revealed that they are
less susceptible than Tatera indica, Meriones hurrianae (Mathur and Prakash 1982) and
B. bengalensis (Sridhara 1979; Brooks and Bowerman 1974). Thus it can be concluded
that 0-005% bromadiolone and brodifacoum is more toxic and active against
M. booduga than warfarin, giving a satisfactory mortality with single day feeding
period.
Acknowledgements
The authors thank M/s Pest Control India (Pvt) Ltd., Madras for financial support and
for supply of warfarin and bromadiolone. They also thank M/s Alkali Chemical
Corporation, Bangalore for supply of brodifacoum.
References
Agarwal R K, Singh K N, Srivastava P K and Girish G K 1979 Relative efficacy of different chemicals against
the house rat, Rattus rattus; Bull. Grain Technol 17 10-13
Brooks J E and Bowerman A M 1974 An analysis of susceptibilities of several populations of Rattus
norvegicus to warfarin; J. Hyg. Camb. 73 401-407
Brooks J E, Htun P T and Naing H 1980 The susceptibility ofBandicota bengalensis from Rangoon, Burma
to several anticoagulant rodenticides; J. Hyg, Camb. 84 127^-135
Buckle A P, Rowe F P and Yong Y C 1980 Laboratory evaluation of 0-025% warfarin against Rattus
argentiventer; Tropical Pest Management 26 162—166
Chandrahas R K 1974 Ecology of the brown spiny mouse, Mus p. platythrix (Bennett) and the Indian field
mouse, Mus 6. booduga (Gray); Indian J. Med. Res. 62 264-280
Gill J E and Redfern R 1980 Laboratory trials of seven rodenticides for use against the cotton rat (Sigmodon
hispidus); J. Hyg. Camb. 85 443-450
Mathur R P and Prakash I 1980 Laboratory evaluation of anticoagulant treated baits for the control of
northern palm squirrel, Funambulus pennanti Wroughton; J. Hyg. Camb. 85 421-426
Mathur R P and Prakash I 1981 Comparative efficacy of three anticoagulant rodenticides against Indian
desert rodents; Prof. Ecol 3 327-331
Mathur R P and Prakash 1 1 982 Evaluation of warfarin against Tatera indica and Meriones hurrianae; Proc.
Indian Acad. ScL (Anim. Scl) 91 463-468
Meehan A P 1978 Rodenticidal activity of Bromadiolone — A new anticoagulant; Proc. Eighth Yen. Pest
Con/. Sacramento California 122-126
Mohana Rao A M K 1980 The Indian field mouse— A serious rodent pest of agriculture and its control;
Pestology 41-3
Mohana Rao A M K, Purushotham K R and Rajabai B S 1978 Baits for the control of Indian field mouse,
Mus booduga Gray; Z. Angew. Zoo/. 65 163-172
Purushotham K R and Mohana Rao A M K 1 979 The distribution of field rodents and their burrowing habits
around Tirupati (Andhra Pradesh); The Andhra Agric. J. 26 85-87
Renapurkar D M and Kamath V R 1982 Laboratory evaluation of brodifacoum, a new anticoagulant
rodenticide; Bull Haffkine Inst. 10 17-20
Soni B K and Prakash 1 1 98 1 Laboratory evaluation of WBA 8119 (Brodifacoum) against Gerbillus gleadowi
Murray; Pestology 5 23-24
Sridhara S 1979 Rodenticidal action of poisons on two rodent pests in India; Pest Control 41 30-31
World Health Organisation 1976 Instructions for determining the susceptibility or resistance of rodents to
anticoagulant rodenticides; WHO Technical Report Series No. 443
>roc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 2, April 1985 pp 99-110
J) Printed in India.
Effects of starvation on respiration and major nutrient stores of the
prosobranch snail Bellamya bengalensis (Lamarck)
D SATYA REDDY and M BALAPARAMESWARA RAO
Department of Zoology, Nagarjuna University, Nagarjunanagar 522510, India
MS received 28 July 1984
Abstract. The effects of starvation on the metabolic rate and the glucose, glycogen and total
lipid contents in the freshwater prosobranch snail Bellamya bengalensis (Lamarck) have been
investigated. Starvation influenced the metabolic rate of B. bengalensis. Although there was an
initial increase, the metabolic rate of both the sexes decreased in the later stages of starvation.
There was a marked difference in the utilization of nutrient stores between male and female
snails starved for 55 days.
Keywords. Bellamya bengalensis; metabolic rate; nutrient stores; starvation effects.
1. Introduction
Studies on the effect of starvation on the oxygen consumption of molluscs are sparse.
Berg and Ockelmann (1959) studied the effects of starvation on the oxygen
consumption of Ancylusfluviatilis, starved for 96 hr. Stickle and Duerr (1970) in Thais
lamellosa, Widdows (1973), Bayne (1973) and Bayne et al (1976) in Mytilus edulis, Mane
(1975) in Katelysia opima and Mane and Talikhedker (1976) in Donax cuneatus studied
respiration in relation to starvation. Heeg (1977) observed the oxygen uptake during
starvation and aestivation in the pulmonate snail Bulinus africanus.
Not only the metabolic rate but also the nutrient reserves decreased during
starvation in a number of molluscs, von Brand (1931) found that in Helix pomatia the
oxygen uptake and also the carbohydrate reserves decreased during starvation.
Emerson (1967) found that the metabolism of the aquatic pulmonate Planorbis corneus
is carbohydrate oriented because the snail utilized 95 % of the original carbohydrate
during 58 days of starvation. Emerson and Duerr (1967) found the herbivorous
prosobranch Littorina planaxis and Stickle and Duerr (1970) found the carnivorous
prosobranch Thais lamellosa to have lipid oriented metabolism, since these snails
utilized lipid during starvation.
It appears that carbohydrate and/or lipid are the main nutrient reserves of molluscs.
Many lamellibranchs and pulmonates utilize glycogen (von Brand et al 1948; 1957;
Martin 1961; Martin and Goddard 1966) whereas, amphineurans utilize lipid (Giese
1966). Some gastropods appear to have carbohydrate oriented metabolism while others
have lipid oriented metabolism.
In the present study an attempt has been made to investigate the effects of starvation
on metabolic rate and glucose, glycogen and lipid reserves of the herbivorous
prosobranch Bellamya bengalensis (Lamarck).
99
100 D Satya Reddy and M Baiaparameswara Rao
2. Material and methods
Animals were collected from a pond at Vengalayapalem village (16°24N:80°33'E).
After clearing the encrustations on the shells, the snails were acclimatized in the
laboratory at 27 ± 2°C for 96 hr.
2.1 Respiratory measurements
Respiratory measurements were made by the method of Ganapati and Prasada Rao
(1960) and the dissolved oxygen was estimated by Winkler's method (Golterman 1 970).
Metabolic rates were measured on individual snails at day 4, 8, 16 and 20 of starvation.
Experiments were run for 2 hr and the time of the experiment (1 100-1 300 hr) was kept
constant to avoid the effect of time of day on the respiration of snails. Each snail was
used only once and the oxygen consumption for 1 hr was taken into consideration.
After the experiment the snails were shelled and the wet weight of the soft parts was
determined to the nearest 0-1 mg. The allometric equation y/x = aX(b~ J) (Davies 1966;
Newell 1970) was used to express the results. All experiments were carried out at
constant temperature (27 ± 1°C) and pH (8-9 ±0-1). Experiments were performed on 10
males and 10 females at each period of starvation except at day 4 where 40 males and 60
females were used.
2.2 Statistical procedure
Regression lines were fitted by the method of least squares. The slopes and intercepts
(elevations) of the regression lines are compared separately between males and females
at each period of starvation and between successive periods of starvation in males and
females using analysis of co-variance (Snedecor and Cochran. 1967).
2.3 Biochemical methods
The glucose, glycogen and total lipid contents of snails prestarved and starved for 55
days were also estimated. The glucose and glycogen contents were estimated by the
method of Kemp et al (1954) and total lipid by the method of Pande et al (1963).
Immediately prior to chemical analysis, the animals were weighed, removed from the
shells and sexed. They were then dissected, foot and visceral mass separated and their
wet weight determined to the nearest 0-1 mg. Experiments were performed on 20
animals and the results are expressed as mg per lOOmg of wet weight.
3. Results
Few of the experimental animals died during the beginning weeks of starvation, only
10 % mortality was observed on day 20. Most mortality occurred between days 40 and
50. Approximately 40% of the experimental animals were alive on day 55.
Figures 1 and 2 show the relationship of the metabolic rate and weight at different
Starvation effects on respiration and nutrients of Bellamya
101
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2 0-2
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cr
0-1
0
-1-9
_L
J_
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-1-6 -1-8 O
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Figure 1. The relationship of log metabolic rate and log wet weight of soft tissues (ST) in
males on different days of starvation.
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Figure 2. The relationship of log metabolic rate and log wet weight of soft tissues (ST) in
females on different days of starvation
periods of starvation in males and females, respectively. The metabolic rate of both the
sexes at all periods of starvation is negative linear in relationship with weight when
plotted logarithmically. The regression equations are given in table 1 . The wet weight of
the soft parts and the metabolic rate ranged from 0-33 to l-24g and 0-63 to l-94mg
O2/g/hr in males and 0-47 to 1-88 g and 0-52 to 2-1 8 mgO2/g/hrin females, respectively.
102 D Satya Reddy and M Balaparameswara Rao
Table 1. Equations of the regression lines: log MR = log a 4- (b - 1) log W, relating weight
(W) in g. to metabolic rate (MR) of B. bengalensis at different periods of starvation.
Period of Regression equations
starvation ~"~~"
(days) Male Female
4 log MR == 0-0224 - 0-4142 log W log MR = 0-0639 - 0-5718 log W
8 log MR - 0-0506 - 0-5487 log W log MR = 0-1098 - 0-6718 log W
12 log MR = 0-0657 - 0-5838 log W log MR = 0-1225 - 0-7842 log W
16 logMR = -0-0292 -0-6630 log W log MR = -0-0508-0-9271 logVP
20 log MR « -0-0953-0-5134 log W log MR = -0-0348-0-7245 log W
n = 10 for each sex except at the period 4 days where 40 males and 60 females were used.
Tables 2-4 show the results of ANCOVA comparing the slopes and elevations of the
regression lines for various sex-starvation combinations. The results presented in table
2 suggest common slopes and also common elevations for the two sexes for the 4th and
16th day of starvation, thereby indicating no marked differences in the metabolic rate of
the male and female snails on these periods of starvation. Further, it is also clear that
common slopes or elevations could not be assumed for the two sexes for the remaining
periods of starvation (8, 1 2 and 20 days) suggesting that the metabolic rate of males and
females differs significantly on these days.
The position of the regression lines in figures 1 and 2 shows that the metabolic rate in
both the sexes is highest on 1 2th day and lowest on 20th day of starvation. The results of
ANCOVA (tables 3 and 4) indicate common slopes amongst different periods of starvation
in both the sexes. However, the elevations were significantly different between 4th and
12th day, 12th and 16th day and 16th and 20th day in males (table 3) and between 4th
and 12th day and 12th and 16th day in females (table 4). The elevations also were not
significantly different between 4th and 8th day and 8th and 16th day in both the sexes
and between 16th and 20th day in females.
The above observations suggest that the metabolic rate between days 4 and 8, 8 and
12 in both the sexes and 1 6 and 20 in female is not significantly different. The difference
in the metabolic rate between 4th and 12th day, however, is significantly different in
both the sexes.
The effects of starvation on the metabolic rate of different standard weights,
calculated from the regression equations presented in table 1, are shown in figures 3
and 4. It is evident from these figures that the general pattern of increase or decrease is
same in both the sexes, but the rate at which the metabolic rate increased or decreased is
different in different sizes of males and females. The percentage increase from 4th to
12th day in the young (0-5 g) and adult (1-25 g) males was 26 and 6 and in the
young (0-5 g) and adult (1-5 g) females was 32 and 6, respectively.
The metabolic rate in both the sexes decreased after 1 2th day. In males it decreased by
10 to 20% and in females 25 to 43% from 12th to 16th day for different sizes.- The
metabolic rate showed further decrease of 17 to 25 % from 16th to 20th day in males of
different sizes while in females it remained more or less constant.
Emerson (1967) while studying the metabolism ofPlanorbis corneus stated that "in
order to estimate the weight loss of dry soft parts during starvation, it is necessary to
calculate the prestarved weights". Therefore, the wet weight, dry weight and entire
Starvation effects on respiration and nutrients of Bellamy a
103
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Days
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Figure 3. Metabolic rate-starvation curves for male B. bengalensis of different body weights.
2-2
cr 1-0
2
0-6
Figure 4.
weights.
12
Days
20
Metabolic rate-starvation curves for female B. bengalensis of different body
weight relations are also calculated and a summary of the results is presented in table 5.
From this table it is clear that the dry weight of the soft parts of animals starved for 55
days is 87 % of the prestarved weights indicating a loss of 1 3 % . This is considerably less
than the weight loss of 23 % reported for the carnivorous prosobranch Thais lamellosa
starved for 53 days (Stickle and Duerr 1970).
Few observations were also made on the metabolic rate of snails starved for 55 days.
The average metabolic rate (n = 5; shell height range 1 -8 to 2-5 cm) in males was 0-96 mg
O2/g/hr and in females 0-71 mg O2/g/hr on 55th day of starvation.
The glucose, glycogen, total lipid contents and their percentage decrease in foot and
viscera of male and female snails starved for 55 days are shown in table 6. The glucose,
glycogen and total lipid in males and glucose and total lipid in females decreased more
in foot than in viscera. Further, the glucose and glycogen contents decreased more in
males than in females whereas, the iipid content decreased more in females than in
males starved for 55 days.
Starvation effects on respiration and nutrients of Bellamy a
Table 5. Weight loss of Bellamy a bengalensis starved for 55 days.
Control group Animals starved for 55 days
(average of 20 animals) (average of 10 animals)
Wet weight of soft parts
of animals expressed as
percentage of entire
weight
Dry weight of soft parts
of animals expressed as
percentage of wet
weight
Dry weight of soft parts
of animals expressed as
percentage of entire
weight
Dry weight of soft parts
of animals after
starvation expressed as
percentage of original
dry weight
Factor 1-15
(Figure in parenthesis indicates standard error).
Factor = Reciprocal of 87%.
Table 6. Glucose, Glycogen and total lipid con-
tent of males and females of Bellamya bengalensis
starved for 55 days.
107
31-00
(±0-7417)
18-19
(±0-5764)
5-64
(±0-2399)
34-36
(±1-8663)
13-99
(±1-1706)
4-89
(±0-6096)
87-00
Male
Female
Foot Viscera Foot Viscera
Glucose (%)
Control
Starved
% Decrease
Glycogen (%)
Control
Starved
% Decrease
Total lipid (%)
Control
Starved
% Decrease
23-75 29-30 21-50 29-30
13-13 22-50 13-75 22-76
44-72 23-21 36-05 22-32
43-75 46-25 23-75 41-25
16-56 26-25 16-56 21-88
62-14 43-24 30-27 46-96
0-51 11-19 0-83 17-44
0-40 9-82 0-55 14-86
20-80 12-20 34-04 14-79
4. Discussion
Decrease in the metabolic rate has been reported in a number of starved molluscs.
Lomte and Nagabhushanam (1971) found that the oxygen consumption of the
freshwater mussel, Parreysia corrugata was reduced to nearly 50 % after starving the
108 D Satya Reddy and M Balaparameswara Rao
animals for 10 days. Three-fifths reduction in the initial value of oxygen uptake of
the limpet, Ancylus fluviatilis was recorded after 96 hr of starvation (Berg and
Ockelmann 1959). There was 50% reduction in the metabolic rate after a period of 8
days starvation in the clams, Katelysia opima (Mane 1975) and Donax cuneatus (Mane
and Talikhedker 1976). In Bellamya bengalensis also starvation affected the metabolic
rate. In both the sexes the metabolic rate showed an initial increase of about 1 5 % from
4th to 12th day of starvation. The metabolic rate decreased from 12th day onwards,
but the fate of decrease was different in males and females. The metabolic rate of males,
from 12th to 16th day, decreased by about 15 % and from 16th to 20th day by another
15 %; whereas the metabolic rate of females, from 12th to 16th day, decreased by about
30 % and from 1 6th to 20th day it remained more or less constant. Thus ultimately there
is only 15 % reduction, from the initial value, in the metabolic rate of both the sexes of
Bellamya bengalensis indicating that this decrease (15 %) is considerably less than that
reported for bivalves, Parreysia corrugata, Katelysia opima and Donax cuneatus (50 %)
and Ancylus (60%).
It is of interest to note that, whereas the oxygen consumption of the pulmonates (von
Brand et al 1948; Duerr 1965) and bivalves (Lomte and Nagabhushanam 1971;
Widdows 1973; Bayne 1973, 1976; Mane 1975; Mane and Talikhedker 1976) decreased
during starvation, the oxygen consumption of the prosobranch, Bellamya bengalensis
increased during the initial days of starvation. This is in agreement with the previous
work on the carnivorous prosobranch Thais lamellosa which also shows an increased
oxygen consumption during starvation (Stickle and Duerr 1970).
After 12th day the metabolic rate of both sexes of B. bengalensis decreased. Stickle
and Duerr (1970) stated that "a decreased oxygen consumption could indicate a
lowered metabolic rate or it could indicate a switch in emphasis from a lipid oriented
metabolism to a carbohydrate or protein oriented metabolism", von Brand et al (1948)
found that in pulmonates the lowered oxygen consumption is an adaptation to
conserve food stores. The increasing metabolic rate of B. bengalensis up to the 12th day
of starvation indicates that this snail does not possess this adaptation in the initial
stages of starvation. However, decrease in the metabolic rate after 12th day suggests
conservation of food reserves or it may indicate a switch in the metabolism as suggested
by Stickle and Duerr (1970). Calow (1975) has shown that on starvation, animals may
have one of two responses; they may decrease their metabolism immediately as a means
of saving energy or they may initially increase their metabolism due to increased activity
caused by searching for food. The present study on Bellamya bengalensis indicates the
possibility of both these responses in starving animals.
Twenty to 40 % of glucose, 30 to 60 % of glycogen and 1 2 to 34 % of total lipid is lost
in snails starved for 55 days. This indicates that the snails utilize more glucose and
glycogen than lipid during starvation. The decrease in the lipid content may be due to its
direct utilization or it may be converted into carbohydrate during starvation. The
results suggest that probably the metabolism of the herbivorous prosobranch, B.
bengalensis is carbohydrate oriented like the other freshwater snails Helix pomatia (von
Brand 1931) and Planorbis corneus (Emerson 1967). It is possible that some of the
protein might have also been utilized during starvation, but no measurements of
protein were made in the present study.
It is also interesting to note that, whereas the metabolic rate of males continued to
decrease from 16th to 20th day of starvation, the metabolic rate of females remained
constant during this period, thereby indicating that the ultimate decrease in the
Starvation effects on respiration and nutrients of Bellamy a 109
metabolic rate of both the sexes remains the same (15%) at the end of 20 days
starvation. Further, when the metabolic rate of males decreased by only about 2 %, the
metabolic rate of females decreased by about 12% from 20th-55th day of starvation.
Furthermore, there was also a marked difference between the male and female snails in
the utilization of glucose, glycogen and total lipid stores. These observations suggest
that there may be differences in the basic physiological and biochemical processes of the
two sexes during starvation and it would be of interest to examine these processes in
detail.
Acknowledgements
The authors wish to thank Prof. G M Branch of University of Cape Town, South
Africa, for his criticism and suggestions on the text. One of the authors (DSR) wishes to
thank the CSIR., New Delhi, for the award of a fellowship.
References
Bayne B L 1973 The responses of 3 species of bivalve molluscs to declining oxygen tension at reduced salinity;
Comp. Biochem, Physiol. 45 793-806
Bayne B L, Thompson R J and Widdows J 1976 Physiology I. In Marine mussels: their ecology and physiology
(ed.) B L Bayne (Cambridge: Univ. Press) pp. 121-206
Berg K and Ockelmann K W 1959 The respiration of freshwater snails; J. Exp. Biol 36 690-708
Calow P 1975 The respiratory strategies of two species of freshwater gastropod (Ancylusfluviatilis Mull, and
Planorbis contortus (Linn) ) in relation to temperature, oxygen concentration and body size and season;
Physiol. Zool. 48 114-129
Davies P S 1966 Physiological ecology of Patella. 1. The effect of body size and temperature on metabolic
rate; J. Mar. Biol. Assoc. U.K. 46 647HS58
Duerr F 1 965 Some aspects "of diet on the Tespiration rate of the freshwater pulmonate snail Lymnaea
palustris; Proc. Dak. Acad. Sci. 44 245
Emerson D 1967 Carbohydrate oriented metabolism of Planorbis corneus (Mollusca: Planorbidae) during
starvationjComp. Biochem. Physiol. 22 571-579
Emerson D and Duerr F 1 967 Some physiological effects of starvation in the intertidal prosobranch Littorina
planaxis (Philippi, 1847); Comp. Biochem. Physiol 20 45-53
Ganapati P Nand Prasada Rao D G V 1960 Studies on the respiration of barnacles: Oxygen uptake and the
metabolic rate in relation to body size in Balanus amphitrite communis (darwin). J. Anim. Morphol.
Physiol. 727-31
Giese A 1966 Lipids in the ecology of marine invertebrates; Physiol. Rev. 46 244-298
Golterman H L 1970 Methods for chemical analysis offreshwaters (Oxford: Black well Scientific PubL), I.B.P.
Hand Book No. 8, p. 166
Heeg J 1 977 Oxygen consumption and the use of metabolic resources during starvation and aestivation in
Bulinus (Physopsis) africanus (Pulmonata: Planorbidae); Malacologia 16 549-560
Kemp A, Adrenne J M and Kitts van Hejningen 1954 A colorimetric micro method for determination of
glycogen in tissues; Biochem. J. 56 646-648
Lomte V S and Nagabhushanam R 1971 Studies on the respiration of freshwater mussel Perreysia corrugata;
Hydrobiologia 38 239-246
Mane U H 1975 Oxygen consumption of the clam Katelysia opima in relation to environmental conditions;
Broteria 44 33-38
Mane U H and Talikhedker P M 1976 Respiration of the wedge clam Donax cuneatus; Indian J. Mar. Sci. 5
243-246
Martin A 1961 The carbohydrate metabolism of mollusca. In Comp. Physiol. carbohydrate metabolism in
heterothermic animals (ed.) A Martin (Seattle: University of Washington Press) 35-64
110 D Satya Reddy and M Balaparameswara Rao
Martin A and Goddard C 1966 Carbohydrate metabolism. In Physiology ofmollusca (eds) K M Wilbur and
C M Yonge (New York: Academic Press) 2 275-308
Newell R C 1970 Biology ofintertidal animals (London: Logos Press Ltd.) pp 539
Pande S V, Parvin Khan R and Venkatasubrahmanyan T A 1963 Microdetermination of lipids and serum total
fatty acids; Anal. Biochem. 6 415-423
Snedcor G W and Cochran W G 1 967 Statistical methods (Calcutta: Oxford and IBH Publishing Co.) pp. 593
Stickle W B and Duerr F G 1970 The effects of starvation on the respiration and major nutrient stores of Thais
lamellosa; Comp. Biochem. Physiol. 33 689-695
*von Brand T 1931 Der Jahreszyklus im Stoffbestand der weinbergchnecke (Helix pomatia); Z. Vergl
Physiol 14 200-264
von Brand T, Noland M and Mann E 1 948 Observations on the respiration of Australorbis glabratus and
some other aquatic snails; Biol Bull. 95 199-213
von Brand T, McMahon P and Noland M 1957 Physiological observations on starvation and desiccation of
the snail Australorbis glabratus; Biol. Bull 113 89-102
Widdows J 1973 Effect of temperature and food on the heart beat, ventilation rate and oxygen uptake of
Mytilus edulis; Mar. Biol. 20 269-276
*Not referred to in the original
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 2, April 1985, pp. 111-116.
© Printed in India.
Influence of distillery effluent on growth and metamorphosis of Rana
malabarica (Bibron)
M A HANIFFA, STEPHEN T DE SOUZA S J, A G MURUGESAN
and BARNABAS XAVIER
Post Graduate Department of Zoology, St. Xavier's College, Palayamkottai 627002, India
MS received 26 September 1984; revised 17 January 1985
Abstract. Increase in effluent concentration reduced the period of limb bud emergence and
tail resorption; it also produced elevated values for length of limbs, tail and body weight of
adult R. malabarica. Analysis of variance (Anova test) confirmed that time (week) has
pronounced effect on morphological parameters (P < 0-05) than treatment (P > 0-05).
Keywords. Distillery effluent; Rana malabarica; growth; metamorphosis.
1. Introduction
To assess the effects of various pesticides and industrial effluents on aquatic life,
toxicologists generally prefer fish and invertebrates and omit amphibians considerably.
A few publications are available on amphibians especially for the effect of pesticides
(Cook 1971; Dial 1976; Greenhouse 1976). Except one or two reports (e.g. Ghate et al
1978) no information is available on the impact of industrial effluents on amphibian
tadpoles. The above authors have reported tail abnormalities, eye defects and odema in
Microhyla ornata exposed to dye factory effluent but omitted the aspects of growth and
metamorphosis in their studies. Since amphibians are the components of food webs in
both terrestrial as well as aquatic communities, Porter and Hakanson (1976) stressed
that preference should be given to amphibians for bioassay studies. The present
investigation is a preliminary report dealing with the effect of distillery effluent* on
growth and metamorphosis of Rana malabarica.
2. Material and methods
The tadpoles of R. malabarica (premetamorphic stage**) were collected from their
natural habitat (Mundanthurai, Tamil Nadu) fed on boiled leaves of Amaranthus
spinosus for 3 days and acclimatized to laboratory conditions. Twenty test individuals
'of equal body length and weight were recruited from the stock and divided into 4 series,
each with 5 individuals. The first group of individuals were reared in dechlorinated tap
water as control, while the remaining groups were exposed to different sublethal
concentrations (0-03, 0-06 and 0-12%) of distillery effluent (Barnabas Xavier 1983).
During the experimental period, fresh concentrations of effluent were prepared in 3 1 of
* Courtesy Trichy Chemicals and Distilleries Ltd., Tiruchirapalli,
** Before the hind limb bud emergence.
112 MAHaniffaetal
water by mixing the required quantity of the effluent with tap water and supplied to the
test individuals daily (Haniffa and Sundaravadhanam 1984). Boiled A. spinosus leaves
were supplied ad libitum to the tadpoles every day. Weekly observations for the change
in length of body, tail, hindlimb and forelimb in cm were made. Every week the tadpoles
were weighed to 0-001 mg after blotting on a cloth towel (Hota and Dash 1981). All
measurements were analysed to standard deviation whereas analysis of variance was
attempted after Zar (1974). Calculations were made for correction factor, total sum of
squares, summation due to week, summation due to treatment and mean square. F
values were separately estimated for time effect (Fj) and treatment effect (F2) on the
length of limbs, tail and body and weight of R. malabarica. F value probability was
taken from the Anova table (table 3, Snedecor and Cochran 1968).
3. Results and discussion
R. malabarica tadpoles reared in tap water took 84 days to complete metamorphosis.
Among the effluent concentrations, individuals exposed to 0-12% took the shortest
period of 50 days to complete metamorphosis (table 1). Body length and tail length
increased during progressive metamorphic stages and after that the body length
remained almost constant whereas tail showed a gradual decrease and was finally
resorbed. Peak values of body length were noticed on 29th, 36th, 36th and 15th day in
R. malabarica reared in tap water, 0-03%, 0-06% and 0-12% effluent respectively.
Among all the test individuals, maximum body length of adults (4*8 cm) was noticed for
the tadpole exposed to 0-03% followed by those reared in 0-06% (4-7 cm), 0-12%
(4-4 cm) and tap water (4-4 cm). During the progressive metamorphic period, the tail
length of tadpoles reared in tap water, 0-03 %, 0-06 % and 0-12% effluent increased from
4-9 to 5-4 cm, 5-2 to 6-2 cm, 5-5 to 5-8 cm and 5-5 to 6-2 cm respectively. Increase in
effluent concentration produced a decrease in the period of tail resorption. Tail
resorption was noticed much earlier (50 days) for tadpoles exposed to 0-12 % followed
by those reared in 0-06% (64 days), 0-03% (71 days) and tap water (85 days). Total
length (body and tail) rapidly increased and reached the peak during the progressive
metamorphic stage and after gradual decrease (retrogressive period) attained a
constant value (table 1).
Hind limb bud emergence was much earlier (1 5th day) for tadpoles exposed to 0- 1 2 %
followed by those reared in 0*06 %, 0-03 % (22nd day) and tap water (29th day). Increase
in effluent concentration produced elevated values for the final length of hind limb, but
the period to attain the maximum length was constant (35 days) at all concentrations
except in tap water. Fore limb bud emergence was also quicker (29th day) for tadpoles
exposed to 0-12 %, whencompared with those reared in 0-06 % (43rd day), 0-03 % (57th
day) and tap water (71st day). The difference in fore limb length was rather perceived
high as a function of effluent concentration taking almost the same duration (28 days) at
the respective concentrations (table 2).
Figure 1 shows the change in body weight of JR. malabarica reared in tap water and
effluent. The control test individuals increased from an initial weight of 7-1 g to 8-5 g on
the 36th day and after that slowly decreased to 6-3 g on the 78th day. The corresponding
changes during the progressive metamorphic period for those exposed to 0-03, 0-06 and
0-12 effluent were from 7-7 to 9g, 6-8 to 8-5 g and 7-1 to 7-6 g on the 29th day
respectively. The final body weight of R. malabarica reared in tap water was much less
Influence of distillery effluent on R. malabarica
113
~H {Ni ~f*l r-i Hhl t*- ~H
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114 MAHaniffaetal
Table 2. Influence of distillery effluent on length of hind limb and fore limb of R.
malabarica.
Hindlimb length (cm)
Forelimb length (cm)
Day Control 0-03% 0-06% 0-12% Control 0-03% 0-06% 0-12%
1
0
0
0
0
0
0
0
0
8
0
0
0
0
0
0
0
0
15
0
0
0
0-5 ±
0
0
0
0
0-06
22
0
H±
0-8 ±
1-2 +
0
0
0
0
0-23
0-20
0-32
29
1-0±
l-8±
l-3±
l-9±
0
0
0
0-2 ±
0-14
0-40
0-26
0-35
0-05
36
1'2±
2-0 ±
2-1 ±
2-5 ±
0
0
0
0-6 ±
0-15
0-06
0-05
0-26
0-10
43
l-9±
2-6 ±
2-4 ±
2-8 ±
0
0
0-1 ±
H±
0-12
0-13
0-25
0-05
0
0-06
50
2-5 ±
2-7 ±
2-7 ±
2-8 ±
0
0
0-6 ±
l-7±
0-06
0-05
0-05
0-06
0-15
0-10
57
2-5 ±
2-7 ±
2-7 ±
0
0-1 ±
H±
0-06
0-06
0-04
0
0-12
64
2-5 ±
2-7 ±
2-7 ±
0
0
0-6 ±
l-7±
0-06
0-06
0
0-06
0-10
71
2-5 ±
2-7 ±
0
0-3 ±
H±
0-06
0-06
0-07
0-06
78
2-5 ±
2-7 ±
0-8 ±
l-7±
0-22
0-32
0-87
0-05
85
2-5 ±
1-8 +
l-7±
0-04
0-17
0-06
Each value represents the average performance of 5 individuals and data reported as ± indicate
the standard deviation.
(6-3 g) when compared with those reared at 0-03% (8-2 g), 0-06% (6-8 g) and 0-12%
(6-8 g; figure 1). At 0-03 % maximum body weight was noticed as two peaks on the 29th
and 50th day whereas the same was noticed as only one peak on the 29th day and 22nd
day for those exposed to 0-06% and 0-12% effluent respectively. These two peaks
correspond to the times of necrosis of old and build up of new tissues in relation to
formation of gut and fore and hind limbs and lungs and resorption of gills, old gut and
tail. The reason for the elevation of the peak from the control in relation to 0-03 % and
its depression in the case of 0-06% and 0-12% is under separate investigation.
According to Hota and Dash (1981) body size in poikilotherms is controlled by
differences in environmental conditions such as food availability and larval density. The
body size and growth rate of Rana larvae are functions of amount of available food
(Wilbur 1 977), density (Brockelman 1 969; Wilbur and Collins 1 973; De Benedicts 1 974)
and temperature (Hota and Dash 198 1). As already cited, most of the reports on growth
and metamorphosis of amphibians deal with influence of food limitation and density.
The few reports which are available on the impact of pesticides (e.g. Cook 1971;
Greenhouse 1976) or industrial effluents on amphibian larvae (e.g. Ghate ct al 1978)
mainly deal with teratogenic and embryological properties and do not reveal any
information on growth and/or metamorphosis. The toxic agents which inhibit or
Influence of distillery effluent on R. malabarica
115
10
"U
o
QQ
6 -
1 Control
o 0.03 7C
A 0-06%
A 0- 1 2 %
15
29
43
71
Day
Figure 1. Influence of distillery effluent on body weight of R. malabarica. Each value
represents the average performance of 5 individuals.
Table 3. Analysis of variance: Influence of time (week) and treatment
(effluent concentration) on length of limbs, tail and body and weight of
R. malabarica
Time effect
Treatment effect
Character
F 1 value Probability F2 value Probability
Adult weight
2-917
P < 0-01
0-434
P > 0-05
Body length
5-857
P < 0-01
10-857
P < 0-01
Tail length
11-729
P < 0-01
0-436
P > 0-05
Fore limb
0-857
F > 0-05
0-357
P > 0-05
Hind limb
2-165
P < 0-05
0-410
P > 0-05
P < 0-05 significant
P > 0-05 not significant.
modify the development of the animal, are likely to be detrimental even if the adults of a
particular species are apparently unaffected.
Limb bud emergence and tail resorption occurred earlier in tadpoles exposed to
0-12% effluent (tables 1 and 2). According to Haniffa and Sundaravadhanam (1983),
Barbus stigma exposed to lower concentrations of distillery effluent showed more food
consumption and growth than those exposed to tap water. The above authors
suggested that the chemical constituents at lower concentrations enhanced the growth
through food consumption. Barnabas (1983) also confirmed this by reporting decrease
in the duration of metamorphosis and an increase in body weight of R. malabarica
exposed to lower concentrations (up to 0-12 %) and vice versa, at higher concentrations
(0-15% and above). Hence it is possible to suggest that the increase in body weight of
116 MAHaniffaetal
R. malabarica at lower concentrations (up to 0-12%) could be due to more food
consumption.
Acknowledgements
This research work was carried out at St. Xavier's College, Palayamkottai and was
supported by a grant awarded to Prof. M A Haniffa, by the CSIR, New Delhi. Thanks are
due to Prof. R K Ramkumar for statistical analysis.
References
Barnabas Xavier 1983 Studies on the effect of distillery effluent on the metamorphosis ofRana malabarica; M.Sc.
Dissertation, Madurai Kamaraj University, Madurai
Brockelman W Y 1969 An analysis of density effects and predation in Bufo americanus tadpoles; Ecology 50
632-644
Cook A S 1971 The effects of DDT, dieldrin and 2, 4-D on amphibian spawn and tadpoles; Environ. Pollut. 3
561-568
De Benedictis P A 1974 Interspecific competition between tadpoles ofRana pipiens and Rana sylvatica: an
experimental field study; Ecol Monogr. 44 129-151
Dial N A 1976 Methylmercury: teratogenic and lethal effects in frog embryos; Teratology 13 327-334
Ghate H V, Dodakundi G B and Leela Mulherkar 1978 Effect of dye factory effluent on the developing
embryos of Microhyla ornata; Indian J. Environ. Health. 20 359-365
Greenhouse G 1976 Evaluation of teratogenic effects of hydrazine, methyl hydrozine and dimethyl hydrazine
on embryos of Xenopus leavis, the south African clawed toad; Teratology 13 167-178
Haniffa M A and Sundaravadhanam S 1983 Effect of distillery effluent on food utilization of freshwater fish
Barbus stigma; Life Sci. Adv. 2 143-146
Haniffa M A and Sundaravadhanam S 1984 Effect of distillery effluent on histopathological changes in
certain tissues of Barbus stigma; J. Environ. Biol. 5 57-60
Hota A K and Dash M C 1981 Growth and metamorphosis ofRana tigrina larvae; effects of food level and
larval density; Oikos 37 349-352
Porter K R and Hakanson D E 1976 Toxicity of acid mine drainage to embryonic and larval boreal toad;
Copeia 2 327-331
Snedecor G W and Cochran W G 1968 Statistical methods (U.S.A.: The Iowa State University Press) pp. 593
Wilbur H M and Collins J P 1973 Ecological aspects of a amphibian metamorphosis; Science 182 1305-1 314
Wilbur H M 1977 Density-dependent aspects of growth and metamorphosis in Bufo americanus; Ecology 58
196-200
Zar J H 1974 Biostatistical analysis (New Jersey: Prentice Hall)
Proc. Indian Acad. Sci. (Anim. Sci.); Vol. 94, No. 2, April 1985, pp. 117-122.
© Printed in India.
Bait preferences of rodents in their natural habitat
NAFIS AHMAD and V R PARSHAD
Department of Zoology, Punjab Agricultural University,- Ludhiana 141004, India
MS received 3 July 1984; revised 9 February 1985
Abstract. Preferences of rodents toward cereal baits have been studied in relation to the
availability of food from their natural habitat in crop fields of groundnut (Arachis hypogaea)
and lentil (Lens culinaris). The experimental area was infested by three rodent species —
Bandicota bengalensis, Tatera Mica and Mus sp. At the podding stage of groundnut crop they
showed a poor response towards plain bait of whole wheat grains, the consumption of which
increased significantly after addition of arachis oil at 1 % concentration. The withdrawal of oil
from the bait had no significant effect on its daily consumption by the rodents. In paired bait
tests in podding groundnut crop, the addition of oil significantly increased the bait
consumption of wheat and millet grains. The differences between daily consumption of millet
grains became more significant when the bait station pairs were shifted to growing lentil crop
which reflect the effect of environment on the feeding responses of rodents. Laboratory tests
with B. bengalensis and T. indica trapped from the experimental fields confirmed the results of
field studies that addition of oil in the cereal bait enhance bait consumption.
Keywords. Arachis oil; bait; crop fields; groundnut; lentil; millet; rodents; wheat.
1. Introduction
Previous laboratory studies have revealed that quality of the oil in the cereal baits
significantly affect the bait preferences of rodents (Barnett 1966; Durairaj and Rao
1975; Kamal and Khan 1977; Kumari and Khan 1978; Ramana and Sood 1982). But no
information is available about the effects of oil on the bait consumption by wild rodents
in their natural environments in relation to the availability of food from the crops.
Therefore, the same have been studied in groundnut and lentil crop fields. Such
information would be useful in improving poison baiting programmes by attracting
larger number of rats to the baits and increasing poison bait consumption.
2. Materials and methods
2. 1 Experimental fields
The field experiments were conducted in the following fields of Punjab Agricultural
University, Ludhiana (30°56'N, 75°52'E).
2. la Field A (figure la): Groundnut (Arachis hypogaea) crop field (podding stage)
including a 3 m broad strip of non-cropped land full of weeds was selected. It was
surrounded by groundnut crop fields on the North and East, a brick-walled water
channel on the west and a field road on the south. A heterogeneous and almost stable
population was observed in the experimental fields by weekly live burrow counts (see
117
118
Nafis Ahmad and V R Parshad
GROUNDNUT CROP
. x
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ROAD
(a)
ROAD
NON CROPPED AREA
(b)
Figure 1. Diagrammatic plans of the a. groundnut crop field and b. lentil crop field
showing the locations of burrow holes *(•) of rodents and of bait stations ( x ).
figure 1). Trapping of rodents in this area showed the occurrence of three species
namely Bandicota bengalensis, Tatera indica and Mus platythrix.
lib Field B (figure Ib): This field was adopted to study the effects of change of crop
and availability of food material on the bait preferences of rodents. Here, lentil (Lens
culinaris) was sown after harvesting of groundnut crop. This field was almost similar to
field A (figure Ib) and the same three species of rodents occurred here.
2.2 Field trials
In the above fields six trials were carried out one after the other, the details of which are
given below. The number and duration of placement of bait stations during each trial
are given in tables 1 and 2. At each baiting point 50 g bait was placed in wooden boxes
(25 x lu x 11 cm) near the burrow openings (figure 1).
Bait preferences of rodents
119
Table 1 . Mean daily bait consumption (g per bait station) of rodents in single bait tests in the
podding stage of groundnut cropfl.
Number of bait
stations
Trial
No.
Bait
Total
Showing
consumption
(Mean ±SE)
Days
Mean
consumption
(±SE)
1.
2. .
3.
Wheat grains
Wheat grains
and oil (99:1)
Wheat grains
13
13
10
2-2 ±0-66
10-3 ±1-06
9-4 + 0-6
5
7
5
0-48 ±0-1 3*
2-46 ± 0-6*
2-64 ±0-54
"Though no marked change in burrow counts has been observed during the single bait test
but slight changes in rodent population during different trials can not be excluded.
bThe differences between mean daily consumption of wheat grain bait (*) with other baits
are significant (P < 0-05).
Table 2. Mean daily bait consumption (g per bait station) of rodents in paired bait
tests in .maturing groundnut and growing lentil crops.
Number of bait
station pairs0
Showing
Trial Crop
consumption
Mean consumption
No.
Total Mean ±SE
Days (±SE)
Wheat Wheat and Oil
4. Maturing
groundnut
10 9-5 ±0-29
4 1-095 ±0-21 3-46 ±0-1 5"
Millet Millet and Oil
5. Maturing
groundnut
10 7-57 ±1-51
7 2-5 ±0.62 3-39 + 0-88"
6. Growing
lentil
10 7-29 ±1-04
7 3-14 + 0-15 7-05 + 0-20"
°The bait stations were exposed to the same population.
bThe differences between mean consumption within a pair are significant
(P < 0-05).
Trial 1: To determine the initial response of rodents towards the plain cereal bait
without oil, wheat grains were placed at 13 baiting points in field A for 5 days. The bait
consumption per 24 hr was recorded.
Trial 2: To study the effect of oil on bait consumption, whole wheat grains smeared
with 1 % arachis oil were offered to rodents as in 'trial 1'.
Trial 3: After enhancing bait consumption with oil in trial 2, 'trial 1' was repeated to
study the effect of withdrawal of oil on plain bait consumption.
Trial 4: Assuming that baits placed at two adjoining baiting points would be available
to the same population of rodents, the box pairs with plain wheat grains and wheat with
120 Nafis Ahmad and V R Par shad
arachis oil (99: 1) separately were placed in fields as shown in figures 1 a, b. The distance
between the two boxes of the pair was about 15 cm. The position of the bait boxes was
altered after an interval of 24 hr to eliminate any side preference.
Trial 5: To study the effects of change of cereal on the preference behaviour of rodents
towards the oily bait, this trial was carried out as 'trial 4' by replacing wheat with millet
(Pennisetum typhoides) grains.
Trial 6: This trial was carried out in lentil crop (L. culinaris) similar to trials 4 and 5
using millet as the cereal.
2.3 Laboratory experiments
For the laboratory test, specimens of B. bengalensis and T. indica were trapped from the
experimental fields and caged individually in 92 x 30 x 25 cm size cages. After
acclimatization for 1 5 days in the laboratory they were offered in choice, 50 g each of
wheat grains with and without 1 % arachis oil in separate food cups. The side of the
food cup was altered daily to eliminate effects of site preferences of rodents on food
consumption.
For comparing the bait consumption between the trials and between two baits within
the same trial, f-test of the difference between means was applied (Sokal and Rohlf
1973).
3. Results
Comparisons of mean bait consumption between the baits with and without oil are
given in tables 1 and 2 and the daily pattern of the mean cumulative bait consumption is
illustrated in figure 2. The rodents showed a poor response (trial 1, table 1) towards
plain wheat grain bait at the podding stage of the groundnut crop in which signs of
considerable damage of developing pods by rodents were also observed. Addition of
1 % arachis oil increased the feeding of wheat grain bait in more number of bait boxes
and resulted in a significant increase in bait consumption (trial 2). After enhancing bait
consumption with oil its withdrawal had no significant effect on the bait consumption
by rodents during the subsequent five days (cf trials 2 and 3). The damage to groundnut
pods continued to occur during trials 2 and 3, as well as during subsequent trials.
When the bait stations were placed in pairs, one containing cereal grains without oil
and the other with oil, the rodents preferred the oily bait (trials 4 and 6, table 2). The
nature of cereal had no effect on the preference of rodents for oily baits as in both cases
they preferred oily cereals over the respective plain cereals. However, more millet grains
were consumed than wheat indicating its preference by rodents. Preference of rodents
for millet with oil over grains without oil (trial 6, table 2) continued in the lentil crop
which was sown after groundnut harvesting. These results indicate that the crop had no
effect on the choice of rodents for cereals containing the oil. However, the differences
between the mean daily consumption of millet grains with and without oil were more
significant in lentil fields than in groundnut fields indicating the effects of the crop on
the feeding responses of rodents. Though rodent damage occurred in lentil crop but no
sign of nibbling or eating of any part of the plant was observed.
Bait preferences of rodents
121
Figure 2. Comparisons of daily bait consumption of rodents in experimental fields between
the cereal baits without oil (W, wheat; M, millet) and with oil (WO and MO, indicates wheat
and millet grains with 1 % arachis oil) a. cumulative bait consumption in single bait trials
in groundnut crop fields, b. in paired bait trial (trial 4) in groundnut crop field where wheat
grains were used as the bait, c. in paired bait trial (trial 5) in groundnut crop where millet was
used as the bait, and d. in paired bait trial in lentil crop field where millet was used as a cereal
bait.
Table 3. Mean daily bait consumption (per 1 00 g body weight) of rodents in bi-choice test in
laboratory.
Mean consumption ± (SE)
No.
of
No. of
Wheat and Oil
test
Species
individuals
Days
Wheat
(99:1)
1.
B. bengalensis
4
6
3-30 ±0-1 3
4-20 ±0-1 5
2.
T. indica
6
6
3-96 ±0-1 6
5-13±0-18*
The differences between mean consumption within a pair are significant (P < 0-05).
Specimens of two species B. bengalensis and T. indica captured from the experimental
fields showed preference (table 3) for wheat grains containing arachis oil over the grains
without oil in a laboratory experiment; thus confirming the results of the field trials.
4. Discussion
The results of the present field studies have shown that addition of 1 % arachis oil in the
bait significantly increases bait consumption of two cereal baits (wheat and millet) by
122 Nafis Ahmad and V R Parshad
rodents in field situations of groundnut and lentil crops. Similar response of rodents (B.
bengalensis and T. indicd) toward oily grains was observed in laboratory experiments in
the present as well as in previous studies (Kama! and Khan 1977; Ramana and Sood
1982). The possibility cannot be excluded that the increased consumption of cereal
grains containing oil in single bait tests may be due to some population change in the
crop fields but the results of feeding during paired bait tests confirm that arachis oil
enhances bait consumption.
Poor response of rodents towards the wheat bait during initial trial at the podding
stage of the crop may be due to their preference for juicy and sweet pods of groundnut,
as field observations showed high damage of developing pods. Inspite of the availability
of the preferred food material from the groundnut crop, the addition of oil in the dry
grains, both in single and paired bait tests, enhanced the consumption of wheat grains.
This response may also be related to the familiarity of the odour or taste of arachis oil
from the bait with that of groundnut pods from the field. Food flavour familiarity
forms an important factor controlling the food preference behaviour of wild rodents
(Barnett 1975; Shumake 1978). No significant change in bait consumption after
withdrawal of the oil (cf. trials 2 and 3), further indicate that the rodents continue to eat
the familiar food material.
In lentil crop also the rodents preferred the oily bait and the total bait consumption
was significantly higher than in the groundnut crop in the same field. This may be due to
the absence of preferred food material from the crop as no sign of its nibbling and
eating by rodents was noticed.
Acknowledgements
This work was carried out under the All India Coordinated Research Programme on
Rodent Control supported by ICAR. Thanks are due to Prof. S S Guraya, Head of
Zoology Department for providing necessary facilities and Dr G Chopra for
suggestions and reading the manuscript.
References
Barnett S A 1966 The feeding of rodents; Proc, Indian Rodent Symp. Calcutta 1 13-123
Barnett S A 1 975 The rat: a study in behaviour (Chicago: Univ. Chicago Press)
Durairaj G and Rao 1 1975 Feeding behaviour of Bandicota bengalensis; Proc. of All India Rodent Seminar at
Ahmadabad, Sept. 23 to 26. 115-117
Kamal A and Khan J A 1977 Food preferences of the Indian mole rat Bandicota bengalensis (Gray); Proc.
Indian Acad. Sci. (Anim. Sci.) B86 329-336
Kumari P V and Khan J A 1978 Food preferences of the gerbil Tatera indica indica Hardwicke; ZooL J. Linn.
Soc. 64 51-58
Prakash I 1976 Rodent pest management, principles and practices Central Arid Zone Research Institute
Jodhpur Publ. pp. 28
Ramana D V and Sood M L 1982 Food and feeding of the lesser bandicoot rat Bandicota bengalensis; Vestn.
Cesk. Spoec ZooL 46 290-297
Shumake S A 1978 Food preference behaviour in birds and mammals; ACS Symp. series No. 67 Flavour
Chemistry of Animal Foods 21-42
Sokal R R and Rohlf F J 1973 Introduction to Biostatistics (San Francisco: Freeman and Co.) pp. 368
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 2, April 1985, pp. 123-127.
(35) Printed in India.
Biochemical correlates of agonistic behaviour in Bandicota
bengalensis: Hepatic cholesterol and ascorbic acid
SHAKUNTHALA SRIDHARA**, T SOMASEKHAR, ELIZABETH
JOHN*, M V V SUBRAMANYAM and A SUNDARABAI
Departments of *Biochemistry, ** Vertebrate Biology and Zoology, University of
Agricultural Sciences, G.K.V.K. Campus, Bangalore 560065, India
MS received 21 January 1984; revised 19 November 1984
Abstract. Social stress was induced in Bandicota bengalensis by staging 1 0 min encounters in
neutral area between male-male, male-female and residents- intruder male for 9 continuous
days. On the 10th day changes in the weight, ascorbic acid, and cholesterol levels of liver were
estimated. In submissive males, females of male-female encounter and intruders, adrenals
hypertrophied. Liver weight remained unaltered. Ascorbic acid levels increased in both
members of heterosexual pair, residents and intruder but decreased in subordinates compared
to controls. Cholesterol increased in subordinate males and stressed females.
Keywords. Social stress; Bandicota bengalensis; adrenals; liver; ascorbic acid; cholesterol.
1. Introduction
Amongst small mammals increased adrencorticotrophic hormones (ACTH) are im-
plicated in crowding and other social stressful situations leading to hyperadrenalism,
increased adrenalin and noradrenalin content of the adrenal glands and enhanced
secretion of adrenal steroids (Barnett 1969). Increased steroid production by adrenals
involves cholesterol as substrate and the reaction is dependent on ascorbic acid. Both
cholesterol and ascorbic acid are synthesized in the liver and hence the levels of these
can be considered as an index and reflection of social stress in rodents. Archer (1969)
regarded adrenal cholesterol and ascorbic acid levels to reflect crowding stress in mice.
This paper examines the significance of changes in hepatic cholesterol and ascorbic acid
content during social stress in Bandicota bengalensis.
Based on inter-species conflict B. bengalensis is considered as the most aggressive
wild rodent in India (Spillet 1968; Sridhara et al 1980). The species also exhibits high
levels of intraspecies strife (Sridhara and Krishnamoorthy 1983). Adrenal hypertrophy
was shown to accompany such interspecific agonistic interactions (Sridhara et al 1983).
The present paper reports hepatic levels of cholesterol, the precursor for adrenal steroid
synthesis and ascorbic acid levels in B. bengalensis exposed to different kinds of social
stress.
2. Materials and methods
Wild B. bengalensis were collected and maintained according to Sridhara and
Krishnamoorthy (1983). Behavioural stress was induced by three ways (i) by allowing
123
124 Shakunthala Sridhara et al
interactions between two adult males for 15min daily (ii) by staging male-female
encounters for 15min daily (iii) by introducing an intruder male into an all male
resident cage for the same duration daily. The paired interactions were staged in a
behaviour chamber described elsewhere (Sridhara and Krishnamoorthy 1983). It
consisted of a 100 x 50 x 50cm galvanized iron chamber with a glass front, glass sides
of sliding type to facilitate introduction of animals and a wiremesh top. A slot in the
centre of the roof enabled insertion of a thin metal sheet thus dividing the chamber into
two equal portions.
Prior to induction of stress the animals were kept in isolation for 1 5 days. From days
16-24 members of hetero- and iso-sexual pairs were introduced into either side of the
behaviour chamber and allowed to habituate to the test environment for lOmin. Later
the metal partition was removed and the two animals allowed to interact for lOmin
daily over a nine day schedule. Similarly three adult males of differing weights were kept
in a single cage (35 x 35 x 50 cm) for 1 5 days. Such animals were considered residents in
contrast to single males kept in isolation. The latter were assigned intruder status as
they were put into resident cages from 16th to 24th days for lOmin each day for 9
continuous days.
Rats of both sexes, kept in isolation served as controls.
On the 25th day both experimental and control animals were weighed and sacrificed
by decapitation. Adrenals, both left and right and whole liver were excised, cleaned of
blood and weighed. The levels of ascorbic acid, and cholesterol from livers of
differentially stressed rats were determined according to Omaye et al (1979) and Sperry
and Webb (1950) respectively.
Adrenal: body weight and liver: body weight ratios were calculated and compared for
different stress situations to see if hypo- or hypertrophy of the organs ensued
consequent to social stress. A total of six male-male, six male-female and six resident-
intruder interactions were staged, each over a nine day schedule. Statistical significance
were arrived at by student t tests.
3. Results
The behavioural aspects of the three different social situations have been reported
elsewhere (Sridhara et al 1983). Briefly one male dominated the other in male-male
encounters. The latter was considered submissive. In heterosexual encounters males
dominated females although females too exhibited aggressive behaviour. Residents
were agonistic towards intruders.
Adrenals hypertrophied in submissives, females of male-female conflict and
intruders (table 1). Females kept in isolation had heavier adrenals than their male
counterparts. The sexes did not differ in the weight of liver. Similarly social stress did
not affect liver weight in any of the experimental situations. Only submissive males and
females of heterosexual conflict registered significantly lower hepatic ascorbic acid
levels compared to dominants and males of male- female pair respectively (table 1).
Compared to control males the liver ascorbic acid levels decreased only in subordinate
rats (P < 0-01) but rose considerably in males of heterosexual conflict (P < 0-01) and in
both residents (P < 0-05) and intruders (P < 0-02). Similarly stressed females had higher
ascorbic acid levels compared to female controls (P < 0-01),
Amongst controls the two sexes did not differ in cholesterol levels of liver (table 1).
Biochemical aspects of aggressive behaviour in B. bengalensis
125
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126 Shakunthala Sridhara et al
Heterosexual and resident-intruder conflict did not affect cholesterol content of liver
while male-male encounters resulted in enhanced levels in subordinate rats (table 1).
Social stress elevated cholesterol levels in the liver of submissive males (P < 0*05).
Similarly females of heterosexual interaction had higher hepatic cholesterol levels
compared to control females (F< 0-001).
4. Discussion
Hyperactivity of adrenals and accompanying enhanced corticosteroid production has
been correlated with density and territoriality (Christian and Davies 1964; Andrews et
al 1972), disorganization of social behaviour (Benton et al 1978) and agonistic
interactions (Bronson and Eleftheriou 1964; Archer 1969): Subordinate and defeated
mice had heavier adrenals (Brain 1 972; McKinney and Pasley 1 979; Sridhara et al 1 983).
Adrenal hypertrophy was also reported in intruders and females of Bandicota
bengalensis during resident-intruder and male-female confrontations respectively
(Sridhara et al 1983). The elevated corticosteroid levels in such situations are believed to
affect population dynamics in small mammals (Christian et al 1965). Cholesterol is the
source of steroid hormones formed in adrenal cortex (White et al 1 978). Increased social
stress was correlated with depleted adrenal cholesterol during male-male, resident-
intruder and male-female encounters (Sridhara et al 1983). Since liver is the major site
of cholesterol synthesis, hepatic levels of cholesterol possibly reflect changed rates of
corticosteroid synthesis. The present finding of higher hepatic cholesterol in submissive
and female rats compared to dominants and males of heterosexual pairs lends credence
to the theory of adrenal hyperactivity during social stress.
Barnett (1969) postulated that social interactions of mammals induces physiological
changes which resemble those resulting from adverse conditions like cold or infection.
Ascorbic acid content of adrenals, testis, liver and kidney of rats increased significantly
during cold exposure (Dugal and Therien 1955). In several mammals ascorbic acid
administration was shown to increase tolerance to cold acclimation and acclimatization
(Lloyd and Sinclair 1953). Additionally ascorbic acid is essential for the synthesis of
adrenal steroids from cholesterol. Liver is the site of ascorbic acid synthesis. In the
current study the hepatic levels of ascorbic acid were higher in subordinates compared
to dominants and females compared to males during male-male and male-female
conflict respectively. Such enhanced synthesis of ascorbic acid may contribute towards
coping with stress and also reflect increased demand for ascorbic acid due to elevated
corticosteroid synthesis during behavioural stress.
Acknowledgements
The authors acknowledge the encouragement and facilities offered by Drs K Krishna
Murthy, K S Krishna Sastry, R Narayana, T S Thontadarya and G P Channa
Basavanna of University of Agricultural Sciences, Bangalore.
References
Andrews R V, Bellknap R W, Southard J, Lorinez M and Hess S 1972 Physiological, demographical and
pathological changes in wild Norway rat population over an annual cycle; Comp. Biochem. Physiol A41
149-165
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Biochemical aspects of aggressive behaviour in B. bengalensis 127
Archer J 1969 Adrenocortical responses to olfactory social stimuli in male mice; J. Mammal 50 839-841
Barnett S A 1958 Physiological effects of 'social stress' in wild rats-I. The adrenal cortex; J. Psychonom. Res.
31-11
Barnett S A 1969 Some physiological consequences of social stimulation. In Proceedings of the International
Symposium on Psychotropic Drugs in Internal Medicine (Amsterdam: Exerpta Medica) pp. 128-134
Benton D, Goldsmith J F, Gamal-El-Din L, Brain P F and Hucklebridge F H 1978 Adrenal activity in
isolated mice and mice of different social status; Physiol. Behav. 20 459-464
Brain D F 1972 Endocrine and behavioral differences between dominant and subordinate male house mice
housed in pairs; Psychon. Sci. 28 260-262
Bronson F H and Eleftheriou B E 1964 Chronic physiological effects of fighting in mice; Gen. Comp.
Endocrinoi 4 9-14
Christian J J and Davis D E 1964 Endocrines, behavior and population; Science 146 1550-1560
Christian J J, Loyd J A and Davis D E 1965 The role of endocrines in self regulation of mammalian
populations; Recent. Progr. Hormone Res. 21 501-578
Dugal L P and Therien M 1955 (Can. J. Res. E25 1 1 3 1947) as reproduced by Burton A C and Edholm O G in
Man in a cold environment (London: Arnold)
Lloyd B B and Sinclair H M 1953 Biochemistry and physiology of nutrition (New York: Academic Press)
pp. 370
McKinney T D and Pasley J N 1973 Effects of social rank in male mice and social disruption in adult male
house mice; Gen. Comp. Endocrinoi 20 579-582
Omaye S T, Turnball J D and Sauberlich H E 1979 Ascorbic acid analysis: Determination after derivitization
with 2-4 dinitrophenylhydrazine In: Methods in enzymology (eds.) D B McComik and L D Wright (New
York: Academic Press) Vol. 62 pp. 7-8
Sperry W M and Webb M A 1950 A revision of the schoehfheimer sperry method for cholesterol
determination; J. Bioi Chem. 187 97-106
Spillet J J 1968 The Ecology of the lesser bandicoot rat in Calcutta (Bombay: Natural History Society) pp. 140
Sridhara S, Narasimhan U and Krishnamoorthy R V 1980 Aggressive interactions among wild and
domesticated rodents; Proc. Indian Acad. Sci. (Anim. Sci.) 89 351-358
Sridhara S and Krishnamoorthy R V 1982 Rhythmic oscillations in non-aggressive behavior of Bandicota
bengalensis; Proc. Indian Acad. Sci. (Anim. Sci.) 91 317-322
Sridhara S and Krishnamoorthy R V 1983 Aggressive behavior of captive Bandicota bengalensis', Proc. Indian
Acad. Sci. (Plant Sci.) 92 185-191
Sridhara S, Elizabeth John, Somasekhar T and Subramanyam M V V 1983 Brain glutamate dehydrogenase
activity in rats (Bandicota bengalensis) belonging to different social status; Physiol. Behav. (com-
municated)
White A, Handler P, Smith E L and Hill R L 1978 Principles of biochemistry (New Delhi: McGraw Hill
Kogakusha Ltd.)
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 2, April 1985, pp. 129-138.
© Printed in India.
Topography of nervous system in two pouched paramphistomes
NEERJA MISHRA and VEENA TANDON
Department of Zoology, North-Eastern Hill University, Shillong 793014, India
MS received 5 September 1984; revised 8 January 1985
Abstract. Using the indigogenic technique for localizing the non-specific esterases, the
complete nerve arrangement in Fischoederius elongatus and Gastrothylax crumenifer, the
paramphistome parasites in the rumen of cattle, has been visualized. In F. elongatus, of
the three pairs of anterior nerves given oft from the cerebral ganglia, two are ventral and one is
dorsal in disposition. The nerves running posterior from the cerebral ganglia include two pairs
of ventral nerves and one of dorsals. Throughout their course the two ventrals of either side are
joined to each other by several loop-like connectives. In G. crumenifer, the overall nerve
arrangement is somewhat similar to that in F. elongatus; the connectives joining the posterior
ventrals of each side are not loop-like. The course of all the nerves and the innervation to the
various parts of the body is traced in both the species.
Keywords. Nervous system; paramphistome; trematoda; digenea; Fischoederius elongatus;
Gastrothylax crumenifer.
1. Introduction
In recent years, the association of non-specific esterases (NSE) with the nervous system
has been successfully exploited by many workers for the demonstration of the fine
nerve arrangements and other components of the nervous system in several trematode
species (Mishra and Tandon 1984). Based on the localization of the esterases, the
nervous organization has been described in some more digenetic flukes, namely,
Encyclometra colubrimurorum, Echinostoma revolutum, Schistosoma spindalis,
Isoparorchis hypselobagri, Singhiatrema najai and Euparadistomum herpestesi (Kishore
and Shyamasundari 1980; Krishna 1981; Rao et al 1982; Simha and Fernandez 1982;
Fernandez et al 1982; Kishore et al 1982).
Among the paramphistome flukes most of the available accounts on the nervous
system are based on histological observations (Gupta and Dutta 1967; Lee 1971).
However, the pattern of nerve distribution has been visualized in toto in Fischoederius
cobboldi, a pouched paramphistome of bovines (Mishra and Tandon 1984). This study
revealed significant deviations from the studies on the sectioned material.
The present communication also deals with the topography of the nervous system in
entire in two more pouched paramphistome species, namely, Fischoederius elongatus
(Poirier 1883) Stiles et Goldberger, 1910 and Gastrothylax crumenifer (Creplin 1847)
Pokier 1883.
2. Material and methods
Live specimens of F. elongatus and G. crumenifer were collected in 0-9 % saline from the
rumen of cattle slaughtered at the local abattoirs. The procured material was fixed and
processed for localization of NSE as described elsewhere (Mishra and Tandon 1984).
129
130
Neerja Mishra and Veena Tandon
3. Observations
Conforming to the commonly observed pattern in many other trematode species, in F.
elongatus also three pairs of nerves run cephalad and three pairs caudad from the brain
mass which comprises two cerebral ganglia connected with a thick band-like cerebral
commissure and lies immediately posterior to the pharynx and dorsal to the
oesophagus (figures 1, 2).
Of the anterior nerves, there is one pair of dorsal nerves and 2 pairs of ventral nerves.
The dorsal nerves arise from the inner border of the cerebral ganglia and supply the oral
tip and the dorsal surface of the pharynx and tegument. The lateral walls of the pharynx
are supplied by the antero-inner ventral nerves that arise from the antero-median facet
of the ganglia. Theantero-outer ventral nerves, arising from the outer lateral side of the
cerebral ganglia, innervate the whole ventral surface of the pharynx. Thin transverse
connectives, completely encircling the pharynx, join all the anterior nerves with one
another. Very fine branches from these are observed petering out into the circum-
pharyngeal tegument.
The post cephalic longitudinal nerves comprise a pair of postero-dorsals and two
pairs of postero-ventrals (figures 3, 4). The postero-dorsals originate from the inner
facet of the cerebral ganglia and are superfically placed nerves. Fine branches of these
nerves innervate the gut, reproductive system and excretory bladder. The postero-
dorsals of the two sides are joined with each other by thin transverse connectives
throughout their course; the connectives, in turn, are connected to one another by still
thinner, 2-3 longitudinal connectives, thus forming a nerve net under the dorsal surface
of the body.
The postero-inner ventral nerves arise from the postero-ventral border of the
cephalic ganglia and terminate in the region of the acetabulum. The postero-outer
ventral nerves arise from the lateral aspects of the brain. Initially they run lateral to the
inner ventrals for some distance but thereafter are seen changing their course and
running inner to the latter at places. A thin, longitudinal lateral connective joins the
Figures la, b. For caption, see next page.
Nervous system in pouched paramphistomes
131
Figures 1-5. Fischoederius elongatus 1. Camera lucida sketch of the whole mount of the
worm (ventral view); a. anterior half; b. posterior half. For clarity sake only the nerve net of
postero-ventral nerves has been shown. 2. Brain mass and the main anterior and posterior
nerves ( x 1 2-3). 3. Posterior longitudinal nerves in the midbody region ( x 1 2-3). 4. Posterior
longitudinal nerves in the gonadal and pregonadal region ( x 12-3). 5. Terminal course of the
posterior nerves and innervation of the acetabulum.
132 Neerja Mishra and Veena Tandon
o
c
a
r-
*
Nervous system in pouched paramphis tomes 133
antero- and the postero-outer ventral nerves of the same side. Along most of their
length the two postero-ventrals (i.e., outer and inner) of either side are joined with each
other by many, rather conspicuous but thin, laterally placed loop-like connectives,
which appear C- or tilted V-shaped. Fine branches from these loops constitute a nerve
net on the midventral side and innervate the tegument and the wall of the ventral pouch.
The tributaries of the postero-ventrals also innervate the reproductive system, vitellaria
and the opening of the ventral pouch. The postero-dorsals are connected only to the
postero-outer ventrals, and not to the inner ventrals, by means of lateral connectives.
While the postero-inner ventrals are deep seated in the parenchyma, the postero-outer
ventrals are superficially lodged.
All the posterior longitudinal nerves join with one another and form a conspicuous
ring-like nerve just in front of the anterior border of the acetabulum. Gradually
tapering branches given out from this nerve and further secondary fine branches of
these innervate the sucker (figures Ib, 5).
Neuroanatomy of Gastrothylax crumenifer, also having three pairs each of anterior
and posterior nerves, follows the same pattern as that off. elongatus (figures 6-8). The
origin, position and naming of the anterior and posterior nerves are the same as
described for F. elongatus. All the anterior nerves of both the sides are joined with one
another by means of thin connectives encircling the pharynx. However, a conspicuous
circumpharyngeal nerve basket is not formed.
The postero-outer and inner ventral nerves send branches towards the median axis,
which ramify and anastomose with one another, thus constituting a nerve net on the
ventral surface (figure 9). Both these nerves also send branches towards the lateral body
wall.
Each postero-outer ventral nerve bifurcates just a little anterior to the acetabulum:
the inner branch meets its fellow of the other side, making a thin transverse connective
(figure 10) many fine branches from which supply the rim of the sucker; the outer
branch innervates the lateral walls and the floor of the latter. Likewise, the postero-
inner ventral also bifurcates, its inner branch joining the transverse connective and the
outer branch joining its counterpart from the postero-outer ventral.
A dorsal nerve net is also constituted by the transverse and longitudinal nerve
connectives of the postero-dorsals (figure 11). Both the postero-dorsals of either side
join with each other posteriorly and innervate the middle and lower edges of the sucker
(figure 12).
The innervation to the various regions and organs of the body is the same as observed
in F. elongatus.
4. Discussion
The neuroanatomy of F. elongatus and G. crumenifer, as revealed by the indigogenic
technique for localization of non-specific esterases, shows many deviations from the
description given for these species by Lee (1971) and Brandes (1898). In the pre-cerebral
region there are three pairs of nerves in both these species. The additional pairs of
lateral and pharyngeal nerves referred to by Lee and Brandes could not be traced in the
present study. Brandes has reported 2 accessory (dorsal and ventral) anterior nerves in
F. elongatus and 3 accessory anterior nerves in G. crumenifer. However, no such nerves
were observed in the present study. Lee has stated the splitting of one of the anterior
134 Neerja Mishra and Veena Tandon
Figures 8-10. For captions, see page no. 138.
Nervous system in pouched paramphistomes
135
Figures 11-12. For captions, see page no. 138.
136 Neerja Mishra and Veena Tandon
ventral nerves into two, thus mentioning the occurrence of 3 ventral branches on both
sides. In the present study all the anterior nerves are found running singly up to the tip;
ring-like transverse connectives, joining the anterior nerves of both the sides of the
system and encircling the pharynx, are conspicuous. Lee has mentioned only a few
poorly developed anterior ventral commissures. Fukui (1929) also mentioned some
transverse commissures in an unspecified Japanese amphistome but did not state which
nerve they connect. Anterior commissures were not figured by Brandes (1898).
The occurrence of three pairs of posterior longitudinal nerves is the most constant
feature of the system in the species investigated. In our findings there is one pair of
posterior dorsal nerves and two pairs of posterior ventral nerves proceeding singly up
to the acetabulum; the posterior inner and outer ventral nerves correspond to the
ventral and lateral nerves in Lee's description. Lee has reported fusion of the lateral and
ventral nerves in the pre-acetabular region and also of the ventral and dorsal nerves in
the acetabular region. Union between the posterior nerves was also reported by
Brandes in G. crumenifer and F. elongatus.
In both these paramphistomes the presence of an additional pair of collateral nerves
resulting from the splitting of the ventral cord has earlier been described (Brandes 1 898;
Otto 1896; Lee 1971). However, no such nerves were observed in the present study.
Fukui (1929) also did not refer to the existence of collateral ventral nerves. In F.
elongatus all the posterior longitudinal nerves join in the immediate pre-acetabular
region to form a thick circular plexus from which many branches emerge in the
posterior direction, supplying the whole wall and floor of the sucker. The presence of
posterior transverse connection between the nerves of the two sides was also reported
by both Brandes and Lee in F. elongatus. According to Brandes, the ventral and
dorsal nerves in the acetabular region split into several branches, some entering the
acetabulum while others participate in the formation of a complete commissural ring
around the acetabulum. Contrary to Lee's (1971) observations that in F. elongatus the
acetabular nerve is solely derived from the ventral cord, ours are in conformation with
those of Brandes in that the acetabulum is supplied by both the posterior ventrals and
dorsal nerves. In the post-cerebral region, conspicuous transverse connectives connect
the three pairs of nerves with one another and with their counterparts establishing a
direct communication between the two sides of the system. Lee has mentioned only a
few ventral commissures and 1 or 2 poorly developed dorsal commissures. Under the
present investigation, the pair of oesophageal nerves (Lee 1971) or a single unpaired
oesophageal nerve (Brandes 1898) were not present.
The genital atrium is innervated by branches of both the posterior ventral nerves. A
pair of transverse genital nerves reported by Lee could not be traced here. Our findings
agree with those of Brandes who stated that these nerves do not occur in the
paramphistomes he examined.
Otto (1896), in his study of amphistomes, found only three pairs of anterior nerves.
The same author stated that the anterior dorsal and ventral nerves of G. gregarius
(Carmyerius gregarius (Loose 1896)) Stiles et Goldberger 1910 occur immediately below
the surface and are probably connected with pharyngeal nerves at the anterior end. He
further maintained that these nerves send off branches posteriorly to connect with the
posterior cords. Fukui 1929 also reported that the same nerves in some Japanese
unspecified amphistome give off paired branches which turn caudad. In the present
study the anterior nerves are situated slightly deeper in the parenchyma and do not give
off branches to join the posterior nerves. Our observation tally with those of Lee (1971)
for F. elongatus and Parorientodiscus magnus.
Nervous system in pouched paramphis tomes 137
A ventro-lateral connective between the antero- and the posteroouter ventrals in F.
elongatus and G. crumenifer seems to correspond to the accessory nerve of anterior
lateral and posterior lateral nerves as reported in P. magnus (see Lee 1971). The ring-like
anterior commissures described in P. magnus show a similarity to those observed by us
in F. elongatus and G. crumenifer.
A comparison of the nerve pattern among the pouched paramphistomes, F.
elongatus, G. crumenifer and F. cobboldi, brings out some notable variations. The nerve
distribution in the pre-cerebral region of F. elongatus and G. crumenifer is simple in not
possessing a conspicuous circumpharyngeal nerve basket which is a prominent feature
in F. cobboldi (see Mishra and Tandon 1984). The C- or tilted V-shaped (= >)
connectives of the postero-outer and postero-inner ventrals in F. elongatus are
conspicuous by their absence in F. cobboldi and G. crumenifer. In F. elongatus all the
posterior nerves join and form a circular connective anterior to the acetabulum and
thus do not terminate individually in the posterior sucker as in F. cobboldi and G.
crumenifer. An interesting aspect of the present study as compared to other trematodes
is the presence of a dorsal and ventral nerve net, thus providing a direct communication
system between all the posterior nerves.
Simha and Rao (1977) and Fernandez et al (1982) have mentioned about the
ganglionated nature of nerves in Singhiatrerna longifurca and S. najai, respectively. No
ganglionated thickening were observed in the parasites under the present investi-
gations. Nevertheless all the anterior and posterior nerves are well developed.
The present observations as well as the so far available descriptions on the nervous
system in paramphistomes clearly reveal that the system cannot be regarded as a
primitively developed one.
Acknowledgements
The authors are indebted to Prof. J P Thapliyal, for laboratory facilities. The award of a
senior research fellowship to one of the authors (NM) by the CSIR, New Delhi, is
thankfully acknowledged.
References
Brandes G 1898 Die Gattung Gastrothylax', Abh. Naturforsch. Ges. Halle 21 183-225
Creplin F C H 1847 Beschreibung zweir neuen Amphistomen-Arten aus dem Zebu-Ochsen; Arch. F. Nat.
gesch. Jahrg. 13 30-35
Fernandez H, Rashetd U and Simha S S 1 982 Nervous system of Singhiatrerna najai', Indian J. Parasiwl. 6
303-305
Fukui T 1929 Studies on Japanese amphistomatous parasites, with revision of the group; Jap. J. Zool. 1
219-351
Gupta N K and Dutta T 1967 On Fischoederius cobboldi — a pouched amphistome from cattle in India; Res.
Bull (N.S.) Panjab Univ. 18 41-52
Kishore B and Shyamasundari K 1980 Nervous system and neurosecretory cells in a trematode,
Encyclometra colubrimurorum (Digenea: Plagiorchidae); Indian J. Parasitol. 3 (Supplement) 1 30
Kishore B, Shyamasundari K and Hanumantha Rao K 1982 Observation on the nervous system of
Euparadistomum herpestesi, Proc. Natl. Acad. Sci. India B52 II 203-209
Krishna G V R 1981 Esterase localization and nervous system in Echinostoma revolution; Indian J. Parasitol.
5 191-193
Lee S K 1971 A light microscopic study of the nervous system of three species of amphistomes (Trematoda:
Paramphistomidae); Zool. Anz. Leipzig 187 1-25
A 5A
138 Neerja Mishra and Veena Tandon
Loose A 1896 Recherches sur la faune parasitaire de Egypta Premiers Partie. Mem. Inst Egypt., Le Caire 3
1-252
Mishra N and Tandon V 1984 Nervous system in bovine pouched paramphistome, Fischoederius cobboldi
(Trematoda: Digenea); Indian J. Parasitoi 8 33-39
Otto R 1896 Bietrage zur Anatomic und Histologie der Amphistomeen; Dr. Z. Naturw. 37 544-550
Poirier J 1883 Descriptions d' helminthes nouveaux du Paionia frontalis; Bull. Soc. Philom. 7th ser 73-80
Rao S L, Krishna G V R and Simha S S 1982 The nervous system and esterase distribution in Schistosoma
spindalis (Trematoda); Curr. Sci. 51 363-365
Simha S S and Fernandez H 1982 The nerve arrangement in Isoparorchis hypselobagri Billet, 1898; Indian J.
Parasitoi. 6 207-209
Simha S S and Rao L N 1977 Distribution of esterases in Singhiatrema longifurca and Paradistomoides
orientalis; Proc. Indian Acad. Sci. 86 311-321
Stiles G Wand Goldberger J 1910 A study of the anatomy of Watsonius (n.g.) \vatsoni of man, and of 19 allied
species of mammalian trematode worms of superfamily Paramphistomoidea, Bulletin of the Hygienic
laboratory, Public Health and Marine-Hospital Service of the United States 60 1-254
Figures 6-12. Gastrothylax crumenifer. 6-7. Camera lucida sketches of the nervous system
in the whole mount of the worm in ventral and dorsal view, respectively. 8. Brain mass and the
main anterior and posterior nerves ( x 72). 9. Postero-ventral nerves and their nerve net as
seen in the bifurcal region ( x 72). 10. Postero-ventrals bifurcating and forming a transverse
connective (arrow) just anterior to the acetabulum ( x 72). 11. Postero-dorsals and their nerve
net ( x 23). 12. Postero-dorsals joining medially (arrow) in the acetabular region
(x23).
(Abbreviations, ad — antero-dorsal nerve; aiv — antero-inner ventral nerve; aov — antero-outer
ventral nerve; eg — cerebral ganglion; pd — postero-dorsal nerve; piv — postero-inner ventral
nerve; pov — postero-outer ventral nerve.)
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 2, April 1985, pp. 139-143.
© Printed in India.
Effect of hypoxia on tissue metabolism of midgut gland of the scorpion
Heterometrus fulvipes
G RAMESH BABU, G N JYOTHf RMAYI and P VENKATESWARA
RAO
Department of Zoology, Division of Pathobiology, Sri Venkateswara University, Tirupati
517502, India
MS received 8 August 1984; revised 31 January 1985
Abstract. The function of the midgut gland of the scorpion Heterometrus fulvipes has been
investigated in relation to hypoxia. Regional differences in the midgut gland became apparent,
one part being more active metaboiically than the other. It is concluded that the midgut gland
might be serving as the liver, as gluconeogenesis is predominant.
Keywords. Heterometrus fulvipes', midgut gland; hypoxia; tissue metabolism; gluco-
neogenesis.
1. Introduction
The gland which performs the function of a 'liver' in crustaceans (Vonk 1960) is referred
to as the "hepatopancreas" inspite of lack of enough evidence to support its
hepatopancreatic function (Phillips et al 1977). Van Weel (1974) has questioned the
usage of the word hepatopancreas for this gland and called it the midgut gland (MGG).
The phylogenetic origin of MGG in the members of the phylum Arthropoda is
puzzling. The presence of a conspicuous digestive gland in the non-tracheate
Arthropods of classes Crustacea and Arachnida, its replacement by a few to many
digestive caecae (hepatic or midgut caecae) in the tracheate insects and its total absence
in the tracheate Myriapods prompted us to investigate the nature and function of this
MGG in the scorpion Heterometrus fulvipes in relation to respiration.
2. Material and methods
Active H. fulvipes females (4-5-6-5 g) in the non-breeding season were used. The MGG
was divided into three parts — anterior (I), middle (II) and posterior (III) in order to
know the regional differences if any. The oxygen consumption and carbon dioxide
production in all parts was studied using the conventional Warburg apparatus as given
by Umbreit et al (1959) and their RQ values calculated.
Fuels, metabolites and end products (glycogen, phospholipids, reducing sugars,
lactate and pyruvate) were estimated in the anterior and posterior parts of the MGG and
the in vitro effect of hypoxia (30 min) on their levels evaluated at 37°C.
Cyanide method of Park and Johnson (1949) was used to estimate reducing sugars.
Glycogen was estimated after ethanol precipitation by the method of Good et al (1933);
Barker and Summerson (1941) method was adopted for lactate estimation. The method
139
A- $
140
G Ramesh Babu, G N Jyothirmayi and P Venkateswara Rao
of Youngburg and Youngburg (1930) for phospholipid and the method of Lu (1939) as
given by Umbreit et al (1959) for pyruvate were employed. Total carbohydrates were
estimated by the colorimetric method of Carrol el al (1956).
3. Results and discussion
Although there appears to be no difference in oxygen consumption or carbon dioxide
production in the three parts of the MGG, RQ values appear to be markedly different,
(table 1). Therefore RQ has a different value for each of the major food components and
serves to determine what substances are being burned (Oser 1954). MGG I with RQ value
of more than 1 is involved probably in the interconversion of carbohydrates and fats.
Table 1. Regional differences in O2 consumption and CO2 production in
the midgut gland.
Regions of
midgut O2 uptake CO2 liberation
gland (/d/gm wet wt/10") (jul/gm wet wt/10")
RQ value
MGG I
71-51 (12)
±24-89
91-31 (12)
±36-42
1-28
MGG II
68-99 (12)
±20-52
71-57 (12)
±19-71
1-04
MGG III
108-38 (12)
±59-34
99-21 (12)
±34:95
0-915
r = 1-99
t = 0-54
(MGG I vs III)
(MGG I vs III)
r value from table = 2-07 (5%).
Numbers in parentheses denote the number of samples studied.
Table 2. Water and protein contents in the midgut
gland.
Regions of
midgut
Percentage of
Protein content
gland
water
(mg/g wet wt)
MGG I
53-50 (6)
72-26 (6)
±5-45
±47-16
MGG II
56-95 (6)
±4-78
56-13 (6)
±15-80
MGG III
60-49 (6)
±16-60
155-33 (6)
±62-25
r = 0-98
t = 2-61
(MGG I vs III)
(MGG I vs III)
t value from table = 2-23 (5%).
Effect ofhypoxia on MGG in H.fulvipes
141
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142 G Ramesh Babu, G N Jyothirmayi and P Venkateswara Rao
MGG II with RQ value of 1 might be carbohydrate oriented in its metabolism. MGG III
with RQ value of 0-9 might be metabolising non-carbohydrates or mixed fuels. MGG III
with a significantly high protein content (table 2) might be the seat of high synthetic or
secretory activity.
Table 3 shows that MGG I loses glucose under hypoxia whereas MGG III maintains
more or less the same level as under normal conditions. Apparently under hypoxia,
glucose production is continuing in MGG III, but not in MGG I The drop in glycogen
levels in MGG III under normal conditions and the higher than the normal hypoxic
levels of glycogen probably further suggests the presence of glycogen synthetic activity
in MGG III. Phillips et al (1977) are of the opinion that the hepatopancreas of the
crustacean Homarus gammurus may not be the site for gluconeogenesis. In vivo studies
reveal gluconeogenesis taking place from lactate in Cherax destructor (Phillips et al
1977). Munday and Poat (1972) suggested MGG as a possible site for gluconeogenesis.
Giles et al (1975) have also expressed a similar view. Maintenance of more or less the
same glucose level under hypoxic conditions in the scorpion MGG might be due to the
gluconeogenetic role of MGG III and gluconeogenesis is therefore one of its important
functions as in the vertebrate liver.
The absence of any change (table 3) in phospholipid content on incubation and lack
of any difference in MGG I and MGG III might indicate the membranous nature of the
gland and these lipids may get involved only on prolonged starvation of the animal
(Reddy and Selvarajan 1 975; Sinha and Kanungo 1 967). The unexpectedly high levels of
these phospholipids in MGG as compared to the levels of total carbohydrate, glycogen,
glucose or lactate, probably suggest the role of the MGG as a storage organ for these
lipids. Similar situation seems to prevail in other arthropods as well (Ravindranath
Gupta 1971; Satyam 1976; Venkata Reddy 1976). The liver mainly functions as a
storage organ for glycogen and lipids. The digestive gland of malacostracan crustaceans
contains glycogen and fat. The stored glycogen is said to be utilized during the
formation of new chitinous substances (Scheer 1957) and during exercise in insects
(Clements 1955). But storage of glycogen alone cannot justify the term 'liver' for this
organ. However, since it also has a gluconeogenetic role it could be suggested that the
MGG of the scorpion serves as a liver.
The higher levels of glucose, glycogen and phospholipid in MGG I when calculated
per milligramme protein (tables 2 and 3) together with the low protein value and lack of
difference in their levels between MGG I and III when calculated on wet weight basis
may indicate synthesis of these fuels in the protein rich MGG III and probably their
storage in the protein deficient MGG I.
References
Barker S B and Summerson W H 1 941 The calorimetric determination of lactic acid in biological material; J.
Biol Chem. 138 535-554
Carrol N V, Longley R W and Row J H 1956 Glycogen determination in liver and muscle by use of authrone
reagent; J. Biol Chem. 22 583-593
Clements A N 1955 The sources of energy for flight in mosquitoes; J. Exp. Biol. 32 547-554
Giles I G, Poat P C and Munday K A 1975 Regulation of pyruvate kinase from the hepatopancreas of the
crab Carcinus maenus; Biochem. Soc. Trans. 3 400-402
Good C A, Kramer H and Somogyi M 1933 Estimation of glycogen in Manometric techniques (eds)
W W Umbreit, R H Bums and J F Stauffer (Minneapolis: Burgess Publishing Co.)
Effect ofhypoxia on MGG in H. fulvipes 143
Lu G D 1939 Estimation of pyruvic acid in Manometric techniques (eds) W W Umbreit, R H Burris and
J F StaufFer (Minneapolis: Burgess Publishing Co.)
Munday K A and Poat P C 1971 Respiration and energy metabolism in Crustacea in Chemical zoology (eds)
M Florkin and B T Scheer (New York: Academic Press) 6 191-21 1
Oser B L 1954 Hawk's physiological chemistry (Bombay, New Delhi: McGraw-Hill Publishing Co. Ltd.)
Park J T and Johnson M J 1949 Estimation of glucose in Manometric techniques (eds) W W Umbreit,
R H Burris and J F Stauffer (Minneapolis: Burgess Publishing Co.)
Phillips J W, Mekinney R J W, Hird F J R and Macnillan D C 1977 Lactic acid formation in Crustacea and the
liver function of the midgut gland questioned; Comp. Biochem. PhysioL B56 427-433
Ravindranath G M 1971 Carbohydrate metabolism in insects with special reference to the effect of selected
agents during development ofCarcyra Cephalonica ; Ph.D. Thesis, Sri Venkateswara University, Tirupati,
India
Reddy A S and Selvarajan V R 1975 Starvation effects on aminotransferases in the scorpion Heterometrus
fulvipes; PhysioL ZooL 40 150-156
Satyam P 1976 Biochemical studies on myriapods, inorganic and organic composition ofhaemolymph and fat
body of the millipede Spirostreptus asthenes; Ph.D. Thesis, Sri Venkateswara University, Tirupati, India
Scheer B T 1957 The hormonal control metabolism in decapod crustaceans in Recent advances in invertebrate
physiology (ed.) B T Scheer (Eugene: University of Oregon Press) 213-228
Sinha R C and Kanungo MS 1967 Effect of starvation on the scorpion Palamnaeus bengalensis; PhysioL
ZooL 40 386-390
Umbreit W W, Burris R H and Stauffer J F 1959 Manometric techniques, (Minneapolis: Burgess Publishing
Co.)
Vanweel P B 1974 Hepatopancreas?; Comp. Biochem. PhysioL A47 1-9
Venkata Reddy V 1976 Studies on mechanisms underlying acclimation to salinity in the freshwater field crab,
Paratelphusa hydrodromus (Herbst); Ph.D. Thesis, Sri Venkateswara University, Tirupati, India
Vonk H J 1960 Digestion and metabolism in Physiology of Crustacea (ed.) J H Waterman (New York:
Academic Press) 1 291-316
Youngburg G E and Youngburg M V 1930 Phospholipid estimation in Practical clinical biochemistry (ed.)
Harold Varley (India: Arnold-Heinemann Publishers) 317-318
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 2, April 1985, pp. 145-151.
© Printed in India.
Male reproductive system of some digenetic trematodes
K SATYA GOPAL, C VIJAYALAKSHMI and
K HANUMANTHA RAO
Department of Zoology, Andhra University, Waltair 530003, India
MS received 4 September 1984; revised 10 December 1984
Abstract. A histological study of the male reproductive system undertaken in the present
study has been concentrated on trematodes with a cirrus sac. Special attention has been
focussed on the terminal genitalia. Five species of trematodes, Fasciola gigantica,
Acanthocolpus liodorus, Stephanostomoides dorabi, Rhynchocreadium singhia and
Prosorhynchus manteri have been considered for the present study.
Keywords. Trematodes; cirrus sac; prostate glands; cirrus.
1. Introduction
The trematode fauna being parasitic has attracted the attention of scientists ubiqui-
tously. Since time immemorial various aspects of this parasitic group have been studied
in detail. Although the biology of this group has attracted the attention for a few years,
presently physio-pathology, histopathology and life cycle studies are in vogue, but
systematics is the only aspect left unimpeded. One particular aspect that should cause
concern to the biologists and systematists, is the most neglected, apparently most
simple, male genitalia of trematodes. Even though one finds a wide array of orientation
in this system, this has not caught the needed attention. It is with this intention a special
attention has been focussed to bring to light the true significance of this system.
Another point of interest is that only anatomical characteristics were observed from the
whole mounts for this system to a large extent. The present study throws light especially
on the importance of histological study of this system. The details which escape the
scrutiny of anatomical study, turn out in many a case to be of systematic importance.
The orientation of the system can also be better understood histologically. In this study
attention has been focussed on trematodes in which the male terminal genitalia are
separate from the female, enclosed in a cirrus sac.
2. Material and methods
The parasites on collection from different hosts were fixed immediately in different
fixatives to accomplish both anatomical and histological details. Some of these
parasites were fixed in FAA under appropriate cover glass pressure and later stained
with alum caramine to study the anatomical aspects. Other parasites were fixed in Susa
and Bouin's fixatives. After dehydration, through graded series of alcohols, infiltration
and sectioning, Heidenhain's Azan and hematoxylin stains were applied to study the
histology of the male genitalia. All the parasites of the present study are of common
146 K Satya Gopal, C Vijayalakshmi and K Hanumantha Rao
occurrence and atleast 1 5 specimens have been examined on an average for each of the
above species. Measurements are in millimetres unless otherwise mentioned.
3. Observations
3.1 Fasciola gigantica Cobbold, 1890 (figure 1)
This parasite was collected from the bile duct of Bos indicus.
The two testes are highly branched, situated in the middle of the body, post-
acetabular, post-ovarian and tandem in position.
The two vasa efFerentia which come from the testes run anteriorly and join each other
above the acetabulum and below the cirrus sac. The common duct, the vas deferens
enters the cirrus sac leading into a bipartite saccular seminal vesicle. The epithelium of
the seminal vesicle lies on a dense, granular basement membrane, below which a thin
layer of interstitial tissue containing circular and longitudinal muscles is present.
Surprisingly in one specimen of F. gigantica a large number of spermatogonial cells
have entered the seminal vesicle. The seminal vesicle narrows and leads into a tubular,
sinuous duct, the pars prostatica which is covered by a syncytial tegument that lies on a
dense, granular basement membrane, below which a thick layer of interstitial tissue is
present containing a single layer of circular muscles and a thick layer of longitudinal
muscles. The base of the syncytial tegument is folded. It is aspinose. Prostate gland cells
open into the pars prostatica, although they are present around the seminal vesicle and
cirrus to a certain extent. Prostate gland cells are pear-shaped and long, with their
narrow duct like anterior part passing through the interstitial tissue and then into the
syncytium of pars prostatica. Nucleus of the cells is conspicuous and round in shape
containing a single nucleolus. Each "prostate gland cell is 0-014-0-017 broad and the
nucleus measures 0-007. The prostate secretions are of two types, globular and granular.
Pars prostatica leads into a highly muscular, tubular, spiny, eversible, cirrus. The cirrus
is lined by a syncytial tegument, which lies on a thick, granular basement membrane,
which is thrown into a number of papillae, the syncytial tegument following the pattern
of the basement membrane. Below the basement membrane lies a thick layer of
interstitial tissue containing a single layer of circular muscles and a thick layer of
longitudinal muscles. A number of conspicuous, stout spines are present embedded in
the syncytial tegument of the cirrus. On either side of the cirrus sub-tegumental cells are
present. The cirrus opens independently into the genital atrium. The cirrus, pars
prostatica and seminal vesicle are enclosed in a muscular, cirrus sac which is covered by
a syncytial tegument. The cirrus sac measures 0-268-0-272 x 0-1 36-0-1 38 and is pre-
acetabular. Metraterm is a muscular, saccular and multi-lobed structure which leads
independently into the genital atrium. It is covered by a syncytial tegument, and is
devoid of spines and is surrounded by subtegumental cells. The genital atrium is simple
and is covered by a syncytial tegument, the nature of which resembles the body
tegument. The genital pore is median.
Discussion
Threadgold (1975a) gave ultrastructural details of the prostate gland of F. hepatica. In
the present study it has been observed that F. gigantica mostly resembles F. hepatica.
Male reproductive system of digenetic trematodes
147
Cs
Figures 1-5. Cirrus sac. 1. F. gigantica, 2. A. liodorus, 3. S. dorabi, 4. R. singhia, 5. P.
manterL
Abbreviations: C — cirrus; Cs — cirrus spines; CS — cirrus sac; G — genital atrium; P — pars
prostatica; Pg — prostate gland cells; SV — seminal vesicle.
148 K Satya Gopal, C Vijayalakshmi and K Hanumantha Rao
Threadgold (1975b) described the duct surrounded by prostate gland cells as the
ejaculatory duct. In the present study this duct has been referred to as pars prostatica.
He also described that the cirrus and cirrus sac are covered by a modified syncytial
tegument. Such a nature has also been reported by Wittrock (1976) in Quinqueserialis
quinqueserialis. Even in the present study it has been observed that the pars prostatica,
cirrus, and cirrus sac are covered by a modified syncytial tegument. Threadgold (1975b)
observed spines in the syncytium of both cirrus and cirrus sac. But in F. gigantica spines
are observed only in the syncytium of the cirrus. Cirrus sac is aspinose. Subtegumental
cells are observed on either sides of the cirrus. These subtegumental cells are of similar
to the subtegumental cells of the general body surface as was observed by Threadgold
(1975b).
3.2 Acanthocolpus liodorus Luhe, 1906 (figure 2)
This parasite has been collected from the intestine of the marine fish, Chirocentrus
dorab.
Testes are elongated, rod-like, situated in the posterior region of the body, post-
acetabular, pre-ovarian, intercaecal, tandem in arrangement, median in position. The
testes are unequal in size, the anterior testis is smaller (0-359-0-373 x 0-097-0-152) than
the posterior testis (0-414-0-483 x 0-097-0-138).
Seminal vesicle is saccular, thin-walled and tripartite. The tripartite nature is well
pronounced with deep intrusion of the walls into the lumen. Pars prostatica is covered
by a syncytial tegument, the underlying interstitial tissue is thick. The base of the
syncytial tegument is much folded. Prostate gland cells are pear shaped, each gland cell
is 10 jum broad and the nucleus measures 3 /*m. The cirrus is muscular, covered by a
syncytial tegument, the base of the syncytial tegument is folded. The cirrus is spiny and
the spines are rose thorn shaped; pointed end of the spine is very long and the base is
bulbous. Spines are numerous and present on either side at the inner surface of the
cirrus touching the base of the syncytial tegument and directed towards the lumen of
the duct. The cirrus leads into a tubular, conspicuous genital atrium. The cirrus sac is
moderately thick and is aspinose and is prominent, elongated, commences above the
ovary and extends anteriorly. The metraterm is saccular and aspinose. The genital
atrium is tubular, muscular and aspinose. Genital pore is in front of the acetabulum.
The syncytium of genital atrium and metraterm closely lines the underlying muscu-
lature and is not folded.
Discussion
In the present study it has been observed that spines are present only in the cirrus. But
Luhe (1906) described spines in the metraterm and the terminal portion of the
hermaphroditic duct. Further the term hermaphroditic duct seems to be vague, as in the
histological study it has been observed that the cirrus and the metraterm open
separately into a common chamber, which is more appropriately called the genital
atrium. Further the cirrus is spinose indicating its involvement during copulation. If we
consider that the metraterm and cirrus unite to form a common hermaphroditic duct, it
has to be considered that the terminal portion of the hermaphroditic duct as eversible.
Male reproductive system of digenetic trematodes 149
It is not justifiable to think that both cirrus and hermaphroditic duct as eversible.
Therefore it has been considered that the cirrus and the metraterm open into a long,
tubular, weakly muscular, aspinose genital atrium. Whenever a hermaphroditic duct is
present, it has been observed that the male genitalia lie free in the parenchyma. So it can
be concluded that since the cirrus sac is also present in Acanthocolpus, it would seem
more appropriate to consider that both cirrus and metraterm open into a common
genital atrium rather than the hermaphroditic duct.
Yamaguti (1971) considered that if the seminal vesicle is bipartite in Acanthocolpus
liodorus and A. luhei, it might serve to differentiate Acanthocolpus fromTormopsolus, in
which the vesicle is definitely unipartite. In the present study it has been observed that
the seminal vesicle is tripartite in A. liodorus.
3.3 Stephanostomoides dorabi Mamaev and Oshmarin., 1966 (figure 3)
This parasite has been collected from the intestine of Chirocentrus dorab.
Testes, two in number, situated in the posterior region of the body, post-acetabular,
post-oyarian, intercaecal, median, tandem in position, smooth elongated and rod-like.
The anterior testis is smaller (0-814-0-820 x 0-220-0-224) than the posterior testis
(0-952-O-958 x 0-179-0-182).
A conspicuous, saccular and tripartite seminal vesicle is present, occuping the basal
region of the cirrus sac. Pars prostatica is covered by a syncytial tegument, the base of
which is much folded. The interstitial tissue of pars prostatica is thick. Prostate gland
cells are pear shaped. Nucleus is round with a single nucleolus. Each gland cell is 10 ^m
broad and the nucleus measures 3 jum. The cirrus is muscular, sinuous, and spiny. The
base of syncytial tegument covering the cirrus is much folded. There are numerous
spines on either sides at the inner surface of the cirrus, the base of the spine touches the
base of syncytial tegument. The spines are rose thorn shaped. The cirrus leads into a
tubular and broad genital atrium. The cirrus sac is sinuous and is thin-walled and
commences above the ovary and measures 0-207-0-210 x 0-1 52-0*1 56. The metraterm is
long, sinuous, broad and spinose. The spines of metraterm are similar to that of the
cirrus spines. The musculature of the genital atrium is not very thick. The genital pore is
in front of the acetabulum. The syncytium of metraterm and genital atrium is folded.
Discussion
It has been observed that the cirrus and metraterm are spiny and there are no spines in
the genital atrium. This observation agrees with the description of Mamaev and
Oshmarin (1966). But they have described the presence of a long hermaphroditic duct.
In the histologicai study, it has been observed that cirrus and metraterm open
separately into a common genital atrium as in Acanthocolpus.
It has been observed that the seminal vesicle is tripartite and not bipartite as reported
by Mamaev and Oshmarin (1966).
3.4 Rhynchocreadium singhia Per shad, 1965 (figure 4)
This parasite has been collected from the intestine of Macrognathus aculeatus.
150 K Satya Gopal, C Vijayalakshmi and K Hanumantha Rao
Testes, two in number, situated in the posterior region, post-acetabular, post-
ovarian, intercaecal, tandem in position, smooth and oval in shape. Testes are unequal
in size. The two testes are widely separated. The anterior testis measures 0-276-0-324
x 0-180-0-240 and posterior testis 0-252-0-360 x 0-192-0-300.
Seminal vesicle is conspicuous, saccular, tripartite and thin-walled. The partitions of
the seminal vesicle are clearly demarcated. Pars prostatica is covered by a syncytial
tegument the base of which is much folded. The interstitial tissue of pars prostatica is
thick. The lumen of the duct is narrow. Prostate gland cells are pear shaped. Nucleus is
round, with a single nucleolus. Each gland cell is big and broad (20-24 /mi). The nucleus
measures 7-10 /mi. The cirrus is muscular. Both cirrus and cirrus sac are covered by a
syncytial tegument. The cirrus sac is situated on the right side, starting in the lateral field
just above the level of the acetabulum. It runs anteriorly up to a short distance and takes
a bend towards the acetabulum in the middle of the body. The wall of the cirrus sac is
relatively thin. The metraterm is muscular, tubular, leading into a muscular, small
genital atrium. Genital papillae are present inside the genital atrium.
Discussion
Srivastava (1 962) in his generic diagnosis of Rhynchocreadium reported that the seminal
vesicle is bipartite. Pershad (1965) also observed a similar condition, but in the present
histological study it has been observed that the seminal vesicle is tripartite. The cirrus is
found to be eversible together with the terminal portion of the cirrus sac. The prostate
gland cells are very big in size in this trematode.
3.5 Prosorhynchus manteri Srivastava, 1938 (figure 5)
This parasite has been collected from intestinal caeca of Trichuris trichuris.
Testes two, situated in the posterior region. Testes are posterior to the stomach, post-
ovarian, situated in the lateral field on the right side. Tandem in arrangement, smooth
and round in shape. Testes are unequal in size. The anterior testis measures 0-193-0-196
x 0-179-0-183 and the posterior testis is 0-166-0-172 x 0-190-0-193.
Seminal vesicle is prominent and bulb shaped. Below the epithelium of the seminal
vesicle there is a slightly thick layer of interstitial tissue. Pars prostatica is covered by a
syncytial tegument, the base of which is much folded. The interstitial tissue of pars
prostatica is thick. The lumen of pars prostatica is narrow. Prostate gland cells are pear
shaped. Nucleus is round with a single nucleolus. The gland cells are small in size,
arranged in a few rows only and are 7 /mi broad. Nucleus is also small and measures
3 /im. The cirrus and cirrus sac are muscular and covered by a syncytial tegument. The
cirrus sac is prominent, it is vertical in position and directed posteriorly. It commences
very close to the posterior testis. The base of the sac is slightly broader and it is in this
region a bulb shaped seminal vesicle is seen. The cirrus sac measures 0-179-0-183
x 0-089-0-097. The seminal vesicle is 0-069-0-072 x 0-028-0-032.
The interstitial tissue of the cirrus sac is thick. The metraterm is narrow. The genital
atrium is very conspicuous and highly muscular and very complicated in nature.
Muscles are present throughout the genital atrium, there being no conspicuous space
inside the genital atrium.
Male reproductive system of digenetic trematodes 151
Discussion
In the members of the family Bucephalidae the genital atrium is highly muscular and
very complicated. As the Bucephalidae lack powerful adhesive organs, the highly
muscular genital atrium helps in powerful union during copulation and also aids in
adhesion when needed. In this trematode the space inside the cirrus sac for the prostate
gland cells is very narrow, whereas the pars prostatica is with a broad lumen.
Acknowledgement
One of the authors (KSG) is grateful to the CSIR for financial assistance.
References
Luhe M 1 906 Trematode parasites from marine fishes of Ceylon; Ceyl Pearl. Oyster Fish Rep. 5 97-1 08
Mamaev I L and Oshmarin P G 1966 Trematodes of the family Acanthocolpidae Luhe, 1901 in herrings of
the North- Vietnam Bay; Helminthologia 7 155-164
Pershad R S 1965 On a new species of the genus Rhynchocreadium Srivastava, 1962 from the fresh water fish
Rhynchobdella aculeata in Hyderabad, Andhra Pradesh; Zooi Auz 175 415-417
Srivastava C B 1962 A new allocreadid trematode, Rhynchocreadium aculeatum gen. sp. n. from the fresh
water eel Rhynchobdella aculeata (Bloch); Indian J. Helminthol. 14 1-4
Threadgold L T 1975a Electron microscope studies off. hepatica. III. Fine structure of the prostate gland;
Exp. Parasitol. 37 117-124
Threadgold L T 1975b Fasciola hepatica: The ultrastructure of the epithelium of the seminal vesicle, the
ejaculatory duct and the cirrus; Parasitology 71 437-443
Wittrock D D 1976 Structure of the cirrus tegument of Quinqueserialis quinquiserialis (Trematoda:
Notocotylidae); J. Parasitol 62 834-836
Yamaguti S 1971 "Synopsis of digenetic trematodes of vertebrates". Vol. I II. (Tokyo, Japan: Keigaku
Publishing Co.)
Proc. Indian Acad. Sci. (Anim. ScL), Vol. 94, No. 2, April 1985, pp. 153-160.
© Printed in India.
Extraretinal photoreception involved in photoperiodic effects on
gonadal activity in the Indian murrel, Channa (Ophiocephalus)
punctatus (Bloch)
S K GARG and S K JAIN
Department of Zoology, Haryana Agricultural University, Hissar 125004, India
MS received 20 June 1984; revised 19 January 1985
Abstract. The effects of blinding on Channa punctatus exposed to LD 14:10, 12: 12, 9:15,
continuous dark (DD) and continuous light (LL) was studied during the winter quiescent phase
of the annual reproductive cycle. Ovarian weights of blinded fish exposed to LD 14: 10, 12: 12 or
LL were higher than those exposed to LD 9:15 or DD. However, no effects of blinding on
testicular recrudescence under any photoperiodic regime were observed. Studies suggest that
in addition to the eyes, other extraocular photoreceptors are also involved for gathering
information on day length.
, Keywords. Channa punctatus; photoreception; extraretinal; extrapineal; blinding; gonadal
response; photoperiod.
1. Introduction
Seasonally changing daylength and temperature have been shown to regulate the
reproductive cycles of many a teleost species (Htun-Han 1977; Sundararaj 1981; Vivien-
Roels 1981). However, at present it is not known whether the effects of light on
reproductive cycles are mediated via retinal pathways and/or by extra-retinal
photoreceptors.
The effects of retinal and extraretinal photoreception in photoperiodic effects on
reproduction has been studied extensively in birds and reptiles (see Menaker and
Underwood 1976; Underwood 1979). However, very little information is available on
their role in the regulation of reproduction in teleosts (Urasaki 1973, 1976; Dalahunty
et al 1979; Hontela and Peter 1980).
Channa punctatus is a seasonal breeder. Gonadal recrudescence in this species is
greatly influenced by the changing photoperiod and temperature (Garg and Jain 1985),
but the role of eyes in gonadal response to photoperiod has not been studied. Therefore,
present studies were undertaken to investigate the role of eyes in gonadal response to
daylength in fish exposed to different photoperiodic regimes during the winter
quiescent phase of the annual reproductive cycle.
2. Material and methods
Specimens of C. punctatus were obtained from the fish dealers of Hissar (Lat 29° 10'N;
Long. 75° 46'E) and were maintained in the laboratory under constant temperature of
25 ± 1° C and a lighting schedule at 12 hr oflight (0800-2000 hr) alternating with 12 hr
of darkness (2000-0800 hr). Fish were acclimated in the above mentioned conditions
153
154
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for a minimum of seven days prior to the initiation of experimental treatments. Fish
were fed on alternate days with fresh meat and liver of lamb and the water in the aquaria
was changed daily.
After acclimation the fish were divided into two groups, one group was blinded and
the other served as control. A blinded and an intact (control) group each of fish was
assigned to each of the photoperiodic treatments at 25°C (LD 14: 10, 12: 12, 9: 15, DDand
LL). Specially made light proof glass aquaria (60 x 30 x 30 cm) were used to keep the
fish. For blinding, the fish were anaesthetized in an aqueous solution (1:4000) of
tricaine methane sulphonate (Sandoz). The complete eye ball was removed with a pair of
iris scissors. To prevent bleeding a small bud of cotton was then inserted into each
empty orbit. Fortified procaine penicillin was occasionally added to the aquaria water
(35000 units/1) as a prophylactic against skin infection. The duration of photoperiod in
each aquarium or chamber was regulated by time switches and the light was provided
by 20 W Philips fluorescent cool daylight tubes. Feeding offish and changing of water
was always done during the day, except the fish maintained in DD, where it was done in
dark using a dim red light.
Fish sacrificed at the beginning of the experiment served as the initial control.
Thereafter, fish subjected to various photoperiod-temperature regimes were sacrificed
at the end of 30 and 45 days (tables 1 and 2). The gonads were removed and weighed to
Table 2. Effects of blinding on testicular recrudescence in C. punctatus
exposed to LD 14:10, 9: 15, 12: 12, LL and DD during the post-spawning
and preparatory periods (1981-82).
Days of
exposure
Photoperiod
GSI*
Intact
Blind
Initial control
40-1 ±6-1
(6)
30\
46-2+10-3
40 ±6
\
(5)
(9)
^ LD 14:10
^
25°C
45-^
76-5 + 6
66+4
(8)
(5)
30\^
85 ± 10-2
69 ±12
^^-^
^ LD9:15
(4)
(6)
45-^""""^
25°C
59 ±7
54+12
(6)
(5)
3(K
42±8
62 ±10
^\
(5)
(5)
^ LD 12:12
^
25°C
45""^
70 ± 10-4
67 ±7
(6)
(7)
30
LL 25°C
84-2 + 9-6
71 ±10
(5)
(6)
30
DD 25CC
63±4
73 ±5
(6)
(7)
*Mean ± SE of mean, Figures in parentheses indicate number offish.
Extraretinal photoreception in C. punctatus 157
the nearest 0-5 mg and fixed in Bouin's fixative for histological studies. For comparison
of data, all gonadal weights were calculated on a lOOg body weight basis (GSI:
gonosomatic index). P values between the experimental and control groups were
calculated by students t test (Snedecor and Cochran 1971).
Three types of primary oocytes were identified from stained ovarian section of
murrel (Garg and Jain 1985) on the basis of nuclear and cytoplasmic characteristics;
stage I (figure 1 A), primary oocytes (diameter 20-130 /zm, mean, 100 ^m) the non-yolky
oocytes present in the ovary during all seasons of the year; stage II (figure IB) primary
oocytes (diameter 140-240 //m, mean: 170 /im) characterized by the presence of a ring
of cortical alveoli, an indication of the onset of vitellogenesis and stage III (figure 1C),
primary oocytes (diameter 360-720 /^m, mean: 510 jum), the fully formed yolky oocytes.
3. Results
At the initiation of the experiments gonads were totally regressed, ovaries had only
stage I primary oocytes, while the testes possessed primary spermatogonia and residual
spermatozoa. Significant differences in ovarian weights or their histology between
blinded and intact fish exposed to LD 14:10 were not observed after 30 or 45 days of
treatment (table 1), however, lower percentage of yolky oocytes (P < 0-001) and higher
number of atretic oocytes were observed in the ovaries of the blinded females under LD
14:10 at the end of 45 days. Under LD 9:15 ovarian weights of blinded group was
significantly lower (P < 0-001) than the intact group after 30 days of treatment, while at
the end of 45 days GSI of blinded fish did not increase appreciably but the ovaries of the
intact fish regressed significantly (P < 0-001). Atretic oocytes were also observed in
both intact and blind fish. No significant differences in the ovarian weights of blinded
and intact fish under LD 12: 12, LL or DD was observed. However, the GSI of blinded fish
under DD was slightly higher compared to the intact control (table 1).
Testicular recrudescence under different photoperiodic regimes though followed
almost similar patterns as that observed in the ovarian recrudescence, statistical
comparisons were not possible (table 2). Also no differences in testicular histology
among different groups were observed.
A review of the results of different treatments indicate that GSI of blinded fish exposed
to LD 14:10, 12:12 or LL was higher than that offish exposed to LD 9:15 or to DD.
4. Discussion
The presence of photoperiodic effects on gonads even after blinding demonstrates the
involvement of extraretinal photoreceptors. The effects of blinding on gonadal
development in C. punctatus are broadly consistent with the findings of Urasaki (1973,
1974), Vodicnik et al (1979) and Borg (1982) that gonadal stimulation can take place in
response to photoperiod even in the absence of eyes. The results obtained on exposure
to short photoperiod however, are in contrast with those obtained by Urasaki (1976) on
Oryzias latipes and by Garg (1981) on Heteropneustes fossilis, where the females
exposed to short photoperiod had higher GSI than that of the intact fish exposed to
similar conditions. It is not clear whether these effects are due to species difference or
due to some other reasons.
158
5 K Garg and S K Jain
Figure 1 . Photomicrograph of transverse sections of the ovary of the Indian murrel, Channa
punctatus (Bloch). (A). Stage I (S^) oocytes, (B). Stage II (S2) oocytes, note the presence of
peripheral ring of cortical alveoli, (C). Stage III (53) yolk-laden oocytes ( x 200).
Extraretinal photoreception in C. punctatus 159
The pineal organ must be given a primary consideration as a site for the extraretinal
photoreception involved in photoperiodic control of reproduction, since the pineal
organ of teleosts contains well-developed photoreceptors (see Oksche and Hartwig
1979 for references) and the removal of this organ affects reproduction in several species
of teleosts (see Matty 1978; de Vlaming and Olcese 1981). However, the effects of
pinealectomy do not necessarily prove that it is the pineal photoreception which is
involved in gathering information on daylength, as the photic information from the
eyes might reach the pineal organ (Hafeez et al 1978).
Saxena (1980) and Delahunty et al (1979) reported that it is primarily the retinal
pathways which mediate the effects of increasing daylength on ovarian development in
goldfish. In H.fossilis, the rate of ovarian recrudescence has been reported to be more
rapid in fish with intact eyes, however, blinding did not prevent gonadal maturation and
this was attributed to the fact that light penetrates the skin and skull and affects the
hypothalamic centres (Sehgal and Sundararaj 1970), suggesting that eyes are not
essential for gonadal development in this fish. In the present studies a higher GSI under
LD 14:10 than that of the intact or blinded fish exposed to LD 9:15, indicate that the
retinal pathways are responsible for gathering information on daylength, but the other
photoreceptors like the pineal or the extrapineal photoreceptors located in the brain
may also be responsible for modulating the seasonal gonadal recrudescence through
the photoperiodic information. The studies of Vodicnik et al (1979) on Carrasius
auratus had suggested that both retinal pathways and the pineal organ are involved in
the gonadal response to the increasing daylength of spring. Hontela and Peter (1980)
have hypothesized that both the pineal and eyes interdependently stimulate ovarian
recrudescence in goldfish under long photoperiod.
The possible involvement of extraretinal and extrapineal photoreception in teleost
reproduction has not been clearly demonstrated, although the presence of such
photoreception in some teleosts has been shown to be present (Urasaki 1976; van Veen
et al 1976), which respond to light stimulus directly and may be involved in gonadal
response to photoperiod in certain teleosts. Lower gonadal weights in blinded C.
punctatus in the present studies perhaps is an indication of the importance of eyes in the
gonadal response to photoperiod and differential response under different photo-
periodic regimes reveal that photoreceptors other than eyes are also involved. Although
these results indicate the involvement of extraretinal photoreception in the photo-
periodic effects on gonads, however, further studies on pinealectomized C. punctatus
are needed to confirm these findings.
References
*.
Borg B 1 982 Extraretinal photoreception involved in photoperiodic effects on reproduction in male three-
spined sticklebacks, Gasterosteus aculeatus; Gen. Comp. Endocrinol. 47 84-87
Delahunty G, Scherck C B, Spencer J, Olcese J, Vodicnik M J and de Vlaming V L 1979 The effects of light
reception on circulating estrogen levels in female goldfish, Carassius auratus: Importance of retinal
pathways versus the pineal; Gen. Comp. Endocrinol. 38 148-152
de Vlaming V L and Olcese J 1981 The pineal and reproduction in fish, amphibians and reptiles. In The pineal
gland: Reproductive effects (ed.) R J Reiter (West palm beach: CRC Press) Chap. 1
Garg S K 1981 Experimental studies on the role of pineal organ in the regulation of reproductive cycles in the
catfish, Heteropneustesfossilis (Bloch) Ph.D. thesis, Delhi University, Delhi, pp. 139
Garg S K and Jain S K 1985 Effects of photoperiod and temperature on ovarian activity in the Indian murrel,
Channa (Ophiocephalus) punctatus (Bloch); Can. J. ZooL 63
160 S K Garg and S K Jain
Hafeez M A, Wagner H H and Quay W B 1978 Mediation of light induced changes in pineal receptor and
supporting cell nuclei and nucleoli in steelhead trout, Salmo gairdneri; Biochem. Photobiol. 28 213-218
Hontela A and Peter R E 1980 Effects of pinealectomy, blinding, and sexual condition on serum
gonadotropin levels in the gold-fish; Gen. Comp. Endocrinol. 40 168-179
Htun-Han M 1911 The effects of photoperiod on reproduction in fishes — An annotated bibliography. Ministry
of Agriculture Fisheries and Food Directorate of Fisheries Research. No. 6. pp. 1-30
Matty A J 1978 Pineal and some pituitary hormone rhythms in fish in Rhythmic activity of fishes (ed.) J E
Thorpe (London, New York, San Francisco: Academic Press) pp. 21-30
Menaker M and Underwood H 1976 Extraretinal photoreception in birds; Photochem. Photobiol. 23 299-306
Oksche A and Hartwig H G 1979 Pineal sense organs — components of photoneuroendocrine systems; Prog.
Brain Res. 52 113-130
Saxena P K 1980 Role of eyes as photoperiodic receptors in the catfish, Mystus tengara (Ham); Indian J. Exp.
BioL 18 903-905
Sehgal A and Sundararaj B I 1970 Effects of blinding and/or total darkness on ovarian recrudescence or
regression during the appropriate periods of the reproductive cycle of the catfish, Heteropneustesfossilis
(Bloch); J. Interdiscip. Cycle. Res. I 147-159
Snedecor G W and Cochran W G 1971 Statistical methods (Ames, Iowa: Iowa State University Press)
Sundararaj B 1 1981 Reproductive physiology of teleost fishes A review of present knowledge and needs for
future research ADCP/REP/81/16 pp. 82. FAO Rome.
Underwood H 1979 Extraretinal photoreception. In The behavioural significance of colour (ed.) E H Burtt
(New York: Garland Press) pp. 127-178
Urasaki H 1973 Effect of pinealectomy and photoperiod on ovoposition and gonadal development in the fish,
Oryzias latipes; J. Exp. Zool. 185 241-246
Urasaki H 1974 The function of the pineal gland in the reproduction of the medaka, Oryzias latipes; Bull Lib.
Arts Sci. Course Sch. Med. Nihon. Univ. 2 11-26
Urasaki H 1976 The role of pineal and eyes in the photoperiodic effect on the gonad of the medaka, Oryzias
latipes; Chronobiologia 3 228-234
Van Veen T H, Hartwig H G and Miiller K. 1976 Light dependent motor activity and photonegative
behaviour in the eel (Anguilla anguilla L); J. Comp. Physiol. 11 209-219
Vivien-Roels B 1984 Pineal control of reproduction in non-mammalian vertebrates. In The pineal organ
Photobiol-Biochron-Endocrinol, (eds) A Oksche and P Pevet (Elsevier: North-Holland Biomedical
Press) pp. 315-334
Vodicnik M J, Olcese J, Delahunty G and de Vlaming V L 1979 The effects of blinding, pinealectomy, and
exposure to constant dark conditions on gonadal activity in the female goldfish, Carassius auratus;
Environ. BioL Fish. 4 173-177
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 2, April 1985, pp. 161-167.
© Printed in India.
Influence of food plants on the food utilization and chemical
composition of Henosepilachna septima (Coleoptera: coccinellidae)
G GANGA, J SULOCHANA CHETTY, R SENTHAMIL SELVI and
T MANOHARAN
Zoological Research Laboratory, Thiagarajar College, Madurai 625 009, India
MS received 24 July 1984; revised 4 February 1985
Abstract. Influence of three food plants, viz Memordica charantia, Luffa acutangula, and
Trichosanthus anguina on the food utilization and chemical composition of Henosepilachna
septima has been studied. The rate of conversion and conversion efficiencies were higher in
T. anguina fed beetles. The rate of conversion was positively correlated with the protein
content of the food plants. The organic constituents of H. septima specially protein and lipid
increased when fed on the different food plants. Greater increase of all the organic constituents
occurred when the insect was fed on the nutrient rich T. anguina than when fed on other food
plants.
Keywords. Food plants; food utilization; chemical composition; Henosepilachna septima.
I. Introduction
Studies on the consumption, digestion and utilization of food plants by insect pests are
important both from fundamental and applied points of view. They provide
information on the quantitative loss brought about by the pests. Several workers have
made such quantitative study of food consumption and assimilation by insects
(SooHoo and Fraenkel 1966; Waldbauer 1968; Latheef and Harcourt 1972; Bailey and
Mukerji 1976; Muthukrishnan and Rajeeya 1979; Ganga and Meenakshi Nagappan
1983). These studies reveal that the quantity-of food consumed is very much influenced
by the type of food. The different feeding activities of insects on different food plants
are influenced by the chemical composition of the diets. Direct relationship between the
protein, carbohydrate, lipid and water content of the food plants and the growth and
conversion efficiencies have been noted in many herbivorous insects (Keller et al 1963;
Dhandapani and Balasubramanian 1980; Manoharan et al 1982) and also the chemical
composition of the insect tissues are modified according to the composition of the diet
(Subbiah et al 1981). Such studies seem to be less in coleoptera and the present work is
designed to relate the influence of food plants on the feeding budget and biochemical
composition of a coleopteran, Henosepilachna septima.
2. Material and methods
The lady bird beetle, Henosepilachna septima were collected from the infected crops of
bitter gourd from the local gardens of Madurai, and maintained on the leaves of bitter
gourd Memordica charantia at 30 ± 1°C and 70 RH. Freshly emerged adults were used
for feeding experiments. Since the individual beetles were small and consumed little
161
162 G Ganga el al
food, a group of 10 insects (5 males + 5 females) was used for the feeding experiment
(Ganga and Meenakshi Nagappan 1983). The experimental groups were reared in
perforated plastic containers (10x8x6 cm). The food plants used in this feeding study
were M. charantia (bitter gourd), Luffa acutangula (ridge gourd) and Trichosanthus
anguina (bottle gourd). The experimental groups were fed ad libitum on weighed
quantities of their respective food for 10 days, and triplicate groups for each food were
maintained. Unfed leaves and faeces were collected daily and dried at 60°C to find out
the percentage of water content. The scheme of feeding budget followed is the slightly
modified IBP formula (Petrusewicz and Macfadyen 1970) represented as C = P-fJR
-f (F + 17), where C is the total food consumed, P the growth (conversion), R the energy
spent on metabolism and F + U the energy loss via faeces including nitrogenous
excretory products; it has been described in detail elsewhere (Muthukrishnan et al
1978). The total protein, carbohydrate and lipid contents of the food plants, freshly
emerged adults and adults after being fed on different food plants for 10 days were
estimated following the methods of Lowry et al (1951), Seifter et al (1950) and Bragdon
(1951) respectively.
3. Results and discussion
3-1 Composition of the leaves
Percentage composition of protein, carbohydrate, lipid and water content of leaves of
the food plants are presented in table 1. The results of the biochemical analysis of the
leaves of food plants reveal that organic constituents and water content are higher in
T. anguina suggesting that the nutritive value of this plant is more than that of the other
food plants used.
3-2 Feeding budget ofH. septima
The feeding budget of H . septima on three food plants is given in table 2.
3.2a Consumption: The total amount of food consumed by a group of 10 beetles for
10 days on three food plants used showed significant variations. The total consumption
amounted to 2210-33, 1043-33 and 674-00 mg when fed on L. acutangula.T. anguina and
M. charantia respectively. The rate of food intake of H. septima significantly increased
from 153-05 mg/g live wt/day on M. charantia to 286-51 mg/g live wt/day on T. anguina
and 532-27 mg/g live wt/day on L. acutangula. Hence the potential damage that this
insect can cause is maximum on L. acutangula and minimum on M. charantia.
Table 1 . Composition of the food plants (in % of dry weight except water)
(X + so).
Food plants Protein Sugar Lipid Water
L. acutangula 15-76 ± 0-38 0-70 ± 0-00 7-73 ± 0-05 75-2 ± 0-37
T. anguina 17-75 + 0-36 1-11+0-02 10-98 ±0-11 81-1 ±0-42
M. charantia 2-90 + 0-00 0-82 ± 0-01 7-56 ± 0-00 78-4 ± 0-26
Influence of food plants on H. septima
163
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164 G Ganga et al
Differences in food consumption may result from a variety of factors. Feeding is
governed by passiveness of food, water content and other physico-chemical properties
of food material (Bhat and Bhattacharya 1978). Continuation of a feeding response in a
feeding behaviour scheme which inevitably, would lead to a greater amount of food
intake by an insect is governed by certain chemical feeding stimulants in its diet (Beck
1965). This possibility of such chemical feeding stimulant in L. acutangula is ruled out
by studies on food orientation behaviour by H. septima (Mary Saroja 1982). The higher
feeding rate on L. acutangula may be due to some nutritional deficiency due to which,
H. septima have to consume more food to fulfil the nutritional requirements or that the
food may be nutritionally unbalanced which increased the feeding rate (Babu et al
1979).
3-2b Assimilation: The total amount of food assimilation was also higher in
L. acutangula (2180*33 mg) than that of T. anguina (1007 mg) and M. charantia
(607-67 mg) in ten days. Of these three plants, L. acutangula has a lower water content
(75-2 %). Maximum assimilation of food having low water content was also reported by
Waldbauer (1968). Total food assimilation was directly related to the total amount of
the food consumed. In Periplaneta americana and Pieris brassicae also assimilation is
found to be proportional to the amount of food consumed (Muthukrishnan and
Rajeeya 1979; Yadava et al 1979). The relationship between the approximate
digestibility and the palatability of food reported by Mathavan and Baskaran (1975)
probably accounts for this direct relationship between consumption and assimilation.
The rate of assimilation of H. septima feeding on L. acutangula, T. anguina and
M. charantia were 525-08, 276-56 and 138-06 mg/g live wt/day respectively. Any two
mean values obtained for total assimilation or for assimilation rate of H. septima on the
three different food plants were significantly different from each other at 1 %
probability level. Assimilation efficiency represents the ability of an insect to digest the
food. The insect fed on L. acutangula has a high assimilation efficiency (98-78 %) than
that fed on T. anguina (96-52 %) or on M. charantia (90-18 %). The lowest assimilation
efficiency of H. septima when fed on M. charantia appears to be related to the lesser
nutrient content present in this food, as nutrient imbalance has been implied to be a
factor inversely affecting digestive efficiency (Waldbauer 1964). However, statistical
analysis has revealed that no significant correlation exists between the amount of any of
the organic constituents of the food and assimilation by H. septima.
3'2c Conversion: The conversion by H. septima fed on different food plants are in the
following decreasing order. L. acutangula (69-06 mg) > T. anguina (61-73 mg) > M.
charantia (37 mg). Total production seems to be directly related to the amount of food
consumed and assimilated on the different food plants. Such direct relationship
between growth and consumption has been observed in other insects also (Mukerji and
LeRoux 1969; Latheef and Harcourt 1972; Sing et al 1975).
The order of food plants in their ability to promote the rate of food conversion
T. anguina (16-88 mg/g live wt/day) followed by L. acutangula (16-6 mg/g live wt/day)
and M. charantia (8-4 mg/g live wt/day). The rate of production is positively correlated
with the protein content of the leaves which is significant at 1 % level (table 3).
Corresponding relationship between the protein content of diet and growth rate has
already 'been reported in many herbivorous insects (Taylor and Bardner 1968;
Schramm 1972; Onuf 1977; Slansky and Feeny 1977).
Influence of food plants on H. septima 165
Table 3. Correlation (r values) between the organic
constituents of the three different food plants and feeding
parameters of H. septima.
Feeding
parameters
Protein
Lipid
Sugar
Consumption
0-6842
-0-1250
-0-4661
Assimilation
0-6891
-0-1184
-0-4596
Production
0-9956*
0-5620
0-2798
Metabolism
0-6770
-0-1353
-0-4757
* Significant 1 % level.
The report of Waldbauer (1964) that low growth rate may be due to low rate of
feeding, nutritional inadequacy of food or a combination of the two, explains the low
production rate of H . septima on M. charantia.
Gross conversion efficiency (ECI) indicates the efficiency with which the ingested food
is converted to body matter. Net conversion efficiency (ECD) represents the efficiency
with which the digested food is converted into body matter. Both ECI and ECD are
highest in T. anguina (5-9 and 6-1) which are due to high nutritious food (SooHoo and
Fraenkel 1966). But surprisingly both ECI and ECD are lowest in L. acutangula fed
groups (3-05 and 3-2 %) and low ECD is due to metabolization of greater digested
material which are not used for structural purposes (Hoekstra and Beenakkers 1976).
Singhal et al (1976) also suggested that the high consumption and assimilation with
lower amount of ECD indicates a greater respiratory consumption in these insects. In
M. charantia, which is the least consumed food and on which the assimilation efficiency
is also lowest, the conversion efficiencies are very high (5-5 and 6-09 %) and comparable
to those found on T. anguina. This may be explained on the basis of the existence of
compensatory mechanism suggested by Duodu and Biney (1981). Poor consumption
and poor digestion of M. charantia is compensated by high conversion efficiencies.
3.3 Influence of food plants on the biochemical composition of the insects
Organic constituents of newly emerged adult H. septima and H . septima fed on three
different food plants are given in table 4. Feeding of the freshly emerged adult on
different food plants resulted in an increase in the biochemical constituents of the insect
to different degrees. In all the adults, the carbohydrate content was uniformly lower
than the other organic constituents. Ramdev and Rao (1979) suggested that carbo-
hydrates are utilized by the insects either for maintenance or for conversion to body
lipid, rather than being stored. Carbohydrates have been reported to contribute to the
building up of protein in Phormia regina (Tate and Wimer 1974). The level of
carbohydrate increased (11-11%) when the insects were fed T. anguina but there was no
increase of sugar when the insects reared on the other food plants. The increase of
carbohydrate content when fed on T. anguina seems to be influenced by the
carbohydrate content of the food plant. In Heliothis zea also the carbohydrate content
of the diet directly influenced the carbohydrate content of the insect (Nettles et al 1971).
There was an increase of protein and lipid in all the fed insects, but the percentage of
increase differed according to the nutritive value of the food plant.
166 G Ganga et al
Table 4. Organic constituents of adult H. septima (in % of dry weight).
Samples of animal tissue
analysed Protein Carbohydrate Lipid
Newly emerged adult
20-49 ±0-24
0-09
18-77 ±0-14
Adults fed on L. acutangula
for 10 days
24-96 ±0-35
0-09
18-91 ±0-00
Adults fed on T. anguina
for 10 days
32-74 ±0-61
0-10
25-38 ±0-30
Adults fed on M. charantia
for 10 days
22-11 ±0-00
0-09
19-87 ±0-18
The percentage of increase of protein was minimum when fed on M . charantia (1- 1 %)
and maximum when fed on T. anguina (59-79 %). The percentage of increase of protein
appears to be related to the percentage composition of protein in the food plants. The
protein content of the different tissues of P. americana showed an increase when fed on
protein rich diet (Senthamil Selvi 1982). Maximum increase in lipid content is observed
when fed on T. anguina (35-22%) and minimum when fed on L. acutangula (0-74%).
Increase of lipid in H . septima is not directly correlated with the lipid content of the food
is suggested by the fact that though L. acutangula and M. charantia have almost equal
amounts (7-73 % and 7-56 % respectively) of lipid, the insects fed in the former showed
an increase of only 0-74 % while those fed on the latter, had an increase of 5-86 %. The
higher food conversion efficiency of H . septima when fed on M. charantia is probably
responsible for this.
From the results it is evident that all the organic constituents of the insect increased
to a greater degree when fed on T. anguina. In the light of these observations, it is
suggested that T. anguina is the most suitable host plant for H. septima, because of its
nutritional value with high protein and water content which allow the insects to convert
the food materials into body tissues.
Acknowledgements
The authors thank the college authorities for the facilities provided.
References
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STATEMENT ABOUT OWNERSHIP AND OTHER PARTICULARS ABOUT
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(Animal Sciences)
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Dated 1st March 1985 • Signature of Publisher
i
Advances in insect behaviour
Foreword
Behavioural studies form an integral part of the biology of insects and one of
the major aspects involved in the concept of integrated control of insects is
behavioural approach. Neural integration, forming as it does an important
parameter, naturally involves the role of chemosensory and other structures which
are known to adequately respond to a variety of chemical substances, whether
attractants or repellants. It is in this light that diverse approaches are being made to
study various components of insect behaviour with reference to phytophagous or
haematophagous insects, not to mention of predators or parasites. The growing
tendency for an indiscriminate use of several categories of insecticides has so
visibly affected the behaviour of many insects, that a proper understanding of
behavioural studies becomes imperative. The recognition of the significance of
biological rhythms in the behavioural activities of several insects, as well as an
understanding of energetics involved in such activities have opened up new
avenues of research. Social insects also offer many clues to an understanding of the
complexities of the behavioural repertoire, and further studies in this area have
gone a long way in enabling us to understand the true significance of the genetics
and evolution of social behaviour. Papers presented in this volume relate to
behavioural rhythms in insects, bioenergetics and behavioural mechanisms in
insects, hormones in insect behaviour, insects and behaviour-modifying chemi-
cals, insect toxicology and behaviour, analysis of behavioural trends in social
insects, host-switching mechanisms and searching behaviour and behavioural
analysis of feeding and breeding in insects.
Communication signals play a salient role in the social interactions of various
animal groups. Despite the prevalence of diverse signals such as visual, acoustic,
tactile and olfactory among higher animals, olfactory cues have certain specific
advantages over other modes of communication. During the recent past con-
siderable attention has been focussed on chemical signals in animals especially in
economically important insects. Sex pheromones, aphrodisiacs, trail markers,
aggregating and alerting pheromones have been isolated in different insects and
diverse factors regulating sex pheromone behaviour have been discussed.
Hormones may directly control the behaviour of insects, or they may modulate
the nervous system in integrating the behavioural repertoire. In neurally-
modulate^ hormonal control of behaviour, hormones may either switch-on new
neural pati^rns of behavioural activity, or by evoking neurophysiological activity, or
hormones may increase or decrease the threshold of stimuli modifying the
behavioural pattern of the animal. Pheromones are of utmost importance in the
hormone-behaviour interaction in insects. Hormones also affect adult behaviour,
including male sexual behaviour, receptivity as well as oviposition behaviour.
Insect migration and orientation as also related behavioural patterns in insects like
locusts, is controlled by hormones.
170 Foreword
Different types of behavioural, developmental and physiological rhythms have
been identified in many species of insects. Daily cycles of activity in endocrine and
nervous systems of insects have attracted attention because of the probable
importance of these systems, in the control of overt rhythms of physiology and
behaviour. Many developmental events in insects have long been known to occur
in a specific part of the day to manifest a population rhythm. Although some
information is available regarding endocrine rhythms in varied groups of insects,
studies on different physiological and metabolic rhythms in relation to the
hormonal activity pattern in insects remain scanty and available information in this
regard is presented. Biological clocks in relation to the eclosion rhythms of
Drosophila, the problem of on-and-off rhythms and their simulations by appropri-
ate high/low or low/high intensity transfers are also discussed.
Food acquisition behaviour in insects includes such energy requiring activity
components as location, gathering and processing. In most insects oviposition is
preceded by a precise estimation of the presence of minimum food supply for
successful completion of developmental stages and emergence. Discussion is
presented on the energy-requiring behavioural activities associated with court-
ship, mating and oviposition in some insects. Like the phytophagous insects,
haematophagous species are also known to possess chemoreceptors that can
detect host specific factors (odours) and non-specific or group factors. Closely
associated with this behavioural aspect is the capacity of the haematophagous
arthropod to 'taste' the blood meal, where contact chemoreceptors come into play
and these aspects have been discussed in detail. The feeding behaviour and the
patterns of host selection in phytophagous insects are conditioned not only by
their ecological requirements but also the general behaviour of the insects
concerned. Though they are generally polyphagous, they are not indiscriminate
feeders and the food plant range is often correlated with their behaviour which is
conditioned by their sensory perception.
Social insects often behave in ways that appear to lower their genetic fitness
while increasing the fitness of other conspecifics. The most extreme examples of
such altruistic behaviour is provided by the sterile workers of social insects, and the
evolution by natural selection of social or altruistic behaviour has long been
recognised as a problem and an incisive discussion is provided on the evolution of
social behaviour. Lamellicorn beetles in particular, the passalids and scarabaeids
are also known to exhibit social behaviour, sound production by stridulation in
both the larvae and adult passalids being attributed to social behaviour permitting
gregariousness. Interesting aspects relating to the whole sequence of bisexual co-
operation in the nesting behaviour of scarabaeids are also discussed.
As conventional methods of control developed for agricultural pests are often
not suitable for forest pests because of the large area covered by the crops, recent
investigations have shown the potentiality of using behavioural studies to greater
advantage in the management of forest insect pests. Information provffled on the
teak defoliator, particularly on moth immigration, a common occurrence In tropical
forests, other behavioural characteristics such as crowding of caterpillars on tree
trunks and aggregation, appear to be useful in the development of suitable
management strategies.
The papers published in this volume were presented at the National Workshop
sponsored by the Department of Science and Technology on 'Advances in Insect
Foreword 171
Behaviour', held at the Entomology Research Institute, Loyola College, Madras
from December 1 4-1 6, 1 984. I wish to express my deep sense of gratitude to the
Department of Science and Technology for sponsoring this Workshop. It is hoped
that this publication comprising as it does diverse aspects of insect behaviour
would stimulate further interest in this upcoming, inter-disciplinary field, of
considerable significance in applied entomological studies.
Entomology Research Institute, T. N. Ananthakrishnan
Loyola College,
Madras 600034.
Proc. Indian Acad. Sci. (Anim. SdL), Vol. 94, No. 3, June 1985, pp. 173-186.
© Printed in India.
Recent advances in animal behaviour
K M ALEXANDER
Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695581, India
Abstract. Ethology, a fast developing field of animal sciences has considerable relevance in
animal husbandry, agriculture, control of animal populations, pest control, medicine, wildlife
biology, etc. It has made vast strides of progress during the past few decades and some of these
trends are reviewed.
Communication signals play a salient role in sociobiology of animal groups. Animals deploy
visual, acoustic, tactile and olfactory signals during their social interactions. Among these,
olfactory cues have certain specific advantages over the other modes concerned. Recently
considerable attention has been focussed on chemical signals in animals, especially those of
economically important forms such as insects, fishes and mammals.
Regarding insects, sex pheromones, aphrodisiacs, trail markers, aggregating and alerting
pheromones have been isolated in various insectan orders. The factors controlling sex
pheromone behaviour and impact of pheromones on control of insect population have been
elaborated.
Investigations on chemical cues of lower vertebrates indicate that fishes, amphibians and
reptiles deploy them in their social interactions. Pheromones modulate the schooling,
reproductive and alarm response behaviour in fishes.
Among mammals, urine, fecal pellets, saliva and secretions of specialised skin glands
function as sources of olfactory cues. Data on histophysiology, and ultrastructure of
specialised skin glands, biochemistry of their secretions have been collected. Osmetrichia, scent
marking patterns and flehmen responses and their hormonal control have been elucidated. The
neuroendocrinological basis of scent marking has been made explicit.
Relatively only very few of the mammalian pheromones have been isolated. The role of
Primer pheromones in modulation of reproductive processes in some of the rodents and
signalling pheromones in social interactions of some mammals have been elaborated.
Data on olfactory cues in human social interactions indicate the presence of social
pheromones.
Visual signals of some insects and their role in reproductive activities have been investigated.
Social postures in some rodent pests and their behavioural relevance have been studied.
Acoustic signals in insects facilitate congregation, sexual attraction, aggregation and alarm
responses. Further various aspects of vocalisations in birds and mammals have been
investigated. Reproductive investment patterns and sex ratios in insects and parental
investment in birds have been elucidated. Play behaviour and their role in behavioural
development has been investigated. Ethological analysis of drug action in aggressive
behaviour in certain mammals has been made.
Keywords. Ethology; applied zoology; communication signals; olfactory cues; pheromones;
aphrodisiacs; vertebrates; chemical signals; schooling and alarm responses; amphibians;
reptiles; secretions of specialised skin glands; histophysiology; osmetrichia; scent marking;
hormonal control; flehmen response; visual and vocal signals; social postures in mice;
vocalisation in birds and mammals; reproductive investment in insects; ethological analysis of
drug action.
1. Introduction
Ethology, the scientific study of animal behaviour is a relatively recent field of animal
sciences, integrating animal ecology, neurophysiology, endocrinology, sensory physi-
ology, etc., and it has made considerable strides of progress during the past few decades.
The contributions of Lorenz (1971), Tinbergen (1951), Frisch (1967), Thorpe (1979),
1 71
174 KM Alexander
Hinde (1970), Muller-Schwarze (1969) to mention only a few, have widened our
concepts and opened up new frontiers in the ever expanding horizons of ethological
investigations.
Communication signals play a salient role in the sociobiology of various animal
groups. In fact a clear insight into the diverse modes of communication is absolutely
essential for having a better understanding of the biology of the animal group
concerned. Generally higher animals deploy diverse modes of communication such as
visual, acoustic, tactile and olfactory (chemical) during their social interactions. Among
these the olfactory signals exhibit certain specific advantages over others in as much as
that they are effective over longer distances, they can be deployed in darkness, their
fade-out time is longer and the presence of the signalling animal is not necessary at the
site of emanation of the signal. In the last mentioned trait, they are comparable to the
written language of human beings.
Considerable attention had been focussed on the communication systems of animals,
especially economically important forms such as insects, fishes and mammals during
the past few years. Due to the limitation of time attention may be focussed only on some
aspects of chemical communication in some of these animal groups.
Ever since Butler (1967) introduced the term aphrodisiac pheromone, it has been
widely used and it pertains to a substance produced by one or the other sex, usually by a
male and often as a part of the complex pattern of courtship behaviour, preparing the
partner for copulation after being brought together by olfactory sex attractants or
other means. According to Shorey (1973) aphrodisiac should influence that par! of the
nervous system (NS) of a female which controls her mating behaviour, thereby
increasing her chances of accepting a male in copulation.
Many insect species deploy sex pheromones acting as stimulants when two sexes
come together (Jacobson 1972; Shorey 1973). However only a few experimental studies
have been made on this aspect.
2. Honey bee queen substances
One of the most versatile pheromone, the queen bee substance of the honey bee, Apis
mellifera is 9-oxydec-trans-2 enoic acid and is produced by the mandibular glands of the
queen and not the workers. Data suggest that it can function as an attractant for
workers in colony, cohesion, swarming, inhibition of queen cell construction and ovary
development in workers, sex attractant and mating stimulant for drones (Butler 1967;
Gary 1970). Experimental studies of Butler (1967) had revealed that both open sting
chamber and odour of the queen bee substance are necessary for the drones to mount a
queen in flight and the queen substance functions as an aphrodisiac.
Apart from the queen bee substance yet another substance released from the
abdominal tergites may induce mounting and copulation. In fact much more remains to
be investigated regarding the queen bee behaviour.
A hierarchy of behaviour in response to stimulation by female sex pheromones has
been demonstrated in a large number of insects belonging to diverse orders (Shorey
1973). For e.g, in Trichopkusia ni a quantitative increase in concentration of female sex
pheromone alone is sufficient for the initiation of each successive step in response
sequence including the release of male copulatory behaviour (Shorey and Gaston 1970).
Such a pheromone is found in many other insects. However it would be much more
Recent advances in animal behaviour 175
logical to confine the term aphrodisiac to substances released after the sexes have been
brought together.
3. Gustatory aphrodisiacs
Sex pheromones acting through the gustatory sense require that the male and female
make contact and hence may possibly have an aphrodisiac function. In many
Orthopteran species virgin females produce sex pheromone which attracts the male.
The courting males themselves produce a pheromone which stimulates the female to
mount and feed on secretion thus attaining the correct position for copulation. Such
male pheromones have been found in Blatta germanica, Blatta orientalis and
Periplanata americana.
Roth and Dateo (1966) had isolated a pheromone from the males of Nauphoeta
cinerea which elicits typical behaviour of a .female attracted to a courting male's tergum.
This pheromone is a polar neutral lipid of low volatility, named seducin for its role in
releasing sexual behaviour.
Regarding Lepidopterans, main female response to male producing aphrodisiac
pheromone tends to be either inhibition of the female's natural tendency to fly or
cessation of flight.
In Noctuidae it has been shown that volatile secretions from the male scent brushes
of moths are used in courtship {Birch 1970). The noctuid males have scent brushes
either on the 8th abdominal segment of Plusia gamma or a pair of brush organs at the
anterior end of the abdomen e.g. Apamea. The male brushes have compounds, generally
simple terpenoids and aromatics — carboxylic acids (Birch 1972; Grant et al 1972).
Electrophysiological studies of Grant et al (1970, 1972) reveal that male scent brushes
do not elicit antenna! responses which are specific to species or sex.
4. Sex pheromones
The complexities of describing species specific pheromone blends have brought the
challenge of pheromone identification from the chemistry laboratory to the field.
Despite the fact that chemical characterisation should still be continued to unravel the
intriguing sex pheromones used by many other species, especially those in families for
which no identification has yet been made, the final duplicating of any natural
pheromone blend can only be accomplished by analysis of various ratios and release
rates under field conditions. Infact an exact reproduction of the pheromone input
should enable man to attract males very efficiently. On the contrary one should bear in
mind the long term use of fairly exact blends could merely serve as an artificial pressure
enabling the insect to further modify its chemical systems.
Reproductive isolation with one or multiple compound systems have been exempli-
fied by many species changing the functional moieties, (acetate, aldehyde or alcohol) the
double bond position (7-11), configuration (cis or trans) or number (1 or 2 sites of
unsaturation) or carbon chain length (12-14 carbons). Apart from these different
release rates the specific circadian rhythms facilitate reproductive isolation.
176 KM Alexander
5. Aggregating pheromones
According to Shorey (1973) aggregating pheromones cause other members of the same
species to aggregate in a particular area. They occur ubiquitously in Coleoptera, family
Scolytidae (Borden and Stokkink 1971). The pheromone functions as a population
aggregation pheromone.
6. Trail markers
Diverse types of odour trails are deployed by a wide variety of hymenopteran species as
an effective means of coordinating the movements of individuals. Seven glands have
been identified as the source of this pheromone and the chemistry and specificity of
these pheromones have been identified.
Ant trail pheromone, methyl-4-methyl pyrrole-2-carboxylate of myrmycine, Alt a
texana is the only ant trail pheromone which has been identified (Tumlinson et al 1972).
The functional aspects of this pheromone especially its role in transmitting cues about
distance and direction have been identified.
1. Alarm pheromones
Considerable amount of data had been collected on the alarm pheromones of ants. The
mandibular glands of meliponine bees are the sources of all alarm pheromones so far
characterised (Blum et al 1970). Many species of ants and bees signal alarm with a large
variety of ketones which function as chemical releasers.
8. Environmental and physiological control of insect sex pheromone behaviour
It has been shown that certain environmental factors viz temperature, intensity of light,
velocity of wind etc. control sex pheromone communication in insects by diverse means
(Bartell and Shorey 1969). Regarding physiological variables, circadian rhythms, age,
mating, previous exposure to pherbmones, population density, hormones etc, are also
important, -
9. Pheromonal control of insectan population
The pheromones play a vital role in control of insectan population. However the true
potential of pheromones as a part of survey devices and as control agents cannot be
easily realised without input of commercial technology. This necessitates a combi-
nation of established and new techniques along with coordination of efforts of
industrial concerns and research centres concerned. Such an exchange is necessary
before educating the end users.
Along with transfer of information, the commercial pheromones developed should
update data, often requiring alterations in protection standards. If pheromones are to
be used directly or indirectly in insect control programmes a registration protocol has
to be established for permitting such use.
Recent advances in animal behaviour 111
In consumers, pheromones and related substances are used in a variety of problems
facing agriculture and forestry. As a survey and detection device, they have already
become an important tool in the overall management of certain pests such as cabbage
looper (Trichoplusia m), pink boll worm, boll weevil, gypsy moth, spruce bud worm
and bark beetles.
10. Vertebrates
Considerable amount of data have been collected on pheromones in fishes especially
those concerned with attraction and recognition of other sex, the offspring or the
parents, maintenance of schooling behaviour and during anadromous migration.
However the exact nature of most of these olfactory signals have not been established.
In fact the only fish pheromone which has been well established is the alarm
substance. The fright reaction was first discovered by Frisch (1938) in the minnow,
Phoxinus phoxinus. The epidermal cells (club cells) concerned with the production of
alarm substance have been localised (Pfeiffer 1 960). The alarm substance of the minnow
is a pterin.
11. Amphibians
The fright reaction has been described in amphibians too. Kulzer (1954) reported this in
tadpoles ofBufo bufo. The source of these olfactory cues have been traced to the giant
cells in the epidermis (Pfeiffer 1966).
12. Reptilia
It has been shown that chemical signals play a salient role in the sociobiology of lizards
and snakes. Snakes have many advantages for chemosensory research in as much as
they rely heavily on chemical senses and most of their responses are regulated by
chemical cues. However very little is known about their reproductive biology. The role
of chemical cues in orientation in chelonians, alarm reaction in snakes, territorial
marking, predation warning and nocturnal behaviour also could be shown.
13. Mammals
Investigations on chemical communication in mammals have revealed that olfactory
cues are deployed by these forms quite frequently. The major sources of body odour are
the urine, fecal pellets, saliva and the secretions of the specialised skin glands. The
specialised integumentary glands have an ubiquitous distribution among various
mammalian orders with over 16 of them exhibiting these glands which are mainly of
two types, holocrine sebaceous and apocrine sudoriferous.
It has been shown that monotremes like Platypus have femoral glands. Some of these
glands produce poisonous secretions which could even kill a dog. Marsupials have
cloacal (anal) glands, frontal and sternal glands.
178 KM Alexander
Studies conducted at the School of Mammalian Ethology, Department of Zoology,
University of Kerala have revealed the presence of specialised skin glands in a large
number of South Indian mammals such as the Indian Musk shrew, rodent pests such as
the wild house mouse, Indian field mouse, South Indian gerbil, common house rat,
palm squirrel and common Indian mongoose (Balakrishnan and Alexander 1984b).
Diverse aspects of histophysiology and hormonal control of these specialised skin
glands have been elaborated. Despite the fact that most of these glands are
hypotrophied by castration and reactivated by hormone administration some of these
glands such as the tarsal glands exhibit a converse effect.
Despite the reports by Stoddart (1976) regarding the absence of behaviourally
relevant specialised skin glands in Mus sp. our studies on the social interactions of these
wild house mouse and Indian field mouse indicate the presence of specific glands at
eyelid, oral angle, perineal and preputial regions in these forms (Alexander et al 1982).
Further the studies on social postures of these forms indicate specific behavioural
responses concerned with olfactory investigations of specific body regions of olfactory
relevance (Santhi and Alexander 1979). The role of gonadectomy in altering the
olfactory status of various interacting conspecifics had also been elaborated.
14. Scent marking
Scent marking in mammals have been reviewed by Ewer (1968), Johnson (1973),
Thiessen and Rice (1976) and Balakrishnan and Alexander (1984). Generally most of
the mammals disseminate their body odour with the aid of specific behavioural
responses known as scent marking, which could broadly be categorised into two,
passive and active.
The scent marking patterns of many of the above mentioned mammals have been
studied. The hormonal control of scent marking in musk shrew had been elaborated
(Balakrishnan and Alexander 1976; Alexander et al 1984). The role of dominance
hierarchy in scent marking of certain artiodactyles such as the spotted deer and the
black buck had also been elucidated (Pillai and Alexander 1984).
15. Functions
It has been shown that chemical signals, similar to other sensory modalities tend to
influence physiology and behaviour and ultimately regulate spacing between
individuals and populations and thus contribute the adjustment of a species to its
resources and general environment.
It has been shown that these olfactory signals generaljy function in the following
functional contexts: maternal, agonistic, social, recognition, physiological state,
recognition of sex, species, sexual attraction and alarm.
Diverse pathways of dissemination of odour has also been studied: (i) Direct release
into air (ii) Secretions may be left on a substrate (iii) Odoriferous substance smeared on
to various parts of the body (iv) Secretions of the body rubbed on other conspecifics.
Scent marking has both individual and social uses for the donor. Regarding the
former, an animal scent marks a novel area for olfactory reassurance, orientation or self
advertisement. As for the latter, viz social uses, it could signal identity or presence of
Recent advances in animal behaviour 1 79
conspecifics and provide valuable data regarding sex, age, identity, reproductive status
etc. of the donor.
Investigations on the olfactory inhibition of scent marking has been conducted on
musk shrew (Balakrishnan and Alexander 1980). Further the effect of cage surface
odours especially urine and fecal pellets on behavioural responses of some rodents have
also been investigated (Nair and Alexander 1984).
16. infrastructure and osmetrichia
The fine structure of the flank gland of the Indian musk shrew had been reported
(Balakrishnan et al 1984b) recently. Further the specialised scent hair had also been
investigated in certain N. American and Indian mammals. It has been shown that these
special tuft of hair, such as flank gland hair of the musk shrew exhibit specific structural
modifications on its scaly surface for holding the odourous molecules (Balakrishnan
and Alexander 1984a). These osmetrichia are used by the musk shrew as scent brushes
for painting the areas to be marked with the odourous secretions of the flank gland,
thereby facilitating olfactory communication.
17. Biochemical assay of glandular secretions and pheromones
Biochemical investigations on the glandular secretions of various mammals such as
wild rabbit, mongolian gerbil, black tailed deer, musk shrew, common Indian
mongoose etc. have been conducted.
However relatively very few mammalian pheromones have been isolated as yet.
In fact, only in seven species of mammals, specific pheromones have been identified.
These are as follows: domestic boar, Sus scrofa (3 alpha hydroxy-5 alpha androst-16-
ene) which stimulates female's sexual behaviour, rhesus monkey (Macaca mulatto)
vaginal secretion, 4copulin' (5 fatty acids, acetic, propionic, isobutyric, n-butyric,
isovaleric acid), prong horn, (Antilocapra americana) sub auricular gland (isovaleric
acid), Mongolian gerbil, (Meriones unguiculatus) ventral gland (phenyl acetic acid),
golden hamster (Mesocricetus auratus) vaginal secretion (dimethyl disulphide), dog
(Canis familiaris) vaginal secretion (paramethyl hydroxybenzoate), black tailed deer
(Odocoileus hemionus columbianuss) (urine in tarsal scent) (ds-4-hydroxy dodec-6-enoic
acid lactone).
18. Primer pheromones
Very interesting data have been collected on the primer pheromones of laboratory
rodents and their role in modulation of reproductive behaviour and processes. The
Bruce effect (pregnancy block), Whitten effect (oestrus suppression in all female
groups) and Vanderbergh effect (induces precocial puberty) are excellent examples of
the role of these priming cues. Vandenbergh (1983) has also demonstrated acceleration
of puberty in heifers by exposure to bull urine.
180 KM Alexander
19. Signalling pheromones
These are frequently used in evocation of many specific hormones modulated and
reproductively related social behaviour such as those occurring in sexual aggression
and maternal behaviour. Mouse signalling pheromones facilitate identification of
individual, species, age, sex, sexual state, presence of fear (alarm pheromone carried in
urine) etc.
20. Neural mechanisms of scent marking
It has been shown that in mongolian gerbil, Meriones unguiculatus, testosterone
implants in the preoptic area, reinstate ventral gland scent marking in castrated males,
whereas cholesterol implanted at the same site has no effect. Testosterone implants in
the cortex, caudate nucleus, amygdala, hippocampus, reticular formation and septum
have also no effect in scent marking. The anterior area of the median preoptic area is the
most androgen sensitive area for stimulating scent marking in the mongolian gerbil.
Recent studies on South Indian gerbil, Tatera indica also indicate similar pattern.
Regarding ovariectomised female mongolian gerbils, they exhibit scent marking
when oestrodiol benzoate is implanted in the anterior hypothalamus preoptic area,
septum but implants in hippocampus, amygdala, thalamus or olfactory nucleus are not
effective.
21. Role of chemical signals in human social behaviour
The probable occurrence of functional human pheromones has been both asserted
(Wilson 1963) and denied (Gleasoen and Reynierse 1969) both without experimental
evidences. However the observation of Michael and Keverne (1970) on 'copulin' in
monkeys and menstrual synchronisation among close friends (McClintock 1971;
Russel et al 1980) has opened up new vistas in this field. It is quite relevant as regards
primer control of human endocrine cycles and reproduction generally and simul-
taneously opens up new prospects in reproductive pharmacology.
Observations reveal that odour plays an important part in psychosexual develop-
ment in infants (Kalogerakis 1963).
Since human male sexual behaviour is non-cyclical and not dependent on female
receptivity, the female-male influence may be a releaser only, except possibly in infancy.
Curtis et al (1971) noted that even a synthetic mixture of acetic, propionic isobutyric,
M-butyric and isovaleric acid evoked a response in the male. The same set of substances
are found in the human vagina and contribute to its attractant odour. The sexually
excitant component of human genital odour is complex involving both musk like notes
and odour similar to trimethyl amine, and is enhanced by alkaline fixatives. An alkaline
component of male genital odour resembles another amine, 1,5, diamino pentane
(cadaverine).
The probable primer and releaser substance in man are all musk odours (steroid,
large ring cycloketones and lactones) (Sink 1967).
The most likely areas of olfactory relevance in man are skin, axillary and pubic
apocrine glands and their tufts. Axillary secretion is most likely the site of human social
Recent advances in animal behaviour 181
pheromone. Prostaglandins also may function as nonolfactory pheromones being
secreted in large amounts in the semen and cause uterine contractions and facilitate the
sperm transport.
It has been shown conclusively that infants can identify their own mothers after the
second week of birth (McFarlane 1975). Humans can also recognise other individuals
by smell. Porter and Moore (1981) have shown that infants are able to identify odours
of siblings and that parents can identify the odours of the T-shirts of their children.
It has also been observed that infants scent-mark blankets, clothes and toys with their
own odour and carry this marked object with them (Passman and Weisberg 1975).
These scent-marks reduce their anxiety.
Studies on menstrual synchrony (Russel et al 1980) of college women indicate the
existence of some types of pheromones in human beings. In fact detailed investigations
are warranted to replicate and extend this work, especially to evaluate the correlation
between menstrual synchrony and ovulation.
22. Reproductive investment patterns and sex ratios in insects
Regarding insects, commitment of resources for reproduction can be dichotomised in
various ways, such as mating vs rearing, pre vs post birth but none of these are mutually
exclusive. The commitment of genetic material per se is trivial in terms of investment
but defines the interests of various individuals on each other in terms of relatedness.
The provision of resources is altruistic if it reduces the ability to produce future young;
parental care is thus altruistic. Relatedness can be assymmetric among individuals and
this together with the facts that parents need not be the main investors in the
production of new individuals leads to the particular complexities of insect societies
with reproductive division of labour. Such complexities can lead to markedly different
preferred sex ratios by workers and reproductives. The effort to determine the level at
which selection acts to set sex ratios of investment in hymenopteran societies in which
males are haploid and which thus have intrinsic assymmetries has run into technical
difficulties and competition for explaining female based sex ratios between the worker-
control and local mate competition hypothesis. Recent findings indicate that the local
mate competition can yield either female or male based ratios, depending on the
dispersal traits of the 2 sexes and that depending on the mode of inheritance, worker
control can yield a variety of sex ratios, not just 3 : 1 in favour of females (Owen 1983).
23. Visual communication in insects
Relatively ethologists have focussed more attention on visual communication. Many
insects emit light signals by virtue of their capacity for bioluminescence, described
mostly in beetles (Coleoptera) fireflies, adult lampyrid and elateid beetles. The
morphological and biochemical aspects of insect bioluminescence had been reviewed
by De Luca et al (1974). Lloyd (1977) has stated the diverse uses for which it is
employed, such as a lure for prey, pair formation, mate identification and location.
In fact in insect courtship the dynamic properties of visual signals are often critically
important in eliciting responses from the opposite sex. One of the excellent examples is
the reproductive behaviour of fireflies.
182 KM Alexander
Considerable work has been done on the sequence of courtship in N. American
fireflies, Photuris and Photinus. In the main sequence of courtship, the male initiates
flashes daring flight at a species specific frequency in well defined habitat areas, with the
female remaining stationary but relies on the flashes of the male and later follows the
male after a brief species specific pause. It has been shown that repeated flashes bring
the two sexes together (Lloyd 1971). It has been shown that flash signals of sympatric
species diff erred significantly, but species which are not located in nearby areas exhibit
similar flash pattern. The mimicking of flash signals of Photinus females by Photuris
females result in the devouring of the former male, by the latter female. According to
Lloyd (1975) female Photuris is very versatile that it can simulate the flash pattern of at
least 4 different species.
It has been shown that dispersion of insects occur by visual signals. Insects direct
signals at each other and these are important in territorial behaviour. Dragon flies are
good examples. They are selective about their breeding sites, with males arriving earlier
than the females and defend their territories by ritualised aggressive displays supported
by physical combat. During aggressive displays the bright silvery abdomen is directed
towards the opponent. Preying mantis also displays territorial behaviour.
24. Alarm signals
Although visual alarm signals are not well developed in insects, the butterflies
(lepidopterans) and paper wasp, Polistes annulans exhibit this reaction.
25. Sexual signals
Visual signals also play an important part in sexual behaviour. These visual signals
mediate a chain of stimuli interactions. It may be possible that in some of the
lepidopteran butterflies the well designed wings may be used as visual signals in
reproductive displays. In certain cases such as the queen butterfly both visual and
chemical signals are involved.
26. Acoustic communication in insects
Despite the fact that acoustic communication has evolved hundreds of times in insectan
species, hearing has been demonstrated only in 5 orders which produce sounds of
higher frequency e.g. Orthoptera, Homoptera, Lepidoptera, Coleoptera and Diptera.
The true nature of insect songs have not been fully understood by human beings,
although the advent of precision recording equipments and sophisticated sound
analysis instruments has facilitated the bridging of the gap between the occurrence of
the insect sounds and their significance.
It has been shown that most of the insects produce sound of communicative value at
some stages of their life cycle, displaying a wide variety with the frictional methods
predominating.
Generally most insect sounds have fewer dimensions than vertebrate sounds and the
insects cannot carry a tune. Their individual song components are called phonatomes
(all sounds produced during one cycle of movement).
Recent advances in animal behaviour 183
Insect sounds function as sexual signals facilitating attraction, courtship, copulation
and post courtship pair formation and also for chorusing and aggregation. Special
courtship signals have been reported for male orthopterans for cicadas and also for
males of many other arthropodan groups, from crabs to Drosophila, occasionally these
insect sounds function as social signals also e.g. wingless cockroaches and gromphadia
of Madagascar, hiss when their culture boxes are disturbed.
27. Vocalisation in birds
Recent studies on acoustic communication in birds had effected an acoustic analysis,
supplemented by x-ray cinematography which resulted in the formulation of a coherent
theory of avian speech (Scanlan 1983). The warbling of the budgerigars (Melospittacus
undulatus) is acoustically similar in many respects to human speech. In fact only slight
alterations are necessary to transform the warbling sound into a speech-like sound. The
nature and extent of this transformation differ among 'talking' species and can be
related to the vocal behaviour and ontogeny of each species. Such transformations are
possible because the birds can best produce just those acoustic cues necessary for
human perception of speech. Thus an important cue for human discrimination of
vowels is the relative formation frequencies and analysis of avian vowels which showed
that the Indian hill Mynah (Gracula religiosa) and several species of Psittacidae can
reproduce these patterns. Temporal cues because of this bird's excellent capability for
time resolution are precisely produced. Some of these acoustic features may be related
to movements of suprasyringeal vocal tract during speech imitation. These movements
have been observed and analysed during x-ray cinematography.
28. Vocalisation in mammals
Very little is known about vocal imitation in mammals. An adult male harbour seal
kept in captivity in New England Aquarium, near Boston, USA has been reported to
reproduce several english words and phrases (Rails and Gish 1983). This harbour seal was
originally brought in as an orphan pup in- 1 97 1 . There were no adult male harbour seals
around for it to serve as acoustic models. It was observed that in 1978 it started
imitating its own name "Hoover" and later on a number of other words such as 'hello',
'how are you' etc.
Adult females rarely vocalise. The rate of male vocalisation increases during breeding
season. Although harbour seals are considered as non-vocal pinnipedes, it has been
shown that they produce a number of growls, groans, hums etc. The quality of their
vocalisation apparently seems to be more superior to that of parrots. A comparison
with human spectrogram showed that Hoover's imitation contained key acoustic
features that the humans use for discriminating lightest vowels sounds.
29. Ethological analysis of drug action on aggression and defence
It has been possible to develop an ethopharmacological analysis of aggression. Specific
attention has been focussed on biologically relevant situations and events engendering
a broad repertoire of species specific agonistic behaviour (Miczek 1983).
184 KM Alexander
The sequential and multicomponent nature of aggressive interactions standardised
means to detect potentially pathological forms of aggression. It also affords the
opportunity to characterize neural and behavioural processes relevant to an organism's
social and environmental adaptations.
In one of the experiments, catecholamine antagonists such as amphetamines
enhanced threat behaviour under certain conditions in rodents but mainly disrupted
integrated aggressive and social behaviour patterns. In attacked animals amphetamines
disengages defensive and flight reactions from their prompting social stimuli.
Amphetamine as well as hallucinogen treated animals are more frequently targets of
attacks than control animals. It appears that engaging in aggressive or defensive
behaviour in the past and at the time of pharmacological intervention alters the
functional state and dynamics of brain catecholamine systems which in turn determines
the nature of drug action. The significance of the profound neurochemical changes
owing to specific behavioural events is further illustrated with experiments on the role
of endorphins in defeat. Mice which are frequently, attacked will ultimately emerge in a
typical physiological and behavioural pattern of defeat. Defeated animals become
immune to pain concurrent with significant changes in brain beta endorphins. This
analgesia is reversed by centrally acting opiate antagonists. Complete cross tolerant to
morphine analgesia further suggests that defeat experience readily activates brain
endorphins.
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© Printed in India.
The Drosophila Orcadian clock*
M K CHANDRASHEKARAN
Department of Animal Behaviour, School of Biological Sciences, Madurai Kamaraj
University, Madurai 625021, India
Abstract. The circadian rhythm in the process of eclosion of the fruitfly Drosophila is the best
investigated with regards to properties such as entrainment, freerun and phase shifts. The
system has been the basis of an important coupled oscillator model, several hypotheses,
landmark papers and a monograph. The PRC for this rhythm has been extensively used in
experiments designed to test the kinetics of the basic clock. The singularity point, signifying a
stimulus that can 'stop' the clock, was also predicted and discovered in this rhythm. Fittingly
the first clock mutant was also discovered in Drosophila,
Keywords. Circadian rhythm; Drosophila', phase response curves.
1. Introduction
The time course in the eclosion process of Drosophila represents perhaps the most
intensively studied and best understood circadian rhythm (Saunders 1976). The system
has stimulated the publication of several landmark papers (Pittendrigh and Minis 1964;
Pittendrigh 1966; Engelmann 1966; Winfree 1970), postulation of several hypotheses
(Pittendrigh and Bruce 1957; Chandrashekaran 1967b) and the writing of a most
stimulating monograph (Winfree 1980). Work on this rhythm has also helped to
analyse the formal properties of circadian rhythms, their response features and kinetics
of responses. Interestingly one of the diagnostic features, the temperature compen-
sation of circadian clocks, was first elucidated in 1954 for the Drosophila rhythm
(Pittendrigh 1954). One of the earliest phase response curves (PRCS) was also worked
out for this system (Pittendrigh 1960). The PRC of the Drosophila rhythm has also been
used as a tool to analyse the process of entrainment (Pittendrigh and Minis 1964) and to
understand the time constants involved during phase shifts (A0) of the basic oscillation
by light flashes (Chandrashekaran 1967a). Perhaps fittingly the first 'clock' mutant — a
single locus — was also isolated for the Drosophila circadian rhythm (Konopka and
Benzer 1971). This paper is a brief review of the author's own contributions to the
gradual unravelling of the formal properties of the Drosophila circadian clock.
2. The eclosion rhythm
In nature, much as other insects do, the fruitflies eclose during the early hours of the
morning and the last of the flies for the day to eclose would have done so by noon. The
entire eclosion of a group of flies lasts over ca 8 hr. Thus the 'eclosion gate' is one third
'Dedicated to Prof. Dr (multi) h c Erwin Dunning, teacher and exemplar to the author, on the occasion of
his eightieth birthday.
188
M K Chandrashekaran
the circadian span. If the flies are raised under light dark (LD) conditions in the
laboratory eclosion peaks some 3 hr after the light comes on. Earlier it was believed that
this eclosion peak appeared in response to the light-on stimulus but it became apparent
that the flies take their cue from the last light-off information. In other words
emergence (eclosion) of flies would start 12 hr after the L/D transition and peak (median
value) 15 hr after L/D. Flies raised in continuous light (LL) or constant darkness (DD) for
one or more generations do not show any rhythmicity. Adult flies eclose at all hours of
the day and night. Nearly all the Drosophila experiments referred to in this short review
were carried out at 20°C. At this temperature it takes the flies about 20-22 days to
300- DD-
3000- DD-
~T 1 -i — ' — i r — •n 1 — ' — i r
30 AO 50 6C 70 80 90 100
Hours
Figure 1. 'On' and 'off* rhythms in the eclosion of pharate adult Drosophila pseudoobscura
flies. The several populations were raised either in DD (upper panel) and then transferred to LL
of intensities in lux indicated or were raised in LL of varying intensities (lower panel) and then
transferred to DD. Time of transfer was on day 20 of the cultures arbitrarily and designated
hour 0. First eclosion peak not shown since synchronization was generally poor in all cases.
The 'on' rhythms illustrated in the upper panel and the 'off' rhythms illustrated in the lower
panel show time courses ca 180° apart relative to each other.
The Drosophila circadian clock
189
progress from the egg stage, through the instars to the pupal stage and eclosion. These
conditions refer to Drosophila pseudoobscura PU301 captured by C S Pittendrigh and
Th Dobhzhansky and used by Pittendrigh and his colleagues (Pittendrigh and Minis
1964; Pittendrigh and Bruce 1957; Pittendrigh 1954, 1960; Engelmann 1966;
Chandrashekaran 1967a, b; Winfree 1970).
In potentially arrhythmic pupal populations of Drosophila pseudoobscura raised in
LL or DD it is possible to induce rhythms by a DD/LL transfer ('on' rhythms) or LL/DD
transfer ('off' rhythms). The so called 'on' or 'off* rhythms are illustrated in figure 1 . The
two rhythms are approximately 12 hr apart (180° displacement) relative to each other.
This has prompted Honegger (1967) to talk of two oscillators, one set in motion at
sunrise and the other set in motion by sunset, and interpret that LD entrained rhythms
resulted from an interaction of these 'on' and 'off' rhythms. It turned out later (Winfree
1 970) that the 'off' rhythm was for all practical purposes very much like LD 1 2 : 1 2 entrained
rhythms, so much so that LD entrainment before allowing the circadian rhythm to freerun
in DD (with a period i of 24-3 hr) was dispensed with. It was sufficient to raise
the pupae in LL and transfer the populations a day or two before the first flies eclosed.
Another difference, was that the 'on' rhythm waned after 4-5 days. Light intensities of
just 0-0001 lux could attenuate the rhythms. Interestingly it was found that the 'on' and
'off' rhythms could be 'simulated' with appropriately higher/lower or lower/higher
intensity transfer of pupae. Figure 2 describes the time course of simulated 'on' and 'off'
rhythms. On the right side the magnitude of intensity differences between Jj (rearing
light intensity) and I2 (transfer light intensity) are shown. These rhythms seemingly
contradict a dictum of Pittendrigh (1966) that the Drosophila circadian rhythm is held
ON :
03-3000
_I
L
JL
-
:10 000
03- 300 :
3-3000
J
L.
1 1000
03- 30 -
30-3000 :
3- 300
_L
-L.
"i
100
03- 3
_!_
*
3- 30 :
300-3000 :
30- 300
JL
~£
10
LD-DD
J
300- 30 -
3- 03 :
•••'-30- 3
_.\,m
JL
10
30- 03 :
-300- 3
-L
L.
~
100
300- 03 :
3000- 3 -
L
-^1.
1000
-
3000- 03 -*-
J_
-10000
OFF -
I
A
-
30
40 5
0 60
70
80
Hours
Figure 2. Illustrates the time course by means of indicating 'median* values of "simulated on'
and 'simulated off' rhythms. Appropriately high and low intensities whose actual value is given
on the left and the factor of simulation is given on the right. All data above LD/DD line are
simulated 'on' rhythms and those below simulated 'off* rhythms. Real 'on"and 'off* rhythm
median values are given in the uppermost and lowermost lines respectively.
190
M K Chandrashekaran
'fixed' at CT 12 phase by L extending beyond the customary 12 hr and will be released
into further motion only by the restoration of £>. Thus peaks would follow on a pattern
of n x T 4- 15 hr in all pupal populations when light was maintained beyond 12 hr. On
the other hand the simulated 'on' and 'off' peaks may describe the behaviour of a slave
oscillator(s) or may be themselves 'transients'.
3. The Drosophila PRC
The circadian rhythm in the eclosion rhythm of Drosophila is sensitive to light
perturbations of 0-5 msec administered against a background of darkness. The
responses of the rhythm assume the form of displacement of the peaks along the time
axis. The peaks either advance or delay relative to unperturbed controls. The direction
of A<£ s and their magnitude are direct functions of the phase being perturbed. Figure 3
describes the standard PRC for the Drosophila rhythm first worked out in all its details
by Pittendrigh and Minis (1964). or 0 hr represents sunrise, CT 12 hr sunset, CT 0-12 hr
subjective day and CT 12-24hr subjective night. The rhythm is refractory to light
stimuli given during the subjective day but responds with increasingly dilating delays
during the first half of the night. At midnight the system switches the quality of its
responses from massive delays to massive advances. The magnitude of advances
ID
O
I
CL
O
C IRCADIAN TIME
HOURS
Figure 3. PRC s for 30 min, 2 hr, 6 hr, 8 hr and 1 2 hr light pulses of 1000 lux plotted against
'onset' of perturbation. The 30 min light pulse PRC is similar in all details to the standard
IS'1000 lux PRC of Pittendrigh and Minis (1964). A<£s are given in hr. Values above 0 line
(control) indicate delay A<£ s and below the line advance
The Drosophila circadian clock 191
diminish in the course of the second half of the subjective night. This is one of the best
worked out PRC s and was somewhat picturesquely described to represent the 'time
course and waveform* of the basic oscillation gating eclosion.
4. Transients and the coupled-oscillator model
The A</> s that follow light perturbations do not express themselves in the same cycle or
in the one after that. It takes the rhythm 3-4 cycles until the altered steady state with the
stable A</> is achieved. The 'creeping' fashion in which the A<£ s express themselves has
been the subject of much discussion and interpretation. One interpretation is given by a
coupled-oscillator model proposed by Pittendrigh and Bruce (1957). This model
postulated an A oscillator which was the pacemaker, light sensitive, temperature
compensated and suffered instantaneous A(/> s and a B oscillator which was the slave,
light insensitive but sensitive to temperature. A influenced B but B had no feedback
influence1 on A. The transients represent the efforts of a B oscillator trying to regain
original phase angle (*F) coupling properties and phases with an instantaneously phase
shifted A oscillator. Even though the model appears in retrospect fanciful and the
transients can be explained even in terms of a single oscillator (Chandrashekaran 1980)
at the time it was postulated it appeared to be an elegant way to explain the
phenomenon of transients. Furthermore one of the main postulates of the model was
stated without ambiguity and lent itself to direct experimentation. The main postulate
was: the instantaneous resettability of the basic light sensitive A oscillator.
Soon after the coupled oscillator model was postulated Running and Zimmer (1962)
gave a different interpretation to transients. They concluded from their studies on the
petal movement rhythm of the crassulacean plant Kalanchoe blossfeldiana that the
transient oscillation of petal movement following light signals reflects the behaviour of
the underlying oscillator. They found the several phases of the transients to respond to
a second light signal in a manner similar to the movement phases of the original rhythm.
This author designed critical experiments with Drosophila to critically test the two
views of transients. In planning the experiments on graph paper i.e. gedanken
experimental phase, the classical phase response curve (Pittendrigh and Minis 1964)
was assumed to really characterize 'the waveform and time course of the basic
oscillation'. The rationale was to administer light pulse 1 at a given -phase and then
follow up with light pulse 2 soon after to check if A$ had already occurred. A0 s would
be large and to scale of the PRC on the assumptions made by the coupled oscillator
model, but small and nearly undetectable according to the assumption of Running and
Zimmer (1962).
Figure 4 illustrates the results of an experiment where pulse 1 (1 5' 1000 lux) was given
at 15-5 cr and pulse 2 at 22 or. Both pulses given individually to two different
populations would have induced 5 hr delay A</> and 5 hr advance A<£ respectively.
According to the Bunning-Zimmer interpretation pulses 1 and 2 should have mutually
counter-acted each other's influence and no A$ s should have shown up. The results
indicate a larger than pRopostulated delay A$ which indicates a summative effect. This
would happen if light pulse 1 had indeed shifted the basic oscillation instantaneously by
an amount postulated by the PRC.
Figure 5 illustrates results of an experiment where it was assumed that light pulse 1
indeed shifts phase instantaneously and light pulse 2 was given to effect then an advance
192
M K Chandrashekaran
l
30-t
e;
O«
all
n
t
20 ^0 60 80 100 120 WO
hours
Figure 4. The effect of two light pulses (Pl and P2) of 15 min duration each and 300 lux
intensity. P1 was given at 15-5 cr and P2 at 22 cr. The curve above the raw data of eclosion
depicts schematically the instantaneous A<£ s effected by pulses. The dotted line indicates the
time course of unperturbed controls. Open circles are calculated medians of peaks and solid
circles indicate positions of medians of experimental populations.
A<£ of 5 hr i.e. at phase 22 or + 5 hr = 3 cr. Now pulse 2 was seen to counteract the
effects of pulse 1 with the result eclosion peaks of experimental and control pupal
populations show the same time course. Figure 6 contains data of a two pulse
experiment whose rationale was the same as in the first two pulse experiment except
that the second pulse was administered in the second cycle. The results are unequivocal.
It was concluded that the assumption of the coupled oscillator model that the basic
oscillator gating eclosion in Drosophila is phase shifted instantaneously by light pulses
holds at least for the Drosophila clock. But it must be mentioned in this context that no
concrete proof has been forthcoming for the so-called J5-oscillator (Chandrashekaran
1980). It is now generally assumed that most organisms, especially animals, may possess
several rhythms generally coupled to each other in a hierarchical fashion (Moore-Ede et
al 1982).
5. Dawn and dusk effects
In the course of experiments with Drosophila certain data tended to indicate that the first
half of the subjective night of the system may shown qualitatively different responses of
light on and light off transitions than the second half. In other words the first half
seemed to respond only to the light 'off ' component of a light pulse, the second half
seemed to respond only to the light 'on' component of a light pulse. This is the gist of the
The Drosophila circadian clock
193
i
70-
12* 2V 12
Ih
-i SCT
80
100
120
hours
1*rQ
Figure 5. The effect on the rhythm of two pulses Pl and P2 of 1 5 min duration and 10000 lux
intensity. Pj at 15-5 cr, P2 at ^2-5 or. Other details as in figure 4.
20
160
Figure 6. The effect of two light pulses of 1 5 min and 3000 lux. Pl was given at 15-5 cr in the
first cycle and P2 at 22 cr of the second cycles. In practice Pj falls 27-5 hr after LD/DD and P2
58 hr after LD/DD. Other details as in figure 4.
194
M K Chandrashekaran
SUBJECTIVE NfGHT
I I ! IL.
ADVANCE
DELAY
A
A
JL
12 18
ClRCAOIAN TIME
f 12
46 0 -6
PHASE SHIP T IN HO URS
Figure 7. Shifting the phase of the Drosophila pseudoobscura eclosion rhythm with light
pulses of 1000 lux and varying duration given in the first and second halves of the subjective
night. The light pulses are represented by the unfilled bars and are arranged in 4 batches on the
'circadian time scale* of Pittendrigh and Minis (1964). In batch 'a' the different populations
experienced the 'on' transition of pulses at different hours but experienced the 'off' transition at
the same phase (18 or). In batch 'b' the populations experienced the 'on' transition at the same
circadian hour (12 or) but the 'off' transition occurred for each population at a different hour.
The pulses of batches la' and 4b* scan the first half of the subjective night. Batches *c* and 'd' scan
the hours of the second half of the subjective night. In batch *c' the 'on' transitions of all the
pulses were in alignment (at 19 cr) with the 'off' transition occurring at a different hour for
each population. In batch 'd' on the other hand the 'on' transitions were systematically
staggered and the 'off' transitions of pulses aligned. The filled triangles represent averaged
median values of eclosion peaks of experimental populations 4-5 days after light treatment,
which responded with delay phase shifts. The filled squares represent averaged median values
of peaks 4-5 days after light treatment showing advancing phase shifts. Apparently the 'off'
transitions of light pulses determine direction and degree of phase shifts during the first half of
the night and the 'on' transitions determine phase shifts during the second half of the night.
dawn/dusk effect model postulated by Chandrashekaran (1967b). The light 'off'
information simulates a dusk or sunset and the light 4on' information acts like dawn or
sunrise. Figure 7 illustrates results obtained in a. later series of experiments
(Chandrashekaran et al 1973). The design was to compare the results of A^ s evoked by
light pulses given during the first half of the night such that they were of varying
durations started at differing phases but went off at the same (18 cr) phase. If 'off' is
indeed the discrete component recognized by the system then all A<£ s must be of equal
magnitude regardless of the duration of pulses evoking them. This is the case. The
opposite design was used for the second half of the subjective night. Pulses of varying
duration started at the same phase (19 cr) but ended at varying phases. Since fcon' is the
The Drosophila circadian clock 195
discrete component implicated all A</> s evoked by the pulses must be of comparable
magnitude. This again is the case. Reciprocal experiments were also performed and the
results further fortified these findings.
6. How to stop the Drosophila clock
Pavlidis (1967) predicted that the Drosophila clock must have a point of singularity on
theoretical considerations. Drawn in the form of a phase plane limit cycle diagram the
singular status will be achieved by a pulse of critical strength S* given at a critical time
T*. Winfree (1970) discovered the values for these two parameters. T* was 6-8 hr after
an LL/DD transfer (18-8 cr)and S* was 100erg/cm2/sec light of 460 nm given over
50 sec. If this treatment is indeed given to pupal populations, eclosion becomes
arrhythmic. Total arrhythmicity according to the formula: number of flies outside the
gate -r number of flies inside the 'gate' x 100 would be 200.
7. The Drosophila clock in contemporary research
Konopka and Benzer (1971) isolated a clock mutant for Drosophila. The clock could
even be surgically transplanted (Handler and Konopka 1979). Engelmann and Mack
(1978) showed that the PRC for the locomotor activity rhythm of Drosophila
pseudoobscura looked very different from the PRC for the eclosion process. This is
indicative of two different oscillators controlling the different rhythms. Work presently
in progress in the laboratory of Engelmann in Tubingen (personal communication)
indicates that the oscillatory pacemaker governing the locomotory activity in
Drosophila is not situated in the optic lobes. The optic lobes are proveniy the sites of the
pacemakers in circadian rhythms of locomotion in the cockroach and crickets
(Saunders 1976). It is to be hoped that the real nature of the elusive circadian clock in
Drosophila might soon be unravelled using the modern techniques of gene cloning and
recombinant DNA.
Acknowledgements
Much of the author's researches on the Drosophila clock was carried out first in the
laboratory of Prof. E. Biinning and later in the laboratory of Prof. W Engelmann at
the University of Tubingen. Fellowships and financial assistance given to him by the
German Academic Exchange Service (Bonn) Alexander-von-Humboldt-Foundation
(Bonn), The Deutsche Forschungsgemeinschaft (Bonn) and the Miller Institute for
Basic Research in Science at Berkeley, University of California made these researches
possible. The author thanks his student Mr P Kumaraswamy for help in preparing the
figures.
References
Biinning E 1973 The physiological clock (revised 3rd English edn) (Berlin, Heidelberg, New York: Springer
Verlag)
196 M K Chandrashekaran
Biinning E and Zimmer R 1962 Zur Deutung der Phasenverschiebungen und 'transients' nach exogener
Stoerung endogener Rhythmen; Planta (Berl) 59 1-14
Chandrashekaran M K 1967a Studies on phase shifts in endogenous rhythms. I. Effects of light pulses on the
ecolosion rhythms in Drosophila pseudoobscura; Z. Vergl PhysioL 56 154-162
Chandrashekaran M K 1 967b Studies on phase shifts in endogenous rhythms. II. The dual effect of light on
the entrainment of the eclosion rhythm in Drosophila pseudoobscura; 2. Vergl. Physiol, 56 163-170
Chandrashekaran M K 1980 Apparent absence of a separate B-oscillator in phasing the circadian rhythm of
eclosion in Drosophila pseudoobscura. In Development and neurobiology of Drosophila (eds) O Siddiqi, P
Babu, L M Hail and J C Hall (New York: Plenum Press)
Chandrashekaran M K and Loner W 1969 The effect of light intensity on the circadian rhythms of eclosion in
Drosophila pseudoobscura; Z. Vergl. PhysioL 62 337-347
Chandrashekaran M K, Johnsson A and Engelmann W 1973 Possible dawn and dusk roles of light pulses
shifting the phase of a circadian rhythm; J. Comp. PhysioL 82 347-356
Engelmann W 1966 Effect of light and dark pulses on the emergence rhythms of Drosophila pseudoobscura;
Experientia (Basel} 22 606
Engelmann W and Mack J 1978 Different oscillators control the circadian rhythm of eclosion and activity in
Drosophila: J. Comp. PhysioL 127 229-237
Handler A M and Konopka R J 1979 Transplantation of a circadian pacemaker in Drosophila; Nature
(London) 279 236-238
Honegger H W 1967 Zur Analyse der Wirkung von Lichtpulsen auf das Schliipfen von Drosophila
pseudoobscura; Z. Vergl. PhysioL 57 244^262
Konopka R J and Benzer S 1971 Clock mutants of Drosophila melanogaster; Proc. Nad. Acad. Sci. 68
2112-2116
Moore Ede M C, Sulzman F M and Fuller C A 1982 The clocks that time us: Physiology of the circadian timing
system (Cambridge: Harvard University Press)
Pavlidis T 1967 A mathematical model for the light affected system in the Drosophila eclosion rhythm; Bull.
Math. BiophysioL 29 291-310
Pittendrigh C S 1954 On temperature independence in the clock system controlling emergence time in
Drosophila; Proc. Natl. Acad. Sci. 40 1018-1029
Pittendrigh C S 1960 Circadian rhythm and circadian organization of living systems: in Cold. Spr. Harb.
Symp. Quant. Biol. 25 159-184
Pittendrigh C S 1 966 The circadian oscillation in Drosophila pupae. A model for the photoperiodic clock; Z.
Pflanzenphysiol. 54 275-307
Pittendrigh C S and Bruce V G 1957 An oscillator model for biological clocks in Rhythmic and synthetic
processes in growth (ed.) D Rudnick 75-109 (Princeton: Univ. Press)
Pittendrigh C S and Minis D H 1964 The entrainment of circadian oscillations by light and their role as
photoperiodic clocks; Am. Nat. 98 261-294
Saunders D S 1976 Insect clocks (New York: Pergamon Press)
Winfree A T 1970 The temporal morphology of a biological clock. In lectures on Mathematics in the life
sciences (ed.) M Gerstenhaber (Providence RI: Am. Math Soc.) 109-150
Winfree A T 1980 The geometry of biological time (Berlin: Springer- Verlag)
Proc. Indian Acad. Sci. (Anim* Sci.), Vol. 94, No. 3, June 1985, pp. 197-205.
© Printed in India.
Hormones in insect behaviour
V K K PRABHU
Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695581, India
Abstract. Hormones play an important role in insect behaviour. These hormones are mainly
the neurohormones of the brain and of the corpus cardiacum, the juvenile hormone of the
corpus allatum and the ecdysone of the prothoracic glands. These produce either releaser
effects or modifier effects. Hormonal modulation of neurophysiological activity controlling
various aspects of behaviour, hormonal influence of reproductive behaviour in the male and
the female insects, their role in migration, as well as hormonal influence of caste determination
and behaviour of social insects, have been discussed.
Keywords. Insect hormones; insect behaviour; insect reproduction; social insects; migration;
neurophysiological activity.
1. Introduction
Insects occupy a unique position in the animal kingdom in that hormones are actively
involved in the control of behaviour in this group more than in any other group of
animals, either invertebrates or vertebrates. This is apparently due, on the one hand, to
the rich behavioural repertoire shown by insects and on the other to the accessibility of
blood containing the hormones, to the neuronal elements of the nervous system. Ever
since the discovery by Bounhiol (1938) that extirpation of the corpus allatum from the
penultimate larvae of the silk worm Bombyx mori led to precocious, miniature cocoon
formation, and especially during the last twenty years there has been a surge of evidence
implicating hormonal control of various behavioural aspects in a variety of insect
species, like courtship, mating, oviposition, circadian rhythm, autogeny and
anautogeny, diapause, caste determination in social insects, feeding behaviour,
migration etc. It is in fact preposterous to attempt to cover all aspects of hormonal
control of insect behaviour in this brief paper and hence it is intended here to touch
upon only a few of the points which are considered rather important.
2. The insect endocrine glands
The insect endocrine system itself consists chiefly of the neurosecretory cells, the
corpora cardiaca, corpora allata and the prothoracic glands, of which the latter
degenerate during adult metamorphosis and hence are present only in the larval and the
pupal instars. In addition, there is considerable mass of evidence implicating ovaries in
ecdysone production in many insects though the exact role played by the ovarian
ecdysone is far from clear. There are also perivisceral neurohaemal organs associated
with the various ventral ganglia (Nayar 1973; Highnam and Hill 1979) whose exact role
is known only very little. The neurosecretory cells are scattered in the cerebral ganglia,
197
198 VKKPrabhu
but mainly in the pars intercerebraiis, distinguishable usually into a median group of
"Gomori positive" cells and the lateral group of phloxinophil cells. There may be other
groups also. The suboesophageal ganglion and the other ventral ganglia also contain
occasional neurosecretory cells. The corpus cardiacum serves as a storage-cum-release
centre for the secretory material elaborated in the brain neurosecretory cells; it also
elaborates its own hormone. As the brain as well as the cardiacum contain
heterogeneous hormones from different sources, removal of these organs resulted in
removal of more than one hormonal principle and it was difficult to pinpoint the role of
individual hormones until recently when many of these hormones have been isolated
and characterized.
3. General effects of hormones on behaviour
The effects of hormones on behaviour have been divided into releaser effects and
modifier effects (Truman and Riddiford 1974). Accordingly, a releaser effect of the
hormone on behaviour is relatively rapid, and is directly triggered by the hormone.
Modifier effect is, on the other hand, slower to appear and results in a change in
responsiveness of the nervous system. Hormones may have either only releaser effect, or
primer effect or both.
An excellent example of the releaser effect of a hormone is the motor effect to phallic
nerve-stimulating hormone, demonstrated by Milburn et al (1960) and Milburn and
Roeder (1962). It is known that in Periplaneta americana, the suboesophageal centres
normally inhibit motor activities involved in copulatory movements. Decapitation
however removes the sub-oesophageal centre from this inhibition resulting in
copulatory movements. When extracts of the corpora cardiaca are injected into male
cockroach, it caused rhythmic movements of the abdomen as characteristic during
copulation, and when the extract is applied to the nerve cord, evoked rhythmic activity
in the phallic nerve. Apparently, the phallic nerve-stimulating hormone acts on the
suboesophageal ganglion to remove the inhibition of the motor centres. Though these
hormones thus perform the same functions as neural pathways from the brain centres
to the lower motor centres, these have the advantage of being sustained in its action,
unlike nervous action.
A good illustration for modifier effect of hormone is the effect of juvenile hormone
on sexual maturation behaviour. For example, in the female grasshopper Gomphocerus
rufus (Loher and Huber 1966), juvenile hormone is necessary for development and
maintenance of receptivity. The immature female responds to the courting male by
primary defence reaction, involving kicking the male and other escape reactions.
Corpus allatum induces maturation resulting in copulatory readiness, the female now
stridulating and moving towards him, resulting in mounting and copulation.
Allatectomy of newly emerged adult female or of sexually mature female results in
maintenance in the animal, or its reversion, to defensive behaviour respectively. Primer
pheromones bring about secretion of a modifier hormone resulting in a change in
behaviour as in the case of maturation pheromone facilitating maturation in male
Schistocerca by JH release, or long term effects of changing environmental stimuli such
as migratory behaviour in Oncopeltus under the stimuli of decreasing day length due to
juvenile hormone secretion or again, co-ordinating behaviour with developmental or
physiological changes of the animal.
Hormones in insect behaviour 199
4. Hormonal modulation of neurophysiological activity
Neurophysiological basis of hormone action has started receiving attention in recent
years. As reported earlier, the effect of the extract of the corpus cardiacum on some of
the inhibitory centres in the nervous system of the cockroach which normally repress
motor programme resulting in copulation, have been demonstrated neuro-
physiologically.
Activation of the adult behaviour in wild silk moths is another interesting example.
Development and differentiation of the adult nervous system is completed in the wild
silk moths (Antheraea pernyi, A. polyphemus and in Hyalophora cecropia) in the pharate
adult even though the pharate adult does not show adult type movement even if the
pupal skin is peeled off, before the normal time of eclosion. That its nervous system is
comparable to that of the adult is shown by the fact that the peeled pharate male
exposed to female sex pheromone under dim illumination shows normal electroanten-
nogram, although it does not show any behavioural response characteristic of the
normal adult male, indicating the inhibition to be likely of central nervous nature. This
behavioural deficiency of the peeled pupa extends to other aspects of behaviour like
lack of tonus, even simple righting reflexes etc. It shows only occasional spasmodic
movements of the legs, abdominal twitches and pupal-like rotary movements of the
abdomen. However, at the time of the day when eclosion normally occurs, the peeled
moth shows pre-eclosion behaviour consisting of a l-25hr long programme of
abdominal movements, followed by eclosion and spreading of the wings. This switch
over to adult behaviour is hormonally controlled. An eclosion hormone is found in the
brain and corpus cardiacum of the pharate moth; when homogenates containing this
extract is injected into pharate animals, normal eclosion and precocious adult
behaviour was observed. At normal eclosion, the eclosion hormone appears in the
blood. Its function thus turns on the adult behaviour. The eclosion hormones turns off
certain other parts of the nervous system, for example those motor centres which
control the dense bands of intersegmental muscles of the abdomen used in eclosion.
The motor neurones which supply these muscles become silent shortly after eclosion,
thus leading the muscles to degeneration. When eclosion hormone is injected into
isolated abdomen of pharate moths, the hormone triggers muscle degeneration, thus
turning off the motor neurones to the muscles.
In Hyalophora cecropia, the pre-eclosion abdominal movements of the pharate adult
consists of three phases with reference to the type and relative frequency of movements
(i) a hyperactive period involving mainly abdominal rotations extending to 30 min; (ii) a
rather quiescent period of about 30 min; and (iii) a period of hyperactivity involving
strong peristaltic waves moving anteriorly along the abdomen. These series of
movements begin 10-30 minutes after injection of extracts containing eclosion
hormone into pharate moth or into abdomens isolated from pharate moth. Truman
and Sokolove (1972) showed that the timing and patterning of these movements are
built into the circuitry of the abdominal gangila, by recording the motor output to the
inter segmental muscles from the deafferented abdominal nerve cord. This preparation
normally showed very low level of firing. On the other hand, addition of eclosion
hormone evoked strong and active motor output, beginning 20-40 minutes after
addition of hormone. This was followed by a decline and then by new frequent bursting
of volleys. The timing and the bursting pattern was such as would result in the pre-
eclosion behaviour of the pharate adults, were the efferent fibres connected to the
200 V K K Prabhu
muscle bundles. The above motor programmes are hence encoded in the abdominal
ganglia, and the eclosion hormone activates the programme.
The flashing of the firefly beetle Luciola is an example to illustrate a case of peripheral
hormone action. Light-induced inhibition of flashing is due to a central mechanism
acting on the pacemaker, and a peripheral mechanism acting on the lantern itself. That
a hormone produced by the testis is involved in this peripheral inhibition has been
shown by connecting the haemocoel of two fire fly beetles and covering the eyes of one
partner by opaque paint. When the other partner is illuminated, flashing by the
"opaque" partner was also inhibited; when the testis of the illuminated partner is
removed, the inhibition of flashing was abolished (Brunelli et al 1968). When the light
organ was electrically stimulated directly, the intensity of the flashing elicited was not
abolished either by decapitation or by denervation of the light organ. On the other
hand, if the eyes of a denervated fly were illuminated, the intensity of the electrically
induced flashing was inhibited, which however was abolished if testis was removed
(Bagnoli et al 1970). Transection of the nerve cord anterior to the ganglion which
innervates the testis, destroyed the inhibitory response to illumination, whereas
electrical stimulation of the nerve to testis led to inhibition of electrically induced
lantern flashing. The flash inhibiting substance stored in and released from the testis is
noradrenalin (Bagnoli et al 1972). The noradrenalin appears to act peripherally, as
application of this substance inhibits flashing, but does not affect the size or frequency
of efferent volleys coming to the lantern.
5. Hormonal influence of reproductive behaviour
Hormonal effects on reproductive behaviour are many and varied; there are vast species
difference also. On the whole, it may be said that in the male, influence of hormones on
reproductive behaviour is either non-existant or unknown in many species; where they
exist, these influences are comparatively simple. Many adults like silk moths display full
sexual behaviour immediately after emergence and remains so for the rest of their adult
life, there being no opportunity for hormones to play any part in the adult life.
However, in some males like grasshoppers and locusts the corpora allata are required
for the maturation of adult sexual behaviour. Perhaps endocrine control of male sexual
behaviour has been best studied in grasshoppers and locusts (Pener 1974). In this group,
information appears to be reasonably thorough in Locusta migratoria migratorioidesy
but rather fragmentary in the other species. Pener concludes that the C-cells of the pars
intercerebralis completely control the sexual behaviour, their effect being direct and
not mediated through the corpus allatum. The C-cells also activate the corpus allatum
completely controlling yellow colour, which exert a secondary effect on mating
behaviour; the corpus allatum influences the intensity of sexual behaviour, but does not
exercise a complete control since their removal does not completely inhibit mating
behaviour.
The corpora allata may be controlled by nervous means by the brain; or the allata
may be controlled by the pars intercerebralis neurosecretory cells of the brain. In the
cockroach, the neurosecretory hormone as reported earlier, elicits abdominal move-
ments acting on the phallic nerves.
In the female, generally speaking the young virgin becomes receptive to male under
the influence of juvenile hormone. This has a direct effect on the behaviour of the female
toward courting male, as opposed to the indirect effect of JH on sex pheromone
Hormones in insect behaviour 201
production and thereby attraction of the male. The control may again be nervous or
neurosecretory, as in the male. In the wild silk moths, the sexually receptive female, in
response to proper environmental stimuli, assumes, calling posture involving protru-
sion of the last two abdominal segments which expose the pheromone glands permitting
pheromone release. This calling behaviour is under the control of the release of corpus
cardiacum's calling hormone under the neural influence of the brain. Injection of blood
from calling female into virgin in the absence of proper stimuli, induces in them, calling
behaviour.
Apparently, under the neurosecretory hormones released by courtship, copulatory
behaviour ensues. Generally > mating is followed by termination of male receptivity and
the females thus become refractory which may however be in some temporary, whereas
in others, permanent. This may be due to a variety of stimuli, like mechanical stimulus,
presence of spennatophore in the female bursa copulatrix or due to the sperms
themselves, which might secrete a "bursa factor" acting on the central nervous system.
As in the mosquito, accessory gland substances of the male, like matrone, may also be
involved. These influences may be coupled with neural influences. Refractoriness may
also be due to withdrawal of juvenile hormone or due to factors from maturing ovaries.
The spermatheca or the bursa of the female may release hormonal factors which may
exert their effect through the central nervous or neuroendocrine system of the female.
There is considerable evidence now that pheromone production in insects is under
hormonal control in many species. Whereas in some insects the corpus allatum
stimulates pheromone secretion, in some the corpus cardiacum is involved in this
activity. However, an inhibitory role for juvenile hormone in pheromone production is
now emerging from some recent studies. For example, it has been known for a long time
that large quantity of juvenile hormone accumulated in the male saturniid moth
abdomen. This store of juvenile hormone is now known to be transferred from the
accessory gland of the male to the bursa copulatrix of the female during mating (Shirk
et al 1980). Webster and Carde (1984) propose that in Platynota stultana and probably
in other similar moths also, this exogenous juvenile hormone transferred in the seminal
fluid to the female might be involved in the switch from virgin to mated behaviour in the
female. Mating in this species resulted in termination of calling, and gradual reduction
of pheromone in the glands comparable to decapitation of virgin females. Mating
apparently terminated neural and hormonal stimuli required for pheromone produc-
tion; exogenous juvenile hormone treatment in virgin females also gave similar results.
Closely connected with phetomone production is pheromone perception; Schafer
(1977) and colleagues have demonstrated that the male adults of Periplaneta have
nearly twice as many olfactory sensillae as female adults; this sexual dimorphism of
adult antennal sense organs appeared only during the adult stage. Treatment of
terminal instar with JH mimics resulted in supernumerary larvae lacking antennal
sexual dimorphism. Inhibitory action of JH prevented the appearance of antennal
sexual dimorphism during normal larval development. Adult males with larval
antennae produced by bilateral treatment with exogenous juvenile hormone mimic do
not respond to the pheromone although they are completely adult in other respects.
Electrophysiological studies involving single unit and electroantennogram recording
confirm that a portion of the receptors added at the adult ecdysis are sex-attractant
receptors, which are not present in the larval or in the adult female antennae in large
numbers, and that topical application of JH mimics to male antennae during terminal
larval instar inhibits their development.
202 V K K Prabhu
In the mated female, release of the neurosecretory material appears to take place,
resulting in not only myotropic activity leading to oviposition, but to oviposition
behaviour as well (Nayar 1958). When ovarian extract or blood from the female during
the oviposition is injected into partially gravid mating females, depletion of neurose-
cretory cells occurred, followed by oviposition. If median neurosecretory cells are
implanted into young females lacking mature oocytes in the ovaries, quivering
movements of the genital plates, simulating oviposition behaviour, occurred. Further
work on ovulation/oviposition has been reported recently by Davey (1984) who found
that in Rhodnius, ovulation is stimulated by a peptide neurohormone originating from
ten large identifiable neurosecretory cells of pars iriter-cerebralis. This neurohormone is
released in response to feeding and the other in response to mating. The latter has been
investigated in detail in this animal. On mating, female Rhodnius releases a sper-
mathecal factor, which is only one of the two factors involved in the release of
myotropic hormone from the neurosecretory cells, as mating precedes ovulation by
some days. A second stimulus, which is provided by the ecdysteroids from the ovary,
also appears to be involved. It has been found that injection of ecdysteroids into
ovariectomized female results in an increase of myotropic activity of the haemolymph
of mated females, but not in virgins. Bursts of action potentials recorded from the
corpus cardiacum during ovulation have been associated with the ten pars inter-
cerebralis neurosecretory cells, the source of myotropin. Isolated brain retrocerebral
complex from mated female have shown the characteristic action potentials in vitro
under the influence of ecdysterone. Ecdysterone action on the neurosecretory cells
appear to be mediated through aminergic neurons of the pars intercerebraiis.
6. Hormone in migration
The case of locusts is one of those which has been studied fairly well especially in
Locusta migratoria and Schistocerca gregaria. Young ones (hoppers) reared in
isolation, show moderate level of activity; the adults tend to be solitary and do not
perform long flights. On the other hand, when crowded, they become highly active,
show marching behaviour and oriented locomotion; their adults become gregarious
and undertake migration. This difference is subsequently shown to be due to the better
developed prothoracic glands in solitary forms. Experiments involving transplantation
of prothoracic glands or injection of their extracts have shown that the gregarious
hoppers or adults could be converted to solitary individuals (Carlisle and Ellis 1963).
Haskell and Moore (1963) have substantiated these findings by demonstrating that
ecdysone reversibly reduced the spontaneous motor output from the metathoracic
ganglion of adult locusts. On the other hand, the corpus allatum has been shown to
stimulate spontaneous locomotor activity as well as sexual activity by a direct action on
the nervous system (Odhiambo 1966).
According to Johnson (1969), migration was triggered when ecdysone was absent
and JH titre was low. Subsequent rise in JH level brought about cessation of migratory
behaviour and onset of oogenesis. Rankin (1974) analysed in detail the causative factors
and hormonal control of flight in the milkweed bug Oncopeltusfasciatus, using a series
of elegant experiments. In this insect, flight is post-teneral and pre-reproductive. It
undergoes an adult reproductive diapause in response to short photoperiods making
available longer time for flights of greater duration. Long photoperiods on the other
Hormones in insect behaviour 203
hand afford favourable breeding conditions, and so it undertakes reproductive activity.
A skillful manipulation of corpus allatum and ovaries of the animal, and by starvation
as well as topical application of JH or by implantation of corpora allata and by
combination of some of these experiments, indicated that the corpus allatum can
stimulate flight behaviour and the effect can be duplicated by application of juvenile
hormone analogue. The flight system may respond to lower titer of juvenile hormone
than does the reproductive system. Rankin and Riddiford (1977) subsequently
confirmed that JH was the primary hormone responsible for stimulation and co-
ordination of migration and reproduction in Oncopeltus by JH bioassay and exogenous
application of JH to experimental animals. It is to be noted that in Dysdercus sp where
starvation stimulates flight, and feeding brings about ovarian development, flight muscle
degeneration and vitellogenesis are induced by corpus allatum which is activated by
feeding and mating (Edwards 1970; Nair and Prabhu 1984a,b) where the situation is
opposite to that of Oncopeltus fasciatus.
The migratory behaviour of cockchafer beetle Melolontha melolontha is character-
ized by reversal of the sense of direction of flight in the female (but not in the male)
which is closely connected to the maturation of oocytes. Stengel (1974) and his
colleagues have shown that the migratory behaviour of this beetle is a good example of
neurohormonal regulation of behaviour which depends upon the existence of two types
of neurosecretory activity separated in time. During the life above ground, the female
adult undergoes two or three ovarian cycles each of which is characterized by oriented
migrations which leads her towards feeding areas consisting of the edge of forest, a
thicket or an isolated tree, constituting "pre-feeding flight" during which the ovaries are
immature; and then back to the egg-laying sites after feeding and mating in the reverse
direction, to the fields whence she came, constituting the "oviposition flight" with fully
mature ovaries, to lay eggs.
The male, however, upon leaving the soil makes an oriented flight to the forest, but is
not capable of reversal in the sense of direction of flight, but moves only in the adjacent
feeding area where it feeds and mates. During the reversal flight the corpus allatum
releases its hormone, which releases the reversal mechanism for the flight sense and at
the same time, blocks oogenesis. The corpus allatum of the pre-oviposition female can
release the reversal mechanism even in the male which does not normally reverse the
flight direction. The corpus allatum of the pre-oviposition female containing oostatic
hormone, if implanted into prefeeding female whose corpus allatum is active in
secreting gonadotrophic hormones, is capable of suppressing its activity and blocking
oogenesis. So it appears, two hormones secreted by corpus allatum are involved here.
Stengel and his colleagues (see Stengel 1974) have also shown that neurosecretory cells
of pars intercerebralis secreted the hormone and they are released by the corpora allata.
7. Hormonal control of social behaviour
According to Luscher (1975), in termites and in the honey bees, the societies use
pheromones which influence juvenile hormone production, which in turn influence
caste development. However, considerable differences exist in the mode of action of
these principles. In termites and honeybees, the development of reproductives is
inhibited by the pheromone of the queen. However, the queen substance of the honey
bee inhibits the corpora allata whereas the pheromone of the termite reproductives
204 V K K Prabhu
stimulates the allatum. Hence the stimulation of replacement reproductives in termites,
by the honeybee queen substance. It has also to be noted that the termite pheromone
acts on the developing larva whereas the queen substance of the bee acts on the adult
worker bees. In bees the pheromone produced by the workers acts upon the larvae,
whereas in termites the queen pheromone acts on them. In both cases the corpora allata
are stimulated to produce more juvenile hormone, causing queen development in bees
and inhibiting development of reproductives in termites.
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and for role of the male gonads in Lucilia lusitanica (Charp); Arch, hal Biol 108 180-206
Bagnoli P, Brunelli M, Magni F and Viola M 1972 The identification of a flash inhibiting substance from the
male gonads of Luciola lusitanica (Charp); Arch. hal. Biol. 110 16-35
Bounhiol J J 1938 Recherches experimen tales sur les determinisme de la metamorphose chez les
Lepidopteres; Bull Biol. F. Belg. (Suppl) 24 1-199
Brunelli M, Buonamici M and Magni F 1968 Effects of castration on the inhibition of flashing in fireflies;
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neurophysiology (New York: Plenum) (eds) A B Borkovec and T J Kelly pp. 93-1 13
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system of the desert locust; Nature (London) 197 56-58
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Arnold) 2nd ed., 358 pp.
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the Study of Social Insects)
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by extracts of corpora cardiaca; Gen. Comp. Endocr. 2 70-76
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americana by extracts of the corpus cardiacum; Biol Bull 118 119
Nair C R M and Prabhu V K K 198£a The role of feeding, mating, and ovariectomy on degeneration of
indirect flight muscles of Dysdercus cingulatus (Heteroptera: Pyrrhocoridae); J. Insect Physioi. 31 35-39
Nair C R M and Prabhu V K K 1984b The role of endocrines in flight muscle degeneration in Dysdercus
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Nayar K K 1958 Studies on the neurosecretory system oflphita limbata Stal. V. Probable endocrine basis of
oviposition in the female insect; Proc. Indian Acad. Scl B47 233-251
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Odhiambo T R 1966 The metabolic effects of corpus allatum hormone in the male desert locust. II.
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Rankin M A and Riddiford L M 1977 Hormonal control of flight in Oncopeltusfasciatus: The effects of the
corpus cardiacum, corpus allatum, and starvation on migration and reproduction; Gen. Comp. Endocr. 33
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and its neuroendocrine regulation; In Experimental analysis of insect behaviour (ed.) L Barton-Browne
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 207-217.
© Printed in India.
Hormonal rhythm and behavioural trends in insects
D MURALEEDHARAN
Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695581, India
Abstract. Circadian rhythmicity in the timing of secretion and release of many of the
neurohormones appears to be a common phenomenon in insects. Involvement of hormonal
components in the locomotor activity rhythm in cockroaches, crickets etc. has not yet been
proved unequivocally eventhough some of the findings along these lines support this. Many of
the physiological events in insects occur only once in each individual's life-time-gated events.
Release of eclosion hormone in insects is determined both by a circadian clock and by the
developmental competence of the insect. Periodic release of PTTH which influence the moulting
process in larvae has been established to be gated. Induction of prodromal signs of pupation as
a result of gated release of FTTH in some insects have been confirmed. Intrinsic neurosecretory
cells of cc release a hormone (calling hormone) in a rhythmic fashion which affect the
pheromone release and subsequent initiation of calling behaviour in some of the lepidopteran
virgin females Production of proctodone by the epithelial cells of hindgut also follows a
rhythm bringing about diapause in some of the insects.
Keywords. Rhythm; endocrines; eclosion; hatching; moulting, bursicon; calling hormone;
diapause.
1. Introduction
As in many other animals, insects are also known to possess internal clocks which can
be synchronised and entrained by daily environmental periodicities. Since many of
these clocks follow a periodicity of about 24 hr, these are designated as 'circadian
clocks' and diverse types of behaviour in insects have been implicated to follow this
cyclic pattern. The performance of behaviour is often directly coupled to the output of
the clock with little or no influence from other stimuli. In many instances, the coupling
between the driving circadian clock and the behaviour appears tabe hormonal. Daily,
seasonal and even annual phenomena are apparently linked to biological clock
functions. Daily and seasonal responses are the results of the effects of photoperiodic
stimulation on the clocks of the neural and endocrine cells that control the behavioural,
physiological or developmental processes (Beck 1980).
Circadian rhythms have a major influence on insectan endocrine activity. Daily
fluctuations of the titers of circulating hormones may reflect daily alterations in the
activity of endocrine organs themselves or they may reflect daily alterations in specific
or general metabolic pathways that serve to clear hormonal activity from the blood
(Truman 1978). Monitoring the daily cycles of activity in the endocrine system of
insects have attracted attention because of the probable importance of this system in
the control of overt rhythms of physiology and behaviour.
2. Endocrine rhythms
2.1 Rhythms in the brain neurosecretory cells
The involvement of circadian rhythmicity in the determination of timing of secretion of
insect neurohormones now appears to be a general phenomenon and a precise
207
208 D Muraleedharan
knowledge of the nature of the clock that controls hormone release is a prerequisite for
a thorough understanding of any developmental problems related to hormone action.
This circadian clock of timing of release of insect neurohormones was demonstrated
unequivocally for the eclosion hormone of silkmoths. a hormone which triggers their
adult eclosion (Truman and Riddiford 1974; Truman 197Ja,b).
The first report of a circadian cyclic pattern of activity in the neurosecretory cells was
that by Klug (1958) in the brain of a beetle, Carabus nemoralis. Rensing (1964, 1966)
used a sort of microspectrophotometer to compare the absorption of neurosecretory
material in the region around the nucleus with that in the axon 'hump* of brain
neurosecretory (NS) cells of Drosophila melanogaster. A maximum accumulation was
noticed near the nucleus around 'dawn' and 'dusk' i.e, some 3 hr after their peak nuclear
size. Larvae also showed similar but less marked changes.
Dutkowski et al (1971) made some ultra-structural investigations to demonstrate the
circadian cycle of neurosecretory activity. Brain NS cells of Acheta domesticus recovered
after sacrificing them 30 min after 'dawn' and 30 min after 'dusk' (i.e. at the time of
minimum and maximum locomotor activity) showed marked differences between the
two groups. The cells of the inactive animals contained extensive ER and secretory
vesicles in the golgi region and in the axon only and nuclei with smooth membranes. On
the other hand cells of the active ones housed only fragmented ER, apparently quiescent
golgi, secretory vesicles in the perikariyon, but not in the axon and nuclei with
undulating membranes. It was therefore concluded that the brain cells of the inactive
animals were synthesising and releasing neurosecretion whereas the cells of the active
animals had ceased (temporarily) to synthesise actively or to release the secretion and
thus accumulated secretion in the perikaryon. The potential value of such EM studies is
revealed from these investigations in elucidating the circadian secretory cycles.
We have noticed a distinct circadian rhythmic pattern of secretory activity in the
PINSC (pars intercerebralis neurosecretory cells) A cells of the late instar larvae (4th and
5th) of castor semilooper, Achoea Janata. During the early hours of the day, the activity
was minimum and around mid-day at peak level and with a gradual drop towards
evening and night. Topical application of an anti-allatotropin, Precocene-II, com-
pletely upsets this rhythm of neurosecretory activity and the level of secretory activity
as well (Mohanakumar and Muraleedharan 1985).
Autoradiographic techniques have been used as another tool in the elucidation of
cyclical metabolic activities of endocrine cells. Cymborowski and Dutkowski (1969)
used this technique in Acheta domesticus to relate neurosecretory cell function to the
control of locomotor activity. They showed that there is a sharp diel rhythm in RNA
synthesis (as indicated by 3H incorporation) in the median NS cells of the brain and the
NS cells of the suboesophageal ganglion of crickets.
Fowler and Goodnight (1966) succeeded in cluturing isolated brains from an
opilionid, Leiobunum longipes for 80 days in L:D at the end of which there was a
distinct residual rhythm of 5-hydroxy tryptamine accumulation. Rensing (1969) also
studied the 24 hr rhythmic pattern of secretory activity in the salivary glands by
measuring the size of nuclei of the gland cells of Drosophila melanogaster which were
cultured.
2.2 Rhythms in the NS cells of ventral nerve cord
In the ventral nerve cord of almost all insects studied, four major categories of NS cells
Hormonal rhythm and behavioural trends in insects 209
are to be noticed. However, the pathways and release sites of these NS cells-products
have not yet been completely elucidated (Raabe 1983). Naturally, very few attempts
have been made to study the activity rhythms of these cell types as well. A reasonably
clear diel change in the nuclear size of NS cells of the SOG (suboesophageal ganglion) was
demonstrated in Drosophila melanog aster (Rensing 1964, 1966; Rensing et al 1965). In
the cockroach Periplaneta americana cyclic changes in the SOG NS cells were noticed
(Cymborowski and Flisinka-Bojanowska 1970). A similar rhythm was also noticed in
the 'C cells of SOG of the stick insect, Clitumnus extradentatus. Marker (1960a, b, c)
traced the rhythm inducing factor of the SOG as two pairs of NS cells located on the
lateral aspects of SOG, one pair on each side. During the active period 'A' type
neurosecretory cells in the ventral ganglia of Leucophaea maderae contained greater
quantities of NS granules (De Besse 1965). In Acheta domesticus a bimodal rhythm in the
SOG NS cells was noticed with a maximum in the midphotophase and midscotophase
(Cymborowski and Dutkowski 1969). Protein synthesis in the NS cells were also found
to be rhythmic. In the light of all the above findings they suggest that in response to
photoperiod, the brain begins RNA synthesis at the onset of light followed by protein
synthesis and subsequent elaboration of neurohormone which is being translocated to
the SOG where it stimulates RNA synthesis and neurosecretion.
2.3 Rhythms in corpus allatum and prothoracic glands
A daily rhythm of nuclear and nucleolar size in the cells of corpus allatum (CA) and
prothoracic glands (PTG) was noticed in the larvae of Drosophila melanogaster (Rensing
1 964, 1 966; Rensing et al 1 965). Ecdysteroid titers in the different tissues like the ovaries,
fatbody and haemolymph of the cricket, Gryllus bimaculatus were monitored by
Hoffmann et al (1982) and it was noticed that ecdysteroid was present in small
quantities in all young organs which increased markedly during ovarian maturation
and decreased again during the last days of adult maturity. Two peaks of haemolymph
ecdysteroids during larval-pupal development, one at the transition from the feeding
stage to post- feeding prepupa and the other in association with pupal cuticle formation
were demonstrated in Manduca sexta by Bollenbacher et al (1975) and the same
observations were later confirmed in Calpodes ethius by Dean et al (1980). Later
Fujishita and Ishizaki (1982) have demonstrated that in the larvae of Samia cynthia
ricini haemolymph ecdysteroid titer begins to rise at 1800 hr of the day preceding the
gut purge under LD 12:12 to reach a maximal level 4-5 hr before the purge.
Eventhough certain amount of work has already been done in investigating the
activity rhythms of endocrines in some of the selected insect species, we are still unaware
of the exact rhythmic activity patterns (whether it is daily modulation of hormone
titers, gating of hormone secretion or photoperiodic control of hormone release) of
most of these hormones especially juvenile hormone, ecdysone etc. in most of the
insects. Even attempts made on neurohormones along these lineis are mostly
observations made on the basis of daily changes in the histological appearance of NS cell
groups or endocrine organs. In certain cases rhythmic changes in the target tissues have
provided evidence for rhythmic endocrine activity. Knowledge about the factors
initiating release of JH, ecdysone, PTTH, JH esterase or proctodone and the time of their
release are prerequisites for the proper interpretation of events controlling meta-
morphosis in insects. One of the methods which can be relied upon is by quantitatively
210 D Muraleedharan
monitoring the titer of a particular hormone by using radioimmunological, mass-
spectrometric and other techniques.
3. Hormones and circadian rhythms
Morphogenesis in insects are known to be controlled by different hormones. Hence it is
not very surprising to see that the timing of larvaHarval moulting, pupation, adult
emergence etc. are also effected via hormones. The circadian control of gated — once in a
lifetime — programmes and the more typical daily, repeated, ongoing behaviour of the
type that is measurable in individual insects appears different. For gated rhythms,
control is in the form of a 'single shot' hormonal release of each unique and largely fixed
behaviour pattern. For ongoing behaviour continuous control must be exerted right
round the clock.
3.1 Locomotion
Marker (1954) reported that rhythmic activity could be induced in arryhythmic
Periplaneta americana by parabiosing them to rhythmic animals. Later Cymborowsky
and Brady (1972) demonstrated in both crickets (Acheta demesticus) and cockroaches
(Periplaneta americana) that headless animals take up the rhythm of the intact animal
stuck on their backs, but significantly more rhythms are induced if the haemocoels are
interconnected than if they are not. Thus some sort of influence affecting rhythmicity in
locomotor activity apparently passed via the haemolymph from the intact donor to the
headless recipient. Marker (1956) showed that the transplantation of SOG from the
rhythmic donor induced rhythm in %a headless arrhythmic cockroach recipient.
Subsequent experiments implied that if two pairs of lateral NS cells of SOG are destroyed,
no rhythms were induced even when the ganglia were implanted. So it was inferred that
these four lateral NS cells of SOG act as an autonomous hormonal clock (Marker 1960c,
1961, 1964). However subsequent work by many others failed to confirm the induction
of activity rhythm in the cockroach itself by any sort of SOG transplant (Leuthold 1966;
Roberts 1966; Brady 1967a). Observations made in the grasshopper, Romalea microptera
and in the beetle, Blaps mucronata also failed to support the above finding by Marker
(Fingerman et al 1958; Thomas and Finlayson 1970). The implicated NS cells could be
successfully removed by microcautery without impairing the periodicity of activity
from the cockroach and even the removal of a great bulk of cell bodies and neuropile
from SOG ventral region — leaving little more than the thorough tracts — did not stop the
rhythm (Brady 1967c; Nishiitsutsujii-Uwe and Pittendrigh 1968). So it becomes almost
clear that SOG NS cells are in no way essential to the timing or control of cockroach
locomotor activity.
Many of the experiments conducted by different workers to establish the role of cc-
CA complex also demonstrated that cc (corpus cardiacum) may be involved in judging
the amount of locomotor activity (Shepard and Keeley 1972; Michel 1972) but appear
not to be involved with its periodicity (Roberts 1966; Brady 1967b; Weber and Gaude
1971; Brady 1971). Attempts were also made to demonstrate the effect of hormonal
principles from the brain NS cells on the cockroach locomotor activity rhythms (Harker
1956; Brady 1967b; Nishiitsutsujii-Uwo et al 1967) and the results showed no influence
Hormonal rhythm and behavioural trends in insects 211
as such on the timing of activity rather than destroying the behavioural integration.
If the compound eyes are severed from the optic lobes, the activity in Leucophaea
maderae remained normally rhythmic but became uncoupled from the environment. In
effect the animals became blind and their rhythm freeran unentrained by light/dark
cycle in which they were kept. On the other hand, if the optic tracts were cut between the
optic lobes and the protocerebrum, the animals became arrhythmic even in LD
(Nishiitsutsujii-Uwo and Pittendrigh 1968). Later this finding was confirmed by
Roberts (1971) by removing the optic lobes along with the compound eyes which
resulted in arrhythmia ty. However, as Brady (1974) suggests a possibility still exists for
the involvement of hormonal components in the cockroach locomotor activity rhythms
since it has already been demonstrated in the NS cells in the optic lobes (Beattie 1971)
and also their axons in the circumoesophageal connectives of locusts (Michel 1972).
Experimental work on crickets (Acheta domesticus) by Cymborowski (1970)
demonstrated hyperactivity and superficial arrhythmicity due to pars intercerebralis
ablation even though autopsy of operated animals showed equivocal stainabie material
in the pars intercerebralis region. So it appears premature to conclude that brain NS cells
ablation in crickets necessarily disrupts its rhythm at least in LD. In some of the noctuid
moths, ablation of median NS cells has a different effect; the normal night disappears,
but is replaced by a burst of activity for an hour or two after dawn.
3.2 Gated events •*
There is a whole class of physiological events that occur once only in each individual's
life time, but which are nevertheless timed by a circadian rhythm. This sort of
phenomenon cannot be detected as a rhythm in an individual; it becomes apparent only
in mixed-age populations. Here the individual completes the morphogenetic aspects of
its development at random with respect to time of the day for its emergence. Thus
although individuals become ready to emerge at all times, they only do so through a
narrow span of time each day, when a so-called circadian gate is open.
3.2a Eclosion: The concept of gating events by a circadian clock grew out of the
studies of the rhythm of adult eclosion in Drosophila by Pittendrigh and Skopik (1970).
In these flies eclosion is restricted to a specific temporal gate, the time of which is
determined by an interaction between the photoperiod and the fly's circadian clock.
Experimental evidence for a triggering effect of hormone on adult eclosion in the moth
Manduca sexta was given in detail (Truman 1970, 1971a, b; Reynolds 1977). When
blood was removed from eclosing animals and injected into pharate moths prior to
their normal gate, the recipients showed precocious eclosion. So also extracts from the
brains or cc of pharate moths contained eclosion stimulating activity which was
depleted during eclosion. This 'eclosion hormone' proved to have a number of actions
on the pharate moth including the behaviour release involved in emergence and wing
spreading, the triggering of the break down of the intersegmental muscles and
plasticising of the wing cuticle. The time of appearance of 'eclosion hormone' was
determined by bleeding pharate Manduca sexta at various times of the day and assaying
each sample for hormonal activity (Reynolds et al 1979). The hormone appears in the
blood only at a restricted time of day about 2*5 to 3 hr before the moth subsequently
emerges. At the gate, eclosion hormone was released as a rapid pulse which is then
212 D Muraleedharan
gradually cleared from the blood. The appearance of the circulating hormone is
complemented by an 85 to 90% depletion of activity stored in the cc.
Truman (1978) concludes in the light of earlier findings, that the gating of the
"eclosion hormone' release assumes that the time of release is determined both by a
circadian clock and by the developmental competence of the insect. Therefore
eventhough the proper circadian time has arrived secretion will not occur during that
gate if development has not been completed. But when the ability of Manduca to
respond to the eclosion hormone was examined, it was found that receptivity appeared
only about 4 hr before the hormone was actually secreted. Thus eventhough the proper
circadian time is arrived at, hormone release will not occur if the animal is not in the
proper developmental state.
3.2b Hatching: Since other developmental events are clearly gated by circadian
clocks, it might have been expected that egg-hatch would also be so. The possibility
have been examined thoroughly only in the pink bollworm, Pectinophora gossipiella
(Minis and Pittendrigh 1968). In this particular species, the hatch rhythm is initiable
until the 12th day of embryogenesis when the first cephalic pigmentation coincides with
some essential link-up in the central nervous system. In Aedes mosquitoes, hatching
occurs as a direct response to environmental amelioration, related only to the effects of
temperature on embryogenesis and the presence of water after some sort of delayed
developmental period (Gillett 1955) and unrelated to any rhythm (Corbet 1966). Pre-
conditioned Aedes taeniorhynchus eggs hatch at any time of day within 15 min of
emersion in de-oxygenated water (Nayar 1967).
3.2c Larval moulting: The release of PTTH to induce the periodic larval moults in
Manduca sexta was established to be gated (Truman 1972). From an analysis of
quantitative and qualitative differences in the responses of neck-ligatured Manduca
larvae at various times of the day, it was found that the PTG needed contact with the
brain for at least 1-5 hr before they were fully activated. Thus a minimal time interval
was necessary for PTTH secretion by the brain. With the opening of the first gate, the
larvae were apparently not competent to release PTTH but some gained this competence
before the gate subsequently closed. The remainder attained competence during the
succeeding day and were able to release PTTH as soon as the next gate opened. Thus the
distribution in the first gate identified the closing of the gate and that in the second gate
identified the time of the opening of the gate. Also the gates for the release of PTTH for
the various instars occurred at essentially the same time of the day and the duration of
gates tended to narrow as the animals grew.
Fujishita and Ishizaki (1981) demonstrated that in Samia cynthia ricini an
endogenous circadian clock controls the timing of larval ecdysis and PTTH secretion
preceding it. The clock upon reaching a specific phase point causes the brain to secrete
PTTH provided that the brain has acquired the secretory competence. Full secretion of
ecdysone occurred 6 hr after PTTH secretion and ecdysis ensued 34 hr thereafter to
complete the ultimate sequence of ecdysis.
3.2d Pupation: The puparium formation in Drosophila is induced by the moulting
hormone, ecdysterone. The process of metamorphosis starts with the puparium
formation and can be regarded as a closed system. In Drosophila lebabnonensis
puparium formation is a rhythmic process which can be characterised as a circadian
Hormonal rhythm and behavioural trends in insects 213
rhythm. The circadian oscillation regulates the timing of the ecdysterone mediated
process of puparium formation. Jan Eeken (1978) opines that the influence of the
circadian oscillation is not at the level of the ecdysterone concentration itself since no
endogenous ecdysterone nor a changed ecdysterone degrading system is present at
different phases of the circadian oscillation. So he suggests the interference system,
coupled with circadian oscillation which determines whether or not the puparium
formation can take place, seems to enforce its action at a level between transcription
and translation.
The release of PTTH by the fifth instar larvae brings about the start of metamorphosis
in Manduca sexta. Ligation experiments showed that the brain was required until an
average time of 1600hr in order to trigger the start of metamorphosis. However, a
careful study of various ecdysone dependent epidermal changes in the larvae indicated
that PTTH release probably began as early as 0100 hr on the preceding night. Thus the
PTTH gate in the fifth instar appears to start at a time similar to that for other instars but
the hormone release in this last case is greatly prolonged over about 1 5 hr (Truman
1978).
Considering the tropic action of brain on the PTG, one might expect that PTTH rhythm
would result in the rhythmic secretion of ecdysone. Using the time of appearance of an
ecdysone sensitive puffin Drosophila, Rensing (1966) postulated a rhythm of ecdysone
release prior to pupariation.
In the last instar larva ofSamia cynthia ridra, the initiation of development towards
pupation as visualised by overt events such as gut purge and wandering occur with
circadian rhythmicity (Ishizaki 1980; Fujishita and Ishizaki 1982), and the involvement
of an innate circadian clock has been demonstrated. Fujishita et al (1982) have
demonstrated that the timed surge of ecdysteroids is responsible for the gated
occurrence of gut purge and that 18 hr before gut purge, larvae acquire the competence
to undergo gut purge in a gated fashion provided that they are exposed to a sufficient
surge of ecdysteroids. A gated release of PTTH was confirmed in the induction of
prodromal signs of pupation in Manduca as well (Truman and Riddiford 1974).
3.2e Bursicon release: Another hormone whose appearance is gated is the tanning
hormone, bursicon. In Manduca sexta this hormone is produced in the abdominal
nerve cord and released from the periviceral organs. Bursicon release occurs during
wing inflation by newly emerged moths and it triggers the tanning of the freshly
expanded wing (Truman 1973). Under normal conditions wing inflation behaviour is
well under way by 15 min after eclosion and thus bursicon secretion was estimated to
occur about 3 hr after the eciosion hormone peak. The time course of bursicon
appearance was followed in individual cannulated Manduca (Reynolds et al 1979) by
means of an isolated wing assay that responded to bursicon by tanning the wing veins.
Within one to two minutes after eclosion, no hormonal activity was detected and 2 min
later substantial increase in bursicon level was noticed in the blood and a peak titer was
reached within 5 to 10 min. Thus the secretion of bursicon also occurs as a large pulse.
The secretion of bursicon is dependent on the prior release of eclosion hormone
(Truman 1973). When moths were induced to emerge early by eclosion hormone
injection, they also showed early bursicon secretion. In fact the release of bursicon by
neurones in the abdominal ganglia of Manduca appears to be a part of the complex
neural programme that is triggered by the eclosion hormone. The gated appearance of
214 D Muraleedharan
bursicon in newly emerged moths is a consequence of the prior gating of eclosion
hormone release.
3.2f Calling hormone: This hormone in moths also show a daily pattern of secretion.
The hormone differs from those discussed above in that its release is not gated but
presumably occurs on a daily basis as long as the female is unmated. In virgin females of
silkmoths, the behaviour involved in pheromone release — calling behaviour — shows a
rhythmic occurrence (Riddiford and Williams 1971) and is triggered by a hormone
released from the intrinsic NS cells of the cc. Blood from calling females when injected
into non-calling individuals readily induced the characteristic behaviour. At other times
of the day, this activity was reduced or absent. Consequently, the rhythmic display of
calling behaviour is apparently triggered by the rhythmic secretion of this hormone
from the cc.
Truman (1978) is of the opinion that in the moth, the cerebral lobe area contains the
gating centre for at least one hormone. Further, the rhythm of hormone secretion could
be a direct or indirect result of an interaction with a circadian clock. The rhythm of
ecdysone and bursicon release are secondary gated rhythms since they result only from
the tropic actions of PTTH and eclosion hormone respectively. By contrast, the latter two
hormones and the calling hormone appear to be primary gated rhythms, the rhythm is
enforced through direct association of the circadian clock (s) and the endocrine courses.
Most likely the rhythmic centres which control the release of the other two hormones
discussed above also reside in the same region of the brain. Whether these centres are
distinct from one another is unknown at this time. Also, there is no evidence to indicate
whether the respective endocrine cells contain the entire rhythmic system.
3.3 Diapause
The epithelial cells of part of the hindgut (ileum) of mature larvae of the European corn
borer were implicated in the production of a hormone (proctodone) involved in the
physiology of diapause (Beck and Alexander 1964). Part of the evidence offered in
support of the postulated endocrine function of the ileal cells was the appearance of
secretory granules within the cytoplasm. The secretory rhythm of these granules had an
8 hr periodicity with phase setting being effected by photoperiod. The cells released
their secretory products shortly after the beginning of the scotophase, after which
cytoplasmic granules would again accumulate and again disappear at 8 hr intervals.
Although the postulated hormonal function of these cells remains questionable, the
rhythmic secretory cycle is striking.
4. Conclusion
As is evident from the foregoing discussion, research work already done on the
endocrine rhythms in insects have been mainly restricted to only a few species of insects.
A thorough understanding about the rhythmic activity patterns of the entire array of
hormones in insects and the controlling centres for the release of these hormones are
immensely useful Once we know the exact activity patterns of all these developmental,
metabolic and other hormones, one can artificially manipulate the hormone titer at the
wrong time in the insecton life resulting in its maldevelopment promising a novel
approach in pest management.
Hormonal rhythm and behavioural trends in insects 215
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© Printed in India.
Behavioural energetics of some insects
T J PANDIAN
School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, India
Abstract. Foraging behaviour of insects includes the following energy-requiring processes:
(i) location and (ii) gathering. Some insects do incur additional energy cost on transporting and
storing food. Energy cost of foraging ranges from 2 to 5 % of the energy gained in bees and
wasps. Initiation of flight, in large and insulated insects obligatorily requires 'warming-up* of
muscle temperature and maintenance of endothermy by over 20°C above the ambient. Over-
heating is avoided by pumping the cooler abdominal blood into the hot thorax. Pollinating
insects include (i) hovering high-energy foragers, which expend more energy and visit more
flowers per unit time and (ii) walking low-energy foragers, which expend less energy and visit
few flowers per unit time. Decreasing of "wing loading" is another strategy adopted by
saturniids, which do not feed as adults. Most bees forage, when flowers are just blooming, and
when they have maximum nectar reward to offer. From the model study on energy cost of
oviposition, it has been shown that Sceliphron violaceum makes greater and greater effort to
complete the process of food provisioning and sealing the larval nest, when it has invested more
and more energy on foraging and provisioning spiders to the larviposited young ones.
Keywords. Behavioural energetics; foraging behaviour; Sceliphron violaceum; high-energy
foragers; walking low-energy foragers.
1. Introduction
A survey on pertinent literature reveals that there is a large number of publications
concerning energetics (Waldbauer 1968; Scriber and Slansky 1981; Muthukrishnan and
Pandian 1983) and behaviour (Saunders 1976) of insects. However, only a few
publications are available on behavioural energetics of insects. Hence, it is chosen to
highlight in this paper only the following aspects: (i) Energy cost of foraging and
(ii) Energy cost of oviposition in selected insects.
2. Energy cost of food acquisition
In all insects acquisition of food involves a series of behavioural responses; while these
responses are related, and perhaps inter-dependent, they are separate processes each
under the control of a set of physical and chemical co-ordination. The processes are
(i) energy cost of maintaining food supply (e.g. ants which maintain aphid population);
(ii) energy cost of locating food supply, (iii) energy cost of gathering or catching food,
(iv) energy cost of processing of food (e.g. conversion of nectar into honey), (v) energy
cost of eating food and (vi) energy cost of transporting and storing food (e.g. bees) (see
Lawton 1973). Of these, processes related to the energy costs of locating and gathering
food are important; some insects such as bees and wasps do invest energy on
transporting and storing food. Interestingly, much work has been done on the energy
cost of transporting and storing food by bees and wasps (Heinrich 1979).
219
220 T J Pandian
2.1 Endothermy and initiation of flight
Mechanical efficiency of the flight mechanism of insects is approximately 10-20 %
(Weis-Fogh 1972). More than 80 % of the energy expended during flight is necessarily
degraded into heat. Curiously those insects, which are large and insulated, retain most
heat in the thorax during flight, also require the highest muscle temperature in order to
maintain sufficient power output to continue flight. The minimum muscle temperature
required to initiate flight varies over the relatively narrow range of 40~45°C (Kammer
and Heinrich 1978). For instance, when the sphinx moth Manduca sexta vibrates its
wings at the rate of 40 times/sec, and produces about 1 J/min of energy, its thorax is
heated to 38°C, and the moth is ready for a take-off (Heinrich and Barthelonew 1971).
Flight activity and endothermy are thus invariably linked in several insects, and
endothermy in flight is a large part an obligatory phenomenon (Heinrich 1974).
Temperate insects such as Bombus sp, invest quite a lot of energy (2-18 kJ/g thorax/hr)
to elevate the thoracic temperature to about 40°C from the ambient temperature of
3-1 6° C, tropical insects such as Schistocerca sp. may require far less energy to elevate its
thoracic temperature to over 35°C from the ambient temperature of 20-25°C.
Information on the energy cost of endothermy and initiation of flight for tropical
insects is almost totally wanting and a comparative study of this aspect for tropical and
temperate insects will be rewarding.
2.2 Thermoregulation during flight
Most insects are small and uninsulated, so that over-heating of the flight musculature is
not a general problem. However, build-up of heat is rapid in the flight muscle of some of
the large, uninsulated insects. In these insects, the over-heating is avoided by
transferring the hot blood from the thorax to the abdomen, when the abdominal heart
(dorsal vessel) beats rapidly and pumps the cool blood through the heated thorax. For
instance, the thoracic temperature of Manduca sexta never exceeds 40° C, and the excess
heat is passed into the cool abdomen (26°C), by adjusting the rate of heat beat. More
than pre-flight heating and endothermy, cooling and thermoregulation during flight
should pose a major problem to the tropical insects. However, no publication is
available on this subject for tropical insects.
2.3 Energy cost of foraging
Measured and calculated energy cost of flying for insects vary over a large continuum. In
general most values fall between 418 and 2090 J/g/hr. They represent 50 to 100 fold
increases over the resting metabolic rate (Kammer and* Heinrich 1978). Necessarily, an
insect may forage by hovering at high energy cost for a shorter duration or by walking at
low energy cost for a longer duration. Table 1 shows the foraging cost of some bees and
wasps, for which information is available. The report by South wick and Pimentel (198 1)
is by far the most complete one for the estimation of foraging energetics of insects. A
colony consisting of 50000 bees (Apis mellifera) is estimated to collect 259 kg nectar
worth 1590680 kJ and 24 kg pollen worth 339066 kJ annually by flying a cumulative
distance of about 13 million km. At an energy cost of foraging as 13-8 J, i.e. 4-6 J/km
Behavioural energetics of some insects
Table 1. Foraging costs in some insects.
221
Predator
Foraging cost
(% acquired
Prey food energy)
Reference
Bombus vagans
Nectar
8-2
Heinrich (1972a,b)
Apis mellifera
Nectar and
pollen
3-7
Southwick and
Pimental (1981)
Delta conoideus
Caterpillar
2-7*
Muthukrishnan and
Senthamilselvan (1985)
Try poxy Ion rejector
Spider
1-6*
Muthukrishnan and
Senthamilselvan (1985)
Sceliphron violaceum
Spider
5-2*
Pandian and
Marian (1985)
*Considering energy cost of flight as equivalent to 418-6 J/g/hr, a value reported for
the wasp Vespa crabro by Weis-Fogh (1967).
(Tucker 1970; Dade 1977; Schaffer et al 1979), a bee travels over 3 km to collect 370-7 kJ
worth nectar and pollen, i.e. the energy cost of foraging is 3-7% (Southwick and
Pimentel 1981). Similar calculation for the estimation of foraging cost of the bumble bee
Bombus vagans shows that it spends about 8 % of the food energy on acquiring it
(Heinrich 1972a,b). Estimations on energy cost of foraging in walking and swimming
insects are totally wanting.
Several species of wasps forage on caterpillars or spiders and transport them to the
nest to provide food for their larvae. Flying a distance of about 68-4 km, Trypoxylon
rejector (Sphecidae) predates and transports 190 spiders (7-22 mg each) worth 24*1 kJ
in about 1 1 hr and 36 min for providing food for larvae developing in 9 cells in a nest.
Covering a distance of 0-64 km in 4 trips Delta conoideus forages and transports 4
caterpillars (71-182 mg each) worth 4*2 kJ in about 2 hr and 57 min for provisioning
one cell with a single larva (Muthukrishnan and Senthamilselvan 1985). Investing
1 -04 kJ on flight for 2 hr and 30 min, Sceliphron violaceum transports spiders worth
13-32 kJ to provide food for its larva developing in an unused hole of electrical socket.
Energy cost of foraging in these wasps amounts to 1-6, 2*7 and 5*2 % whereas T. rejector
and D. conoideus have to invest another fraction of their respective food energy on nest
building activity, S. violaceum has avoided the investment of nest building by choosing
unused holes.
2.4 Metabolic strategies of flower foragers
In the extremes there are two basic metabolic strategies of harvesting food energy from
flowers: (i) Hovering high-energy foragers, which expend more energy and visit more
flowers per unit time and (ii) Walking low-energy foragers, which expend less energy
and visit few flowers per unit time. Hovering flight places heavy energy demands on
insects (836 J/g/hr; Weis-Fogh 1972). This mode of foraging increases the rate of intake
of food energy. For example, hovering flies Bombilius spp visited 21 flowers of
Houstania caerulea/min; whereas Syrphus spp which do not hover at flowers, visited
222 TJPandian
only 5/min. Hovering moths Hemaris spp. visited 50 Kalmia angustofolium flowers/min,
whereas non-hovering Bombus spp visited only 1 5/min. The food rewards of a
composite inflorescence for example, are generally individually too small to be
economically harvested by hovering. But they can be gathered by a butterfly or a bee
that lands on the flowers and reduces its energy expenditure. Low-energy food sources
can generally not be harvested by high-energy foragers, which can make much more
rapid energy profits from high-energy food sources (Kammer and Heinrich 1978).
Large wings allow insects to fly with a low wing beat frequency and allow some insects
to initiate flight without prior endothermic warming-up and to continue flight by
gliding; the energy expenditure of locomotion is considerably reduced in such insects.
Thus, the third strategy of reducing the energy cost of flying is to decrease 'wing loading'
by increasing the wing area per unit body weight. Some saturniid moths and sphinx
moths, which do not feed as adults, represent the extreme examples of this kind of
strategy. Having relinquished energy intake, and having to rely only on the fixed energy
reserves, they have minimized the energy cost of flying by decreasing the 'wing loading'
as much as possible (Nachtigall 1966; Pringle 1974).
2.5 Blooming times
Flower density is another important factor that affects the energy cost of foraging.
Although the flower density is ultimately determined by population density of the
plants; it is altered by the time and duration of blooming. Synchronous blooming of the
flowers of a species in a given plant population would minimize the temporal and energy
costs of flying between plants (Heinrich and Raven 1972). Besides, flower density,
(i) daily time of blooming, (ii) amount of energy reward provided, (iii) type of flower
product (nectar or pollen or both), and (iv) structures affecting access nectar or pollen
are some factors that may modify the energy cost of foraging. Thus, Bombus spp., which
can forage at ambient temperatures of 5°C or less, forage at an energy cost two or three
times greater than that at 26°C (Heinrich 1972asb). Hence, flowers which are pollinated
at low temperatures should either provide more energy rewards than those blooming at
high temperatures (perhaps one reason for the low efficiency of honey production in
tropical bees) or be denser so that they can be visited in rapid succession (Heinrich and
Raven 1972).
While foraging in the early morning at an ambient temperature of 2°C from flowers
of manzanita Arctostaphylos otayensis, Bombus edwardsii (0-1 g) has a thoracic
temperature near 37°C. The energy cost of maintaining this thoracic temperature is
3-3 J/min. Each flower of A. otayensis provides nectar equivalent to 6-3 J in the early
morning and the nectar reward dwindles to 1*3 J by noon. Thus, it is energetically
advantageous for B. edwardsii to forage in the morning, when there is little competition
for nectar, or the rate of nectar production is high (Heinrich and Raven 1972). Likewise,
Chilopsis flowers provide the largest amount of nectar (24 ml/flower) in the early
morning and as the result of foraging by Bombus, nectar volume declined to
0-3 ml/flower by 0930 hr. By taking into account the time required to suck up nectar
and the energy cost of foraging at different times of the day, Witham (1977) calculated
that in the early morning Bombus that took only the pool nectar was making a net
foraging profit of 51 J/min, whereas that which went for both groove and pool nectars,
could make a profit of only 414 J/min. In a country like India, where oil-seeds are in
short supply, research work on pollination ecology of legumes deserves priority.
Behavioural energetics of some insects
3. Energy cost of opposition
223
For want of pertinent publication, the presentation on the energy cost of oviposition
has been restricted to information collected by Pandian and Marian (1985) for
Scelipheron violaceum. Male S. violaceum predates, stings, paralizes spiders belonging to
Argiope pulchella, Cyrtophora cicabrosa and C. citricola and deposits them into unused
holes of electrical sockets. When the male has deposited spiders equivalent to 68 ± 9 mg
in about 35 min the female oviposits a single egg. Subsequently the male continues the
process of spider deposition and seals the hole. From experimentations and observa-
tions lasting over 3 years, Pandian and Marian (1985) noted that the wasp deposits
spiders equivalent to 200 ±10 mg. The male brings spiders weighing 3-60 mg/trip, and
within 10-30 trips, he deposits 200 mg. Marian et al (1982) reported that spiders
equivalent to 68 and 110 nag are the minimum requirements for the successful
completion of larval and pupal stages respectively.
To test the ability of the wasp (i) to recognize its own prey, (ii) to add more prey and
(iii) to keep in memory the quantity of prey provided at any stage, the process of spider
deposition was interfered by Pandian and Marian (1985) by way of adding or removing
Table 2. Interference with the deposited spiders and response of the wasp Sceliphron
violaceum (from Pandian and Marian 1985).
Deposited
spider wt
(mg)
Addition ( + ) or
removal ( — ) of
spider (mg)
Wasp response
Before oviposition
18 4- 58 ±14 All the wasps recognized and removed the added spiders; 60% wasps
continued depositing spiders up to 200 mg but the others abandoned the
hole
42 +19 ±3 All the wasps recognized and removed the added spiders; 70% wasps
continued depositing spiders up to 200 mg but the others abandoned the
hole
After oviposition
122 4-83±3 .100% wasps recognized and removed the added spiders; 80% wasps
closed the hole but the others abandoned the hole
112 +21 ±3 100% Wasps recognized and removed the added spiders; 80% wasps
sealed the hole; but the others abandoned the hole
1 06 + 1 9, 63 * 1 00 % Wasps recognized and removed the added spiders but abandoned
the hole
149 - 38 ± 3 50 % Wasps added spiders up to 200 mg and sealed; the others, which
have seen the interference, abandoned the hole
Nearing the sealing
182 +74 ±22 50% Wasps identified and removed the added spiders and sealed the
hole; others abandoned the hole
193 +17+4 100% Wasps removed the added spiders and sealed the hole
203 -53 ±6 Ignored and sealed the hole
201 -201 Ignored and sealed the hole
*These spiders were added accommodating them in between the originally deposited spiders.
224 T J Pandian
spiders. Their intention was to study the response of the wasp (i) before oviposition,
when spiders weighing less than 68 mg were deposited, (ii) after oviposition, when
spiders weighing more than 110 mg were deposited and (iii) before the closure of the
hole, when spiders weighing about 1 80-200 mg were deposited. From their obser-
vations presented in table 2, the following may be inferred: (i) an individual wasp is able
to recognize its own prey from that of others, (ii) with increasing energy cost of spider
deposition and hence food provisioning, a higher percentage of the wasps makes a
greater effort to continue and to complete the process of spider deposition, and seal the
hole, (iii) the wasp was capable of doing addition and its memory lasted atleast for one
day and (iv) the wasp was not capable of realising the removal of spiders from the hole.
Briefly, greater the energy cost of providing food for its larva, greater is the effort by the
wasp to successfully complete oviposition and provision of food for its larva. Energetics
of oviposition behaviour is a woefully neglected area and requires immediate attention
atleast for those pests, which are being considered for biological control.
References
Dade HA 1977 Anatomy and dissection of the honey bee; Int. Bee. Res. Assoc. Bucks, U.K. p. 281
Heinrich B 1972a Temperature regulation in the bumble bee Bombus vagans: a field study; Science 175
185-187
Heinrich B 1972b Energetics of temperature regulation and foraging in a bumble bee Bombus terricola Kirby;
J. Comp. Physiol 77 49-64
Heinrich B 1974 Thermoregulation in endothermic insects; Science 185 747-756
Heinrich B 1979 Bumble bee economics (Cambridge: Harvard University Press) p. 245
Heinrich B and Bartholomew G A 1971 An analysf$-of pre-flight warm-up in the sphinx moth Manduca sexta;
J. Exp. Biol. 55 223-239
Heinrich B and Raven P H 1972 Energetics and pollination ecology; Science 176 597-602
Kammer A E and Heinrich B 1978 Insect flight metabolism; Adv. Insect Physiol. 13 133-228
Lawton J 1973 The energy cost of food gathering. In Resources and population (ed.) B Bayamin, P R Cox and J
Peel (London: Academic Press) pp. 59-76
Marian M P, Pandian T J and Muthukrishnan J 1 982 Energy balance in Sceliphron violaceum (Hymenoptera)
and use of meconium weight as an index of bioenergetics components; Oecologia (Bert) 55 264-267
Muthukrishnan J and Pandian T J 1983 Prediction of bioenergetics components of lepidopterous larva;
Prqc. Indian Acad. Sci. (Anim. Sci.) 92 361-367
Muthukrishnan J and Senthamilselvan M 1985 Oviposition energetics of some wasps: an analysis of
behaviour (under preparation)
Nachtigall W 1966 Die Kinematik der schlagfliigelbewegungen von Dipteran; Z. Vergl Physiol. 52 155-21 1
Pandian T J and Marian M P 1985 Oviposition energetics of Sceliphron violaceum: an analysis of behaviour;
(under preparation)
Pringle J W S 1974 Locomotion: flight. In The physiology ofinsecta (ed.) M Rockstein (New York: Academic
Press) Vol. 3 433-476
Saunders D S 1976 Insect clocks (New York: Pergamon Press)
Schaffer W M, Jensen J B, Hobbs D E, Gurevitch J, Todd J R and Schaffer V M 1979 Competition, foraging
energetics and the cost of sociality in three species of bees; Ecology 60 976-987
Scriber J M and Slansky F J 1981 The nutritional ecology of immature insects; Ann. Rev. Entomol 26 183-21 1
Southwick E E and Pimentel D 1981 Energy efficiency of honey production by bees; Bioscience 31 730-732
Tucker V A 1970 Energetic cost of locomotion in animals; Comp. Biochem. Physiol. 34 841-846
Waldbauer G P 1968 The consumption and utilization of food by insects; Adv. Insect. Physiol. 5 229-288
Weis-Fogh T 1967 Respiration and tracheal ventilation in locusts and other flying insects; J. Exp. Biol. 41
561-587
Weis-Fogh T 1972 Energetics of hovering flight in hummungs birds and mDrosophila; J. Exp. Biol. 56 79-104
Witham T G 1977 Coevolution of foraging in Bombus and nectar dispensing in Chilopsis: a last dreg theory;
Science 197 593-595
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 225-238.
O Printed in India.
Behavioural analysis of feeding and reproduction in
haematophagous insects
R S PRASAD
Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695581, India
Abstract. The only common factor the haematophagous arthropods share among themsel-
ves is the blood sucking habit. This habit which ties them down to an unnatural assemblage,
confers on them certain parallelism even in their natural diversities. Behavioural activities of
haematophagous arthropods, like those of many other animals, centre around 3 major aspects:
searching for a suitable host and feeding on it; meeting of the sexes and finding a suitable place
for oviposition.
Behaviour of blood sucking insects assume importance because these insects act as vectors
of many blood-borne infections of man and animals. In this article, feeding and reproductive
behaviours of haematophagous insects are analysed on certain hierarchy of events like:
motivation; search and consummation.
Keywords. Haematophagous insects; behaviour; feeding; reproduction.
1. Introduction
Feeding and reproduction are largely interconnected performances. This would
become clear from the flow diagram (figure 1) which shows in a very general and
simplified way how a hungry, motivated insect searches for a host and after finding a
suitable one, takes a blood meal (consummation) leading to satiation. This further leads
to ecdysis and/or ovarian maturation and egg laying (in hemimetabolous insects like
bugs and lice which are blood feeders during all the stages of their life cycles, feeding
results in ecdysis during immature stages, while in adults, feeding results in egg laying).
This brings our insect back to the beginning of the cycle i.e., searching for a host. The
flow diagram is qualified as 'metabolic homeostasis' because the whole cycle deals
fundamentally with the energy flow into and within the arthropod (see Gelperin 197 la,
for the terminology).
Treating feeding and reproduction as separate entities would be an over simplifi-
cation. However, in the present context these are treated as independent performances
each having motivational activities leading to search of a host or a mating partner; sign
stimuli from a host or a mating partner which releases a behavioural reaction and finally
the consummatory act. In this article, analyses of both functions and mechanisms are
dealt together emphasising the influence of endogenous and exogenous stimuli.
Study of the feeding and breeding behaviours of haematophagous arthropods
assumes importance in two respects. While on the one hand the study is important for
its own sake, on the other it is of relevance from epizootiological/epidemiological and
disease or insect control angles. It is an established fact that many of the haemato-
phagous arthropods act as transmitters (vectors) of blood-borne pathogens of
vertebrates including humans. The type of cycle given in figure 1 becomes epizootio-
logically/epidemiologically involved, when it is repeated several times during the life of
225
226
R S Prasad
Phase I
Gul
Biochemical changes m j
internal milieu j
Salt & water content of i
haemolymph i •
Sense organs rectptiw to I
host odours j
Hunger / Thirst L
Locoinokit activity selectively
'-lir»''tf'-''l to ovipovtionui sity
r..:.:dy '".uf n,,'piuc
;;nd ,/ur
(•j(')ru.id:J*rnph!i.' cycles i
Initiation of
locomotor
activity
1
I
H. tivity
Phase II
Feeding drive
Host location
Probing
Sensing suitability of hlouci
Consumption
d'j not (C'Cdiptivf j
Figure 1. Metabolic homcostasis and its relationship to behaviour of hacmatophagous
insect.
a vector. Many of the factors such as: whether the vector is zoophilic or anthropophilic;
whether it rests outside the house or inside it; whether it is the 1st feed or subsequent
ones; whether the time lag between phase III and phase I (through phase IV) (see
figure 1) is sufficient for the growth and development of the pathogen in the vector and
so on and so forth, are important in deciding the vectorial status of a blood sucking
arthropod. A proper understanding of the behaviour of the vertebrate host, the vector
and the pathogen — the three elements involved in vector borne diseases- is a must for
effective vector/disease control operations. For example, effective vector or disease
control operations can be directed only if host preferences, resting habits, choice of
breeding areas etc., of vectors are known.
2. Feeding behaviour
In feeding behaviour the chain of events can be described as: motivation leads to the
search for a host and sign stimuli from the host help in host recognition; probing,
sampling of blood and sustained feeding which further leads to termination of feeding
(Chart 1).
Motivation
I
Search for Host
Sign stimuli j
Host recognition
Sampling Blood
Sustained feeding
Stretch receptors j, of abdomen to brain
Termination of feeding
Chart 1. Sequence of events in feeding behavi.
Behavioural analysis of blood-sucking insects 227
One of the most interesting yet poorly understood aspects of feeding behaviour is the
causes which motivate the insect to search for a blood meal (induction of biting drive).
Both external and internal factors can motivate the insect to feed. "The internal
motivational factors can either be endocrinal or nervous. Nervous excitation may have
its origin in excitation of enteroreceptors sensitive to the internal conditions of the
animal and /or in spontaneous activity of nervous centres" (Markl 1974). Only limited
studies have been made on the biochemical changes of the haemolymph which induce
feeding drive. Garcia (1959) has presented a unique observation, which states that a
mosquito seeks a blood meal to satiate the lowering of the levels of serotonin and
norepinephrine in its haemolymph. Experiments on blood avidity of mosquitoes have
shown that presence of developing oocytes inhibit biting (Khan and Maibach 1970;
Edman et al 1975). However Armstrong (1968) suggested that ovaries have a secondary
role in the control of feeding activity in that they remove protein from the haemolymph;
the primary control is the protein reserves (as measured by the amino acid level) and
high energy phosphate bonds (AMP). He showed that when the total free amino acid
level in the haemolymph was raised above that found in the unfed female there was no
feeding. He also suggested that there exists a system (probably neuroendocrine in
nature) to measure amino acid level and high energy phosphate bond availability and to
regulate feeding activity. Such a correlation between the haemolymph protein
concentration and blood avidity was detected in rat fleas Xenopsylla cheopis and
X. astia also. Injection of 50 % bovine albumin into the haemocoel of fleas reduced
blood intake of teneral females, but injection of 10 % albumin was sufficient to cause a
reduction in blood intake of 7-day old female fleas (Geetha Devi and Prasad 1980). The
concentration of haemolymph protein of 7-day old fed female fleas was found to be at
least two times greater than that of newly emerged unfed female fleas (Narayana Pillai
1983). Klowden (1981) suggested the involvement of 2 humoral factors in the inhibition
of host-seeking behaviour of gravid Aedes aegypti. The first factor produced by the
ovaries which causes the release of a second, from another site (site to be identified),
which is responsible for the inhibition of host seeking. Involvement of 20-hydro-
xyecdysone in the development of host-seeking inhibition in Anopheles freeborni was
proposed by Beach (1979). But Klowden (1981) does not believe the substance involved
in host-seeking inhibition in A. aegypti to be ecdysone. Meola and Petralia (1980)
suggested juvenile hormone to be involved in post emergence development of biting in
Culexpipiens and Cx. quinquefasciatus. Though in most cases biting drive is directly
related to gonotrophic cycle, in certain diapausing mosquitoes a process called
'gonotrophic dissociation' results in no egg development and laying eventhough the
mosquito continues to feed (Washino 1977). Maybe in such cases the reserves built up
in the haemolymph as a result of blood meal are channelised for build up of fat body
reserves rather than for oocyte maturation. The end result in both cases, as one should
guess, would be the depletion of reserves from the haemolymph. The actual link
between the haemolymph changes and the endocrinal or nervous controlling
mechanism still remains to be fully elucidated in haematophagous insects. Extensive
work done on the feeding behaviour of the blowfly Phormia regina has thrown light on
the various internal components controlling feeding drive. The components involved
are identified as: the locomotor centre of the thoracic ganglion and the stretch receptors
of the foregut and abdomen. The foregut stretch receptors monitor peristalisis of a
restricted region of the foregut (and so control gut filling) whereas the abdominal
stretch receptors monitor crop volume. The activity of these receptors regulates
228 R S Prasad
centrally the threshold of acceptability for food by setting taste threshold. The
locomotor centre of the thoracic ganglion which inhibit feeding is under the control of a
humoral factor from the corpus cadiacum, which in turn is controlled by the stretch
receptors monitoring foregut filling. Food uptake in Phormia can only begin when the
internal inhibitory stimulus ceases (Dethier and Bodenstein 1958; Dethier and Gelperin
1967; Gelperin 1966a,b; 1967; 1971a,b; Gelperin and Dethier 1967; Getting 1971;
Getting and Steinhardt 1972; Green 1964as b; Nunez 1964; Omand 1971).
Motivation is followed by search for a host which may be called 'appetitive
behaviour*. This may use simple or complicated stereotyped movement patterns
together with release controlling mechanisms. It is possible to categorise haemato-
phagous insects into three groups (i) those that live as permanent ectoparasites on their
hosts (e.g. lice), (ii) those that live in close quarters of the host and visit them frequently
to obtain blood meal (e.g. fleas) and (iii) those that are free-living in one sense of the
term, yet are dependent on the host for blood meal and visit the host only occasionally
as and when the need for a blood meal arises (e.g. mosquitoes). Sensory equipment that
aid in distant perception of host show maximum development in the third group and
the least in the first. The cues that can release a 'host-attacking reaction' with reference
to haematophagous arthropods are: host specific odours; non-specific or group factors
like CO2, convection currents, water vapour etc., and visual cues. Visual cues are
important for day-biting insects (Gatehouse 1972; Gillies 1972). As a matter of fact
Gatehouse (1972) found that when tsetse flies were not presented with any visual
stimulus, their response to calf odours was low. Convection currents of IR emanations
from the body of the warm blooded vertebrate would be important for close range host
detection. Non-specific factors are universal features of all vertebrates, but quantity
of CO2 given by large and small-sized hosts would vary. In general it may be stated that
odours provide the best information possible about the host at all ranges (Gillies 1 972).
The insect recognises the presence of the host when it gets into a 'host-stream' (the
plume of host-conditioned air drafting downwind from the host) during its random
dispersal flight from the breeding areas. Once they are in the host-stream, the flight
becomes oriented towards the host (Daykin el al 1965; Gillies and Wilkes 1969). The
efficacy of various substances — CO2, water vapour, fatty acids and derivatives,
ammonia, amines, amino acids etc. — have been tested on mosquitoes for release of
host-attack reaction (Brown 1966). CO2 activates the insect to fly while water vapour
and warmth induce target orientation (Kellogg and Wright 1962). As mentioned earlier
these three stimuli emanate from all warm blooded vertebrates and so would be non-
specific. Then what causes specific host selection? Discrimination lies, it appears, with
fatty acids and their derivatives. Among fatty acids, lactic acid plays an important role
and this was shown to be an attractant of female Ae. aegypti in the laboratory by Acree et
al (1968) and Muller (1968). The degree of attractiveness of mosquitoes varies with the
quantity of lactic acid secreted by each individual host. Three types of olfactory
sensillae are recognised in mosquito antennae; the sharp trichoid (type Al), short blunt
trichoid (type A2) and smaller, thorn-like basiconic (type A3). Lacher (1967) showed
that the type Al sensillae of Ae. aegypti are sensitive to fatty acids but essential oils
depress their activity. A2 are excited by higher fatty acids but are depressed by lower
fatty acids. According to him the type Al sensillae should be considered as odour
specialists. Once the insect has located and settled on the host, it starts probing for
blood. Several phagostimulants have been discovered in the blood of vertebrates which
trigger sustained feeding. Many nucleotides and amino acids have been shown to act as
Behavioural analysis of blood-sucking insects 229
phagostimulants for mosquitoes, tsetse flies, Rhodnius, fleas and ticks (Friend 1965;
Friend and Smith 1971, 1975, 1977, 1982; Galun 1966, 1967; Galun and Kindler 1968;
Galun and Margalit 1969, 1970; Galun and Rice 1971; Galun et al 1963, 1969; Hosoi
1958, 1959). Contact chemoreceptors are of importance in sampling the blood meal.
These are distributed on the tarsi, mouth parts and cibarium. Electrophysiologically
these contact chemoreceptors are little known. A review on the chemoreceptors of
haematophagous insects is presented by Lewis (1972).
Stretch receptors of the gut play an important role in termination of feeding
(Klowden and Lea 1979). Messages sent to the brain from stretch receptors of the
abdomen via the ventral nerve cord would tell the insect when to stop feeding (Maddrell
1963; Jones 1978). Gwadz (1969) showed that hyperphagy can be induced in mosquito
by cutting the ventral nerve cord in the 2nd abdominal segment. Maybe termination of
feeding is also effected at a critical level of back pressure from the abdomen as suggested
by Bennet-Clark (1963) in Rhodnius. Hyperphagia was shown to be induced in the
blowfly by transecting the recurrent nerve posterior to the brain (Dethier and
Bodenstein 1958; Dethier and Gelperin 1967; Green 1964b).
3. Reproductive behaviour
It is possible to describe reproductive behaviour also in the same sequence of events as
described for feeding behaviour (Chart 2).
A sexually motivated insect searches for a receptive partner leading to mating
(consummation); feeding (may or may not precede mating) and oviposition. As to
motivation, endogenous factors motivating the insect to search for a mate are not fully
understood. As suggested by Anderson (1974) they may include; (i) prior feeding on
carbohydrate/protein; (ii) maturation of sperm in the male and partial or full
maturation of primary follicles after feeding and the resulting receptivity of the female
and (iii) secretion of specific hormones. In any event sexual maturity and the
reproductive physiology of the two sexes must be synchronised.
Excepting for the cases where parthenogenesis is seen (lice, Bovicola bovis; certain
mites and certain strains of tick spp.) all other haematophagous insects are bisexual and
the sexes are to meet somewhere. With regard to haematophagous insects which live on
the host itself or in the near vicinity of its nesting quarters (in other words, those like
lice, fleas and pupipara, where the individuals of a population are not widely scattered
as in mosquitoes or other free-flying blood-sucking diptera) meeting of the sexes offers
little problem. In the flea Ceratophyllus gallinae, for example, an accidental collision
Motivation
I
Search for partner
J
Partner recognition
Mating
Matting may or
may not precede
feeding
Feeding
Chart 2. Sequence of events in reproductive behaviour,
230 R S Prasad
between the male and the female results in mating (Humphries 1967), whereas in
dipterous insects an aggregation or assembly of males called swarming into which
females occasionally enter serves as one of the means of meeting of the sexes. In
swarming, the assembly typically builds up over a visual land mark which is recognised
by both sexes (Downes 1955; 1958a, b; 1969) or males wait in a zone which is likely tp be
traversed by females. For example, above a food source as some simuliids the males of
which swarm over cattle and catch the females as they come to feed (Wenk 1965). Not
that all diptera swarm before mating. An unusual mating system among mosquitoes is
that of Deinocerites cancer. Adult male of D. cancer walk over water and locate female
pupae and stand guard over it and mate with the female as soon as it emerges. The
antennae are used to locate female pupae. Pupal attendance in D. cancer may be
triggered by chemical cues emanating from the pupa (Conner and Itagaki 1984). The
only other mosquito which shows pupal attendance is Opifexfuscus, but this has not
evolved the ability to distinguish the sex of the pupa as D. cancer has. Such species of
mosquitoes which do not swarm have lost the long hairs with auditory function which
characterise the male antennae of most species of mosquitoes (Clements 1963). It was
originally thought that the females in a swarm are recognised in close range by their
wing sound in many culicids, but researches have shown the presence of contact
pheromone which are active only over short distances (a few centimeters). Such
pheromones are thought to be involved in sex recognition ofCuliseta inornata (Kliewer
et al 1966), Culex tarsalis, C. pipiens and C. quinquefasciatus (Gjullin et al 1967); Aedes
aegypti, Ae. mascarensis, Ae. albopictus and Ae. polynesiyensis (Nijhout and Craig 1 97 1 ).
Presence of contact pheromones active over short distances have also been suggested in
Ceratophyllus gallinae (Humphries 1967); Rhodnius prolixus (Baldwin et al 1971);
Arnblyomma americanum, A. maculatum and Dermacentor variabilis (Berger 1972;
Berger et al 1971; Chow et al 1975; Gladney 1971).
Mating may take place exclusively on the host; on and off the host or exclusively off
the host depending on the blood sucking insect's life style. For example, anoplura and
pupipara which are permanent residents on the host, mate exclusively on the host,
whereas mating of free-flying forms like mosquitoes and bugs takes place off the host.
Strain variations are seen in laboratory colonies of mosquitoes with regard to their
requirement of space for mating. Eurygamous strains require large space for mating
because copulation is initiated in flight, while males of stenogamous strains will
approach and mate with resting females and can do so in confined areas such as a small
cage or a test tube.
In most cases of haematophagous diptera (Nematocera and Brachycera) the
hierarchy of behavioural responses would be: host seeking, mating and blood feeding.
But in certain cases blood feeding may precede mating (Teesdale 1955). Females of
most Cyclorrapha take a blood meal before mating. Similar variations are also seen in
fleas, ticks and bugs. In some cases like ticks, mating may take place while the female is
feeding.
Oviposition behaviour is rhythmic in nature and finds a correlation with feeding
cycle. In the case of permanent ectoparasites like lice which do not leave the body of the
host and all the stages are blood suckers, eggs are laid on the body of the host itself and
eggs are cemented to the hair /feather. In those like fleas, eggs are laid in the nest of the
host. In the event of laying while the flea is on the host, eggs will roll down into the nest
of the host and would not stick to the body of the host as in the case of lice. Free-flying
dipterous blood-sucking insects lay off the host in sites suitable for the development of
Behavioural analysis of blood-sucking insects 231
their immature stages. Oviposition site preference is a ritual in most mosquitoes. Two
phases may be recognised — a general reaction to the environment and final selection.
Site selection not only involves "finding the water and laying eggs, but also selection of
environment, whether shaded or open, stream, rice field, pond, tree hole or artificial
container, whether water is moving or still, polluted, saline or fresh" (Clements 1963).
Vision and chemoreceptors of the legs are thought to play important roles in site
selection for oviposition in these cases. Some mosquitoes even 'taste' the water to assess
suitability.
4. Rhythms in adult activities
Locomotor activity is normally accompanied by other behavioural manifestations like
feeding, mate seeking and oviposition. All these behavioural manifestations are known
to be rhythmic in nature. In addition to these, pupation, adult emergence, erections of
antennal fibrillae in the males of some spp of mosquitoes; the start of migration of some
spp., of mosquitoes, nectar feeding and unspecific flight activities are all known to be
rhythmic. These rhythms, as typical of biological rhythms, are entrained by or
synchronised to a cyclical environmental cue called Zeitgeber.
The diel pattern of adult emergence of a given species tends to be correlated with the
locomotory activity rhythms and the reproductive behavioural pattern of that species
(Beck 1968). Adult emergence rhythms depicts a synchrony of developmental processes
among individuals belonging to a population. This synchrony, possibly has a basis in
oviposition rhythms. As has been mentioned before, adult emergence is closely
followed by swarming and finding a mate in diptera on which extensive research has
been made. Swarming is a circadian endogenous phenomenon, entrained by environ-
mental photoperiod. Light intensity plays a role of releaser of swarming habit, though
temperature, wind etc., do play important roles. Swarms were induced only when
transition from light to dark (or vice versa) was gradual. The possible role of visual
adaptation in swarming of C. tarsalis was shown by Harwood (1964). He used 'eye
index1 — defined as the average cornea diameter: iris diameter ratio — to study visual
adaptation. According to him fully light adapted mosquito has an eye index of 21
whereas in fully dark adapted ones it was only 4 and swarming occurred when the eye
index reached 5.
One of the few rhythmic biochemical changes studied in mosquitoes is the synthesis
of glycogen in Cx. pipiens (Takahashi and Harwood 1964). Peak glycogen level is seen
towards the end of photophase and this gets depleted as the insect becomes active
during the scotophase. Photoperiod has some influence on the synthesis of glycogen
which in turn is under the control of neurosecretory system of the insect (Handel and
Lea 1965).
Both embryonic and adult diapause have been described in mosquitoes. The former
is caused by the photoperiod experienced by the parental generation rather than the
eggs themselves with one exception of the case of Ae. triseriatus in which case diapause
occurred in response to short-day photoperiods experienced by the embryos from 5 to
8 days after egg deposition (Kappus 1965). With regard to larval diapause, the studies
on An. barberi and Ae. triseriatus showed that short-day photoperiods induced diapause
and long-day photoperiods terminated it. Photoperiod sensitive stages varies widely
among different species. Larval diapause may be determined in most cases by the
232 R S Prasad
photoperiod to which the larval stages have been exposed but in the case of Ac.
triseriatus, larval diapause appeared to be determined by the photoperiod experienced
by the parental generation (Love and Whelchel 1955). Similarly, pupal diapause was
determined in response to short-day photoperiods experienced by the female
progenitor (Depner 1962).
In-depth studies have been made on feeding rhythms of mosquitoes but not in other
haematophagous insects. Each mosquito species has a preferred feeding time and only
at such times would it become receptive to host odours. This is true for both day-biting
and night-biting mosquitoes. Feeding and egg laying are correlated. For e.g., Ae. aegypti
which shows a feeding peak late in the afternoon and egg laying peak almost at the same
time 3 days after. Inborn endogenous 24 hr rhythms of activity and rest which use onset
of darkness as?an external time-cue for the timing of the cycle have been detected in
mosquitoes. Such circadian rhythms not only serves to brief the mosquito on its take-
off time, but determines the duration of flight period keeping it within the normal fuel
range. Temperature, light and wind can inhibit activities normally controlled by
circadian rhythms, but very little is known about the interaction of climatic factors and
endogenous rhythms in determining the population activity. Most behavioural
patterns require releasing stimuli from the environment. Light intensity may be such a
releaser for mosquito biting behaviour, although other exogenous stimuli perhaps
host-borne may also be involved. Photoperiodic entrainment may play a role in
determining the responsiveness of the insect to the releasing stimulus.
Another synchronised rhythmic behaviour between the reproduction of the host
rabbit and its flea ectoparasite Spilopsyllus cuniculi was described by Rothschild (1965).
The flea starts its reproductive activities only when the host becomes pregnant and the
level of ACTH in the blood rises. Mating of these fleas takes place on the newly born
young rabbits. Thus there is a rhythmic synchronisation of the breeding of rabbits and
that of their flea ectoparasites.
5. Behavioural genetics
In general it may be said that genetics of behaviour of haematophagous arthropods is a
very poorly studied aspect. Mattingly (1962) states that "despite its great importance,
this difficult field is one of the most neglected in contemporary biology". Mattingly
(1967) draws out the few studies carried out under genetics of: mating behaviour; host
choice; irritability and photoperiodism; oviposition behaviour and environmental
selection.
The recessive autosomal gene causing yellow eye in Ae. aegypti enhances mating
efficiency (Adhami and Craig 1965). Tate and Vincent (1936) found stenogamy ex-
pressed in F1 progeny of cross between eurygamous Cx. pipiens and stenogamous Cx. p.
var. molestus and this was maintained in the subsequent generations also. Bates (1941)
showed heritable changes in swarming behaviour of hybrids between members of the
An. maculipennis complex. Mating vigour is another aspect on which some studies have
been made among members of Cx. p. complex and found marked differences (Rozeboom
and Gilford 1954; Parker and Rozeboom 1960). Gillies (1964) was able to select distinct
zoophilic and anthropophilic strains of An. gambiae. Heritability of enhanced irritability
and increased positive phototropism of mosquitoes under insecticidal pressure was
demonstrated by Gerold and Laarman (1964) in An. atroparvus in the laboratory. Wood
Behavioural analysis of blood-sucking insects 233
(1961, 1962) was able to show a possible genetic variation in oviposition site preference
and variation in the length of time elapsing between the blood meal and oviposition
among different strains of Ae. aegypti. Strain variations have also been reported in Ae.
aegypti with regard to their requirement of mating for egg laying (Gillett 1955, 1956).
6. Evolution of haeraafophagy
Haematophagy is exhibited by Ixodidae (hard ticks), Argasidae (soft ticks) and a few
families of Mesostigmatid mites among Acarina; Anoplura, Thysanoptera and
Hemiptera among the Hemimetabola; Lepidoptera, Diptera and Siphonaptera (all the
three belonging to the Panorpoid complex-Hinton 1958) among the Holometabola
(Hocking 1971). Blood sucking behaviour can probably be traced back to the Permian
when land vertebrates emerged. Hoogstraal (1965) has concluded that ticks have arisen
in association with reptiles in the late Paleozoic or early Mesozoic. By Triassic
Psychodiform, Culiciform and Tabaniform have been well differentiated and insects in
a form nearest to Psychodiform appear during the Permian (table 1) and these were
contemporaries of Theromorpha — the warm blooded reptiles (Downes 1971).
This would mean: (a) that blood-sucking habit originated much before the
emergence of mammals as an ecologically important group; and that (b) the blood
sucking forms had the whole lot of land arthropods and vertebrates to choose as hosts.
Feeding on poikilotherms is still a habit noticed among many of the blood sucking
insects and arachnids. Hocking (1971) states that "the most readily available blood
initially was probably arthropod blood, that many different groups today feed on this
suggests that it may have been an early development". There is even a report of Aedes
mosquitoes feeding on mantis and laying viable eggs in the laboratory (Mathews and
Mathews 1978). Hocking (1971) concludes that "in the Acarina and more or less
contemporaneously in the Hemimetabola and Holometabola, trends toward fluid
feeding seem to have developed. It may be supposed in each instance the initial
adaptation was to feeding on exposed fluids, but that as plants and animals generally
became better adapted to life on really dry land, by developing substantially
impermeable skins and cuticles, a taste for nutrient fluids demanded a combination of
suction with cutting or penetration". Among diptera a dichotomy exists in feeding
habits i.e., sugar meal t;s blood meal. Evidences strongly support the assumption that
blood sucking at least in most cases is a secondary development. This duality of feeding
habit has a foundation in Mecoptera — the probable ancestors of Diptera and
Siphonaptera. But insects like Siphonaptera, which are more advanced in parasitic
habits, are exclusive blood suckers. Animal tissue feeding appears to have evolved
independently in different groups through four routes. In mites, lice and fleas this could
have evolved through lair or nest associations. Scavenging on debris within lairs,
burrows or nests of vertebrates could have developed into feeding on blood. For
example psocids which are found in large numbers on animal habitations. Hopkins
(1949) believes that lice were derived from psocid-iike ancestors. Fleas in their larval
stages, still continue to be scavengers, in the nests of rodents, feeding on debris. A
second possible mode would be predation. Blood sucking triatomine bugs illustrate
how predatory habit could have developed into blood feeding on vertebrates. A similar
situation may be seen in the dipteran family Rhagionidae. Some of them are blood
suckers, attacking mammals, while some others are predaceous on insects. It may be
234 R S Prasad
Table 1. Appearance of blood sucking arthropods in relation to their host.
Era
Period
Dominant life
Blood sucking
forms
Pleistocene
Cenozoic Tertiary
(60)
Pliocene
Miocene
Oligocene
Eocene
Paleocene Mammals
Late (120) Cretaceous
Jurassic
All the main
1
Early (155)
Insect orders
N
Early (155)
known today except
S
Lepidoptera
Triassic
Birds and flying
Psychodifonn
reptiles
Culiciform
Tabanifonn
Permian
Theromorpha
Forms nearest to
Late (215)
Land vertebrates
Psychodiform.
Carboniferous
First Insects
Ticks
U
Middle
Devonian
Arthropoda, land
a
o
(350)
plants, amphibia
3
Silurian
brachiopoda,
&
scorpions
Early
(550)
Ordovician
Cambrian
First fish
Mollusca,
Trilobite
noticed that predation in the cases cited consists of feeding on liquid contents of the
prey rather than consuming the whole prey. Feeding on proteinaceous secretions could
as a further step lead to haematophagy. The feeding habits of the eye frequenting
lepidoptera Arcyophora and Lobocraspis spp. which feed on the lachrymal secretion of
cattle, sambar and other large mammals are examples to this. Secondary
haematophagy-feeding on blood oozing out from wound — could be a fourth route to
development of more purposive blood sucking habit (James and Harwood 1969).
7. Conclusion
In conclusion it may be stated that the little that is done on the behaviour of
haematophagous insects centre round mosquitoes, leaving out the several other groups
practically untouched.
Factors which induce feeding drive in the majority of haematophagous arthropods
still remain unknown. Host and oviposition site preferences need further attention. It is
Behavioural analysis of blood-sucking insects 235
known that kairomones emanating from the water bodies attract mosquitoes to reach a
suitable oviposition site. It is also known that micro-organisms especially bacteria have
a role to play in the production of such attractants. The feasibility of altering sites
suitable for egg laying by genetic or other manipulations of the bacterial flora has not
yet been experimented.
Very few studies have been made on human influence on behaviour of haeinato-
phagous arthropods, an aspect which has importance from epidemiological angle. One
of the few studies made show that exophilism and exophagy are induced among
mosquitoes as a result of spraying insecticides in houses. As a result of spraying the
walls, endophilous mosquitoes seldom remain on the walls inside houses and show
positive phototropism. Such an induced exophilism would result in deviation to
alternate host especially cattle. This would bring in a dangerous situation with regard to
zoonotic diseases (Trapido 1952).
Much emphasis need be laid on genetics of behaviour especially those of host
selection, feeding (exophagy, and endophagy), mating, oviposition site selection and
such other behavioural aspects. The full impact of gene manipulations on the above
mentioned behavioural aspects is yet to be fully explored. With the development of gene
splicing and gene transfer technologies several alterations at population levels can be
thought of.
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C) Printed in India.
Feeding and ovipositional behaviour in some reduviids (Insecta-
Heteroptera)
E T HARIDASS
Entomology Research Institute, Loyola College, Madras 600034, India
Abstract. Feeding behavioural studies of many exclusively predatory species exhibit clearcut
stimuli-response mediated sequences and these can be categorised into distinct sub-units like:
search and location of prey -> approach and attack of prey -* immobilisation of prey
-+ transportation of prey to safe place -*• consumption of prey. These feeding behavioural
activities differ among reduviids particularly with respect to prey types. These bugs are
endowed with many structural, physiological and behavioural adaptations for efficient
predation.
The ovipositional behaviour of reduviids in different habitats also shows considerable
variation and their reproductive strategies include selection of suitable sites to assure
successful emergence and development of young ones and so far very few egg predators and
egg parasites have been reported for these terrestrial insects.
Keywords. Feeding behaviour; ovipositional behaviour; reduviids.
1. Introduction
The family Reduviidae is one of the terrestrial groups of bugs well represented in
tropical and subtropical regions of the world. They are known to colonise a wide variety
of habitats, such as from under stones, on low herbage or lower foliage or on trees, to
the most unusual ones like ant-hills, termitaria, .cobwebs, bird nests, rat holes and
human dwellings. Naturally, they exhibit a wide range of structural, physiological and
behavioural adaptations for an exclusive predatory habit, feeding on a variety of
arthropods, including millipedes, termites, bugs, beetles, caterpillars, ants, bees etc.
Members of the Triatominae alone have specialised for haematophagy , engorging the
blood of birds and mammals.
2. Feeding behaviour
Feeding behaviour of reduviids, as it is true for several other predatory insects, shows
many distinct events, and these stimuli-response mediated sequences can be
conveniently divided into: location of prey -» approach and pounce on prey
-* immobilisation of prey -+ transportation of prey to safe place -> feeding.
2.1 Location
Being exclusive predators, visual stimuli appear to be of primary importance to these
and with the possession of well-developed compound eyes and ocelli they easily locate
239
240 E T Haridass
and capture various prey types. Visual stimuli from the moving prey initiate subsequent
predator-prey interactions. The importance of visual stimuli for predatory reduviids
has been well documented (Odhiambo 1958a; Edwards 1962; Parker 1969, 1971, 1972;
Livingstone and Ambrose 1978a; Louis 1974; Haridass and Ananthakrishnan 1980a).
Species like Haematorrhophus nigroviolaceus (Reuter), Guionius nigripennis (Fabr.) and
Ectrychotes pilicornis (Fabr.) as well as other members of the subfamily Ectrichodiinae
feed exclusively on millipedes (Cachan 1952; Miller 1971; Haridass 1978) and these
predators are aroused from a state of akinesis only after receiving the stimuli from the
moving millipedes. Even artificial baits like paper, rolled like millipedes, or dead and
dried millipedes, also initiated feeding responses in them. Artificial objects or dead
bodies of their prey dragged in front of piratine species like Pirates affinis Serville,
Ectomocoris tibialis Distant, £. ochropterus Stal, and Catamiarus brevipennis Serville,
elicited similar responses. Arousal of feeding responses by optic stimuli has also been
noticed in several other reduviids, including termite feeding Rhaphidosoma atkinsoni
Bergroth (figure IF) and ant feeding Acanthaspis pedestris, Stal (figure ID) A. siva
Distant, and caterpillar feeding Sycanus collaris Fabr (figure IE), Rhinocoris
marginatus Fabr and Sphedanolestes rubicola Distant. Exceptions to this are the
members of Triatominae, majority of which have taken to haematophagy and in forms
like Triatoma rubrofasciata De geer (figures 1G, H), Linschcosteus costalis Ghouri
(Haridass 1978; Haridass and Ananthakrishnan 1980a) and Rhodnius prolixus Stal
(Friend and Smith 1977), it is the temperature gradient from the vertebrate hosts that
aroused the starved insects. Blinding of their eyes does not deter them from locating
their correct hosts and in these haematophagous insects the antenna are the primary
sense organs.
2.2 Approach and pounce on prey
Successful location is followed by the quick approach to the prey located and pouncing
on them for subsequent immobilisation. Several species exhibit a definite preference for
a particular prey type. Ectrichidiinae show a preference for spirostreptid millipedes
(figure 1A) and never attempt predation on polydesmid species. Similarly P. affinis
prefers carabid Omphora pilosa Klug and 0. atrata Klug than any other ground beetles
and £. tibialis and E. ochropterus prefer gryllids to any other insects (figure 1C). Fast
runners like Piratinae, Ectrichodiinae, Acanthaspidinae, Reduviinae etc., quickly reach
the prey and pounce on them gripping them tightly with tibial pads (figures 1 A, B). The
presence of tibial pads on the fore- and/or mid-tibiae is a characteristic feature of this
family (Gillette and Wigglesworth 1932; Miller 1942; Edwards 1962; Bahadur 1963;
Haridass and Ananthakrishnan 1980b). These tibial pads with their oil secreting tenet
hairs, enable the predators to increase the gripping efficiency during prey capture. The
tibial pads enable the insects to withstand static tension, on rough and smooth surfaces
to an extent of 20-27 g (Haridass and Ananthakrishnan 1980b). In contrast to the fast
running habits, members of Harpactorinae, Emesinae, and Rhaphidosomatinae exhibit
slow gait and use the long legs and rostrum to reach the prey and gripping of the latter is
never involved (figures 1G, H). Some reduviids like the species of Apiomerinae and
Ectinoderinae make use of the resin coated legs to capture prey like fly-paper (Miller
1971). The fore-legs of the thread-legged emesine bugs are also raptorial (Wygodzinsky
1966). The camouflaging of the body surfaces with particles of mud and debris as seen in
Feeding and ovipositional behaviour in some re
Figure 1. Feeding behaviour in some reduviids. A. Hacmai^t r h* »| t*t** t „
on millipede prey. B. Pirates affinis (5th nymph) immobilism? 1*11 .»?>)«! *»r -
tibialis feeding the gryllid prey. E>. Acanthaspis pedcstn\ frvtfiiij* *! -
E. Sycanus collaris dragging the immobilised caterpillar pic\ I Kn* s
with termite worker (arrow) hanging from the rostral up <„. /TJ¥ m/
orienting towards the hand of a sleeping person. H. /ritjium*. n*f » *
engorging blood from the body of a dog.
< I, m
many Acanthaspidinae and Triatominae (Odhiambo 195Hb; I.ivmtrsi.M».-.uul \H)b».-,.-
1978a,b;Zeledon etal 1973), the cryptic body colouration and nnniu k n,F dr > i» u-, ud
grass stems as m Rhaphidosomatinae and Emesinae are some v>t the »,-.hn .ul-irsa^.-r ,
of this family for efficient predation.
2.3 Immobilisation of prey
After prey capture, the behaviour of these bugs is to search for a .„„., Wr ,!r ,,.r ,,v !,,
insertion and injection of toxic salivary secretions The
Sly,e«s are usually ,«d in ,he pleura, membrane o
242 E T Haridass
the anterior legs or at the antennal bases (Haridass and Ananthakrishnan 1980a). When
small, like termites and young caterpillars, the prey is lifted off the ground by the rostral
tip of the predators to escape the violent encounters with the prey (figure IF). But in
most cases, involving larger prey species like carabid beetles, millipedes and caterpillars,
the predation always entails a violent reaction and following unsuccessful salivary
injection, the prey often escapes. Under these conditions, predation is attempted
quickly for a second time, invariably with success. Often in Ectrichodiinae, improper
gripping of the millipede always resulted in the entwinning of the prey around the
predator, and the latter allows itself to be dragged for long distances. It is noteworthy
that a majority of them, particularly those of Ectrichodiinae, Piratinae,
Acanthaspidinae and Reduviinae that encounter such violent reactions from their preys
are endowed with tough body coverings to withstand the rough treatment meted out to
them during predation. In all cases of successful insertion of stylets and injection of
salivary toxins, the prey including larger ones like millipedes, caterpillars and beetles,
become totally paralysed and killed within 20-30 sec. The caterpillar feeding
Harpactorinae immobilise and kill their prey by stabbing the stylets and by injecting the
saliva two to three times in quick succession.
The stylet structure of Reduviidae exhibit a wide range of modifications and
evolutionary progression, involving elaborate barbs, teeth and tubercles in their
mandibular and maxillary stylets (Cobben 1978). The salivary system of reduviids is
also very complex (Baptist 1941; Edwards 1961; Southwood 1955; Haridass 1978) and
the anterior lobes of the main glands are concerned with the secretion of neurotoxic
substances involved in the paralysis and death of the prey (Haridass and
Ananthakrishnan 198 Ib).
While succumbing to death, millipedes, caterpillars, beetles and ants secrete copious
secretions of obnoxious, and irritant exudations like formic acid, p-benzoquinones, and
other phenolic compounds (Eisner et al 1962, 1963; Roth and Eisner 1962). When such
repellants are secreted, the immobilisation of the prey is followed by a distinct
behaviour of the predators, where they spend considerable time in cleaning the body
and antennae, using the fore legs and by rubbing the body surfaces on the ground
(Haridass and Ananthakrishnan 198 la).
2.4 Transportation of prey
Transporting the immobilised prey to a safe place for consumption is yet another
distinct unit of the feeding behaviour. By inserting the stylets at suitable places, usually
at the bases of the mouth parts, or antennae or anterior legs, the prey is dragged beneath
the body as the predator walks, or by pulling the long bodied prey while the predators
move backwards (figure IE).
2.5 Feeding
Feeding on the immobilised prey is the last unit of the behavioural sequence and it lasts
for about l£ to 2 hr. Since the mouth parts are of the stylet-type, the predigested food
from the prey body is flushed out by the watery secretions of the accessory glands, while
the posterior lobes of the main glands secrete enzymes for the digestion of the prey
Feeding and ovipositional behaviour in some reduviids 243
(Miles 1972; Miles and Slowiak 1976; Haridass and Ananthakrishnan 1981b). The
salivary glands of haematophagous triatomine bugs secrete anticoagulants to facilitate
sucking of large quantity of vertebrate blood without the danger of the blood being
clotted (Baptist 1941; Haridass and Ananthakrishnan 1981a, c). While blood feeders
engorge from the same feeding site, the predatory forms manipulate the body of the
prey with the fore legs and change the feeding sites as and when a particular part of the
prey's body contents are emptied (figure 1 A) and in this fashion they completely suck
out everything leaving behind only the rectal regions and the empty exoskeleton.
Sharing of the food is not observed in any of the adults, though smaller nymphal
stages of Ectrichodiinae and Harpactorinae not only share the same millipede or the
caterpillar respectively, but also jointly attempt predation of larger prey. Cannibolism
is very prevalent in reduviids, the males often succumbing to females and small
nymphal stages falling victims to large ones. The feeding behaviour of Triatominae is
comparatively less complicated and the host location is by the temperature gradients
emanating from the vertebrate host (figure 1G). On locating a suitable feeding site,
considerable time is spent in probing with rostral tip and in the sampling of blood. The
mechano- and chemo-receptors of the rostrum are believed to get signals from
nucleotides of the host's blood about the suitability of food source (Pinet 1968; Bernard
et al 1970; Friend and Smith 1977). Once selection is made feeding continues from the
same site and terminates only after satiation, due to the stretching of the abdominal
stretch receptors affecting critical abdominal volume (Maddrell 1963; Anuzel 1972).
Nymphs of triatomine bugs also exhibit cannibalism, fully fed older nymphs are
attacked by younger ones, the latter pierce the swollen blood filled abdomen of the
latter and feed. Such victims do not suffer from any ill effects (Ryckman 1951; Haridass
and Ananthakrishnan 198 IcX
3. Ovipositional behaviour
A wide variety of habitats are colonised by reduviids and an important aspect of their
reproductive strategy is the selection of a suitable site for oviposition to ensure
successful emergence and development of young ones. Sexually mature females resort
to multiple matings, exhibiting either an-end-to-end or a riding type of copulatory
posture characteristic of several Heteroptera (figures 2 A, E, G, H and K). Ground
dwelling piratine species like P. affinis, E. tibialis, E. ochropterus, E. cordiger and C.
brevipennis, deposit their eggs in the soil (Readio 1926; Miller 1953, 1971), using the
plate-like ovipositors. The gravid female assumes a slanting posture with raised head
and thorax and with the apex of the abdomen alone touching the ground makes side-to-
side, twisting, and downward thrusting movements to insert one egg (figure 2D). The
exposed part of the egg (figure 2F) is then covered with small particles of sand and mud
by the manipulation of the hind legs. Ectrichodiinae like H . nigroviolaceous, Guionius
nigripennis and Ectrychotes pilicornis deposit their eggs in clusters. While the latter two
species glue the eggs in the crevices of bark of trees (figure 2C), the females of the former
dig slanting tunnels (7-8 cm) in the ground to deposit eggs loosely (figure 2B). After
oviposition they spend considerable time to refill the tunnel with excavated mud using
mid- and hind legs, and finally press the closed tunnel with the abdomen. Unlike the
plate-like ovipositors of the species that insert their eggs in the soil or in crevices, these
structures of Ectrichodiinae are very much reduced and stublike. Members of
244
E T Haridass
Figure 2. Ovipositional behaviour in some reduviids. A. Copulating adults of Haemator-
rhophus nigroviolaceus. B. Loose egg cluster of H. nigroviolaceus inside a tunnel excavated in
the ground. C. Guionius nigripennis attaching eggs to the crevices on tree trunk.
D. Ectomocoris tibialis inserting single egg into the ground. E. Copulating adults of
Catamiarus brevipennis. F. Oviposited egg of Pirates affinis. G. Copulating adults of P. affinis.
H. Copulating adults of Sycannus collaris. I. Compact egg mass of S. collaris glued to the
surface of tree trunk. J. Eggs of Rhaphidosoma atkinsoni attached to grass stem.
K, Copulating adults ofTriatoma rubrofasciata. L. Eggs of T. rubrofasciata inserted into the
crevices of dry cow-dune rak-p«
Feeding and ovipositional behaviour in some reduviids 245
Acanthaspidinae like Acanthaspis siva, A. pedestris, A. quinquispinosa (Fab.) and those
of Salyavatinae like Lizarda annulosa Stal and Petalochirus indicus as well as these of
Triatominae like T. rubrofasdata and L. costalis oviposit the spheroidal or sub-ovate
eggs loosely scattering them on the ground, under stones or in crevices (figure 2L).
Rhaphidosoma atkinsoni found usually among grasses, glue the bases of the flask shaped
eggs to the stems, the eggs projecting at an angle (figure 2J). This oviposition is very
similar to those already described for other Rhaphidosomatinae (Miller 1953),
The most elaborate ovipositional behaviour is exhibited by species of Harpactorinae
and this is known for a large number of forms (Kershaw 1909; Muller 1937; Cheriyan
and Kylasam 1939; Bose 1951; Wallace 1953; Miller 1953, 1971; Odhiambo 1959;
Edwards 1962, 1966; Parker 1969; Nyiira 1970; Swadner and Yonke 1973a,b). The
gravid females ofRhinocoris marginatus, Sycanus collaris and Sphedanolestes bowringi
Distant attach a cluster of large number of eggs on the under surface of big boulders, or
on the stems of trees and plants. While placing the eggs the females work from the
margins to the centre of the egg mass always in precise "chevron' pattern, gluing the eggs
in vertical but oblique rows. Each egg is attached to the substratum as well as to the
previously laid one giving a polygonal shape to the completed egg mass (figure 21). The
females cover such egg masses with copious secretions from their accessory glands
transforming the egg masses into almost an ootheca (Southwood 1956; Miller 1971;
Hinton 1981). Though parental care has been reported in many harpactorine bugs
(Bequart 1912; Odhiambo 1959; Parker 1965; Miller 1971; Ralston 1977) this has not
been noticed in any of the species observed.
The success of the ovipositional behaviour of Reduviidae is evident from the total
absence of egg predators for this group as well as from the fact that so far very few egg
parasites have been reported for reduviid eggs (Odhiambo 1959; Swadner and Yonke
1973a; Masner 1975; Sankaran and Nagaraja 1975).
References
Anuzel R 1972 The structure and properties of an abdominal receptor in Rhodnius prolixus stal (Hemiptera-
Reduviidae); J. Insect Physioi 18 2143-2153
Baptist B A 1941 The morphology and physiology of the salivary glands of Hemiptera-Heteroptera; Quart. J.
Micros. Sci. 83 91-139
Bahadur J 1963 Rectal pads in Heteroptera; Proc. R. Entomol. Soc. London A38 59-69
Bequert J 1912 L'instinct maternal chez Rhinocoris albopilosis Sign. Hemiptera- Reduviidae; Rev. Zooi Afr. I
293-296
Bernard J, Pinet J M and Boistel J 1970 Electrophysiologie and des recepteur des stylets maxillaire de
Triatoma infestans action de la temperature et de la teneur en eau de i'air; J. Insect Physioi. 16 21 57-2180
Bose M 1951 On the biology of Cor anus spiniscutis (Reuter), an assassin bug (Fam. Reduviidae); Indian J.
Entomol. 11 203-208
Cachan P 1952 Etude de la predation chez les reduviides de la region ethiopienne 1. La predation en groupe
chez Ectrichodia gigas H. Sch; Physioi. Comp. Oecol. 2 376-395
Cheriyan M C and Kylasam M S 1939 On the biology and feeding habits of Rhinocoris fucipes (Fab.)
(Heteroptera-Reduviidae); J. Bombay Nat. Hist. Soc. 61 256-259
Cobben R H 1978 Evolutionary trends in Heteroptera Part II. Mouthpart structures and feeding strategies;
Meded. Landbouwhoge sch., Wageningen pp. 407
Edwards J S 1961 Action and composition of the saliva of an assassin bug, Platymeris rhadhamanthus Gerst.;
J. Exp. Biol. 38 61-77
Edwards J S 1962 Observations on the development and predatory habits of two reduviid Heteroptera,
Rhinocoris carmelita Stal and Platymeris rhadhamanthus Gertst.; Proc. R. Entomol. Soc. London 37
89-98
246 ETHaridass
Edwards J S 1966
Gillette J D and Wigglesworth
sial
T N
89
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«*
A33
Feeding and ovipositional behaviour in some reduviids 247
Parker A H 1971 The predatory and reproductive behaviour of Vestula lineaticeps (sign)
(Hemiptera-Reduviidae); Bull. Entomol. Res. 61 119-124
Parker A H 1972 The predatory and sexual behaviour of Phonoctonus fasciatus (P et B) and P. subimpictus
Stal (Hemiptera-Reduviidae); Bull. Entomol. Res. 62 139-150
Pinet J M 1968 Donnees ultrastructure sur 1'innervation sensorille des stylets maxillaires des Rhodnius
prolixus Stal.; C. R. Acad. Sci. Paris 267 634-637
Ralston J S 1977 Egg guarding by male assassin bugs of the genus Zelus (Hemiptera-Reduviidae); Psyche 84
103-106
Readio A 1926 Studies on the eggs of some reduviids (Heteroptera); Univ. Kansas Sci. Bull. 16 157-179
Roth L M and Eisner T 1962 Chemical defences in arthropods; Ann. Rev. Entomol. 7 107-136
Ryckman R E 1951 Recent observations on the cannibalism in Triatoma (Hemiptera-Reduviidae); J.
Parasitol. 37 433-434
Sankaran T and Nagaraja H 1975 Observation on two sibling species of Gyron (Hymenoptera-Scelionidae)
Parasitic on Triatominae (Hemiptera) in India; Bull. Entomol. Res. 65 215-219
Southwood T R E 1955 The morphology of the salivary glands of terrestrial Heteroptera (Geocorisae) and its
bearing on classification; Tijdschr. Entomol. 98 77-84
Southwood T R E 1956 The structure of the eggs of terrestrial Heteroptera and its relationships to the
classification of other groups; Trans. R. Entomol. Soc. London 108 163-221
Swadner S O and Yonke T R 1973a Immature stages and biology of Apiomerus crassipes
(Hemiptera-Reduviidae); Ann. Entomol. Soc. Am. 66 188-196
Swadner S O and Yonke T R 1973b Immature stages and biology of Zelus socius (Hemiptera-Reduviidae);
Can. Entomol 105 231-238
Wallace H R 1953 Notes on the biology of Coranus subapterous De Geer (Hemiptera-Reduviidae); Proc. R.
Entomol. Soc. London A28 100-110
Wygodzinsky P 1966 A monograph of the Emesinae (Hemiptera-Reduviidae); Bull. Am. Mus. Nat. Hist. N. Y.
133 1-613
Zeledon R, Valerio C E and Valerio J E 1973 The camouflage phenomenon in several species of Triatoma
(Hemiptera-Reduviidae); J. Med. Entomol. 10 209-21 1
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 249-264.
© Printed in India.
A behavioural assessment of the impact of some environmental and
physiological factors on the reproductive potential of Corcyra
cephalonica (Stainton) (Lepidoptera: Pyralidae)
S S KRISHNA and S N MISHRA
Department of Zoology, University of Gorakhpur, Gorakhpur 273001, India
Abstract. Results of a study of certain specific environmental and physiological variables
affecting the reproductive activity (specially egg yield and egg hatchability characteristics) of
Corcyra cephalonica (Stainton) (a pyralid pest damaging a variety of stored edible com-
modities) are considered, from a behavioural point of view, in this contribution. The
environmental cues examined are (a) light, (b) population density, (c) space availability and (d)
host presence. The physiological factors tested are (a) age, (b) sex ratio, (c) nutrition and (d) time
of mating. The findings obtained in this investigation provide a basis to comprehend more
meaningfully the complex, delicate and varied effects produced by these factors on the
reproductive performance of this moth in relation to its establishment on jowar, one of the
stored products naturally infested by this pest.
Keywords, Rice moth; mating; oviposition; egg viability; environmental factors; physio-
logical factors. Corcyra cephalonica.
1. Introduction
It is well-known that the degree of establishment of an insect on its host is determined
not only by its competency to survive and grow but also by its ability to breed on that
host. In the rice moth, Corcyra cephalonica (Stainton) — a major pest of stored
commodities (Piltz 1977) — , despite availability of appreciable amount of useful
information on various aspects of nutrition (review of Bhattacharya and Pant 1965;
Srivastava and Krishna 1976, 1978), our knowledge concerning the reproduction of this
insect, specially with regard to the influence of environmental and physiological factors
on mating and ovipositional programmes of the female, is still far from adequate and is
mainly derived from the accounts published by a few workers (Seshagiri Rao 1954;
Mammen and Vishalakshi 1973; Singh and Sidhu 1975, 1976a, b; Krishna and Narain
1976; Sehgal and Chand 1978; Mishra and Krishna 1979, 1980, 1981; Russel et al 1980;
Chakravorty and Das 1983a, b). The acquisition of more information in these areas
becomes important and necessary for a proper appreciation of the ecological and
physiological relationships between the pest and its host. The present communication,
based on this objective, specifically includes our observations on the moth's mating, egg
laying and egg fertility correlated with photoperiod, space availability, population
density, host presence (all environmental), time, age, sex ratio and nutrition (all
physiological).
2. Material and methods
Newborn caterpillars, obtained from eggs collected from a laboratory stock culture of
the rice moth maintained at 23-27°C and 90-95 % (relative humidity) on coarsely
249
250 S S Krishna and S N Mishra
ground jowar (Sorghum vulgare Pers.) containing 5 % (w/w) powdered yeast (Krishna
and Narain 1976), were allowed to develop singly, unless otherwise stated, inside
muslin-capped glass vials (10 mm diameter; 50 mm height) on similar dietary medium
(except in certain specifically arranged trials described herein) into moths for eventual
utilization as experimental animals in the various tests included in this investigation.
The basic set up of an oviposition test conducted here consisted of pairing a freshly
emerged male and a female individual, both associated with the same experimental
regimes, inside a glass container (35 mm diameter; 100 mm height) serving as the
oviposition chamber whose top open end was covered by a piece of black muslin held
by elastic bands. Number of eggs deposited inside this chamber was monitored daily for
5 days (when the females were generally prolific in their egg laying) and the hatchability
of these eggs was also ascertained. All experiments were adequately replicated and were
performed within the temperature and humidity ranges selected for maintenance of
culture of this insect. Wherever desirable, the data were subjected to appropriate
statistical analysis (Paterson 1939) for interpretation. Since these lepidopterans do not
feed as adults, no food was provided to them during experimentation.
Specific features of the experimental outlay connected with each variable considered
in this enquiry are outlined separately below:
2.1 Effect of photoperiod
The following light regimes were arranged for a study of this aspect:
(a) 24 hr L : 0 hr D (The illumination provided by a 1 5 W electric bulb was either white
light or blue, green, yellow or red light rendered possible by employing appropriately
coloured bulbs of similar wattage)
(b) OhrL:24hrD.
2.2 Effect of variation in space availability
Two types of experiments were designed to examine this issue. In the first type, adults of
either sex belonging to a pair enclosed in an oviposition chamber were raised singly
inside one of the 3 kinds of glass containers whose diameter and height were (a) 10 mm
and 50 mm or (b) 35 mm and 100 mm or (c) 70 mm and 90 mm respectively. In the
second type, males and females, individually reared in glass vials (10mm diameter,
50mm height), were single paired within glass containers (functioning as oviposition
chambers in this set up) whose height measured up to 50, 1 00, 1 50 or 200 mm although
its diameter always remained 35 mm.
2.3 Effect of population density
Here the moths tested were developed from individually reared caterpillars or from
hose mamtamed in batches of 5, 20 or 40 indmduals per batch for the first 25 daysTf
nabelr,8 r°mainer^(70mm *"**"' 9°mm hei«ht) and *" ^olated to
enable each larva to commue it. growth solitarily into an adult in the same container
Rice moth reproductive behaviour 251
2.4 Effect of host presence
Consideration of this aspect entailed the conduct of two different series of experiments.
The first series of tests were arranged with single paired freshly emerged males and
females held inside oviposition chambers (as described in the basic set up) within each
of which was already placed a small glass tube (1 5 mm diameter; 50 mm height) covered
at the top with black muslin and half filled with normal or water- or ether-extracted
jowar supplemented with yeast (host material).
Extraction of jowar, carried out in distilled water or in diethyl ether, was performed
separately employing a soxhlet extraction assembly. For each extraction, 500 g of jowar
was first properly ground in dry form in an electric grinder to yield the flour. This was
later wholly transferred to the soxhlet assembly containing 500 ml of one of the above
mentioned solvents and subjected to repetitive extractions within the apparatus to
ensure removal of all soluble constituents. The extracted flour was subsequently taken
out of the assembly and air-dried to eliminate completely the odour of the employed
organic solvent or any excess moisture (in case of water-extracted flour) present in them
before placing them as host material inside the glass tube.
Number of eggs deposited by the mated females on the muslin cloth piece covering
the main oviposition chamber and on that closing the inner glass tube having yeast-
added normal jowar were determined separately. Also, egg output recorded on the
cover of this glass tube was compared with egg yields obtained on the same site when
the tube contained water- or ether-extracted jowar mixed with yeast. As in previous
tests, egg hatchability was ascertained in every case.
The second series of trials, with normal jowar enriched with yeast placed inside the
small glass tube, was planned along similar lines to compare the moth's egg output and
egg viability at the two oviposition locations between mated females possessing both
antennae and those devoid of these head appendages. The technical procedure
concerning removal of antennae and time allowed for the females to overcome the post-
operational trauma were identical to that reported earlier (Krishna and Sinha 1969).
2.5 Effect of time of mating
Information into this area of the reproductive biology of this moth was obtained by
confining single pairs of newly eclosed male and female adults inside oviposition
chambers for one of the three 4 hr periods (1830 through 2230 hr— first quartet; 2230
through 0230— second quartet or 0230 through 0630 hr— third quartet) on the first day
and continued similarly for 4 successive days. The reason for choosing the nocturnal
part of the daily time-cycle to hold these insects in "couples" is because of the observed
pronounced high sexual activity of both sexes leading to mating, like in a number of
other lepidopteran members (Engelmann 1970), during the night hours. For the
remaining part of the normal day-night rhythm, when the sexes were not paired, they
were, however, allowed to enjoy the company of each other only through a metallic
wire-mesh partition installed in a manner basically identical to that reported for Farias
fabia (Shahi and Krishna 1979) or for Tribe/mm castaneum (Singh and Krishna 1980). In
addition to procurement of egg output and egg viability data, continuous observation
was made to record the number of matings and the length of each copulatory act per
female during a quartet of every experimental day when the insects were in the paired
state.
252 S S Krishna and S N Mishra
2.6 Effect of age
Mating potential and subsequent reproductive performance of females were assessed
by pairing a newly emerged male or female moth with an adult individual of the
opposite sex belonging to one of the following ages (expressed in days counted from
emergence): 0, 3 and 6.
2.7 Effect of sex ratio
There were 6 distinct sex ratio groups with two densities of 1 : 1 sex ratio (1 male: 1
female; 1 male : 5 females; 2 males : 4 females; 3 males : 3 females; 4 males : 2 females and
5 males : 1 female) on which this study was based. In all the tests conducted here, the
glass container functioning as oviposition chamber was a relatively larger one (40 mm
diameter; 200 mm height).
2.8 Effect of nutrition
The reproductive competency of this moth was evaluated separately in relation to its
single rearing on (a) yeast-added normal jowar or (b) one of the two different strains of
rice IR 8 or IR 20 enriched with yeast or (c) normal jowar fortified with yeast up to the
first 15 days of the caterpillar's life and later on jowar extracted with water, 100%
ethanol, chloroform or diethyl ether and then supplemented with yeast. Extraction of
jowar with one of these solvents was carried out as mentioned earlier.
3. Observations
3.1 Effect of photoperiod
Complete darkness or coloured lights providing red or yellow illumination in place of
only blue facilitated C. cephalonica females, subjected to variable light experiences, to
release significantly higher number of total and viable eggs at the same time (P < 0-01
or <0-05) (table 1). Nevertheless, a 24 hr scotophase situation specially favoured,
amongst all the light regimes tested here, a marked augmentation in egg hatchability in
these females (P < 0-01),
3.2 Effect of variation in space availability
Females produced from larvae and pupae raised individually on jowar supplemented
with yeast inside glass containers (70 mm diameter; 90 mm height) laid significantly
greater number of total and viable eggs than single-reared counterparts whose post
embryonic development occurred in relatively smaller containers (35 mm diameter;
100mm height or 10mm diameter; 50 mm height) (P < 0-01) (table 2).
From the account given above, it is clear that provision of greater accommodation to
the rice moths during their postembryonic development enables emergence of females
Rice moth reproductive behaviour 253
Table 1 . Estimates of oviposition and hatchability of eggs in C.
cephalonica held on various light regimes during their adult lives
(data pooled from five females)*.
Light regimes
Mean number of
total eggs laid
Mean number of
viable eggs laid
DD
216-6 a
172-8 a
LL
(i) Red
223-0 a
104-2 b
(ii) Yellow
204-8 a
92-6 b
(iii) White
183-6 ab
73-8 be
(iv) Green
152-2 b
73-0 be
(v) Blue
150-4 b
45.0 c
Mean
188-4
93-6
LSD(1%)
71-3
44-8
(5%)
52-6
33-1
* Means in the same vertical column followed by the same
alphabet do not differ significantly at the 1 % or 5 % level by the
least significant difference (LSD) test.
DD = Continuous Darkness; LL = Continuous Light.
Table 2. Estimates of oviposition and hatchability of eggs in C. cephalonica raised on
jowar supplemented with yeast in glass containers of varying dimensions (data pooled
from five females)*.
Dimensions of glass
container (in mm)
Mean number of total
eggs laid
Mean number of viable
eggs laid
Diameter x Height
'
70x90
499-0 a
433-6 a
35x100
388-4 b
320-6 b
10x50
336-8 b
272-0 b
Mean
408-1
342-1
LSD(1%)
72-6
81-2
(5%)
51-8
57.9
* Means in the same vertical column followed by the same alphabet do not differ
significantly at the 1 % level by the LSD test.
whose egg output and egg hatchability become decidedly greater. Will alterations made
in spatial accommodation only during the adult lives of these moths affect the
reproductive capacity and, if so, to what degree? Answers to these questions were also
obtained in this investigation. Males and females housed in glass containers having
maximum vertical space facility (200 mm height) resulted in females depositing only
slightly higher mean number of total and viable eggs which, based on aggregate egg
scores relating to both these determinations, were about 12% and 10% respectively
more than the values obtained similarly for females coupled with males and enclosed in
containers whose height measured up to only 50 mm (figure 1).
254
S S Krishna and S N Mishra
280
UJ
_J
^200
120 -
O
UJ
UJ
_
-
LU
260 ^
""""•
n
-
LL
"
—
-
180 t
-J
_
-
5
-
100 §
UJ
i
UJ
20 2
B
D
Figure 1. Histogrammic representation of mean total and viable eggs laid by C. cephalonica
in relation to confinement of both sexes during their adult lives in glass containers having
uniform diameter (35 mm) but of varying heights (data based on 5 replicates per test):
A -50 mm; B- 100 mm; C- 150 mm; D- 200 mm. Full height of each bar in the figure
represents mean number of total eggs laid/female. Level of horizontal line within the body of
each bar shows the value pertaining to the mean number of viable eggs laid/female.
3.3 Effect of population density
Egg deposition and egg hatchability progressively decreased when these pyralids,
instead of being reared singly all through, were allowed to develop, even in the midst
of abundant food provisions, in groups of 5, 20 or 40 larvae per batch for the first 25
days of the caterpillars' lives and subsequently individually up to the eclosion of adults
(figure 2).
3.4 Effect of host presence
Number of total and viable eggs laid by C. cephalonica on the cloth covering the small
glass tube holding the larval food and kept within the larger glass container
(oviposition chamber) were significantly higher than what were recorded on the muslin
roofing the latter (P < 0-01) (table 3). However, oviposition by these moths on the cloth
of the diet tube and fertility of these eggs sharply fell when the tube contained ether-
instead of water-extracted or normal jowar (P < 0-01 or < 0-05), though between the
latter two test conditions egg hatchability values were significantly different (P < 0-05)
(table 4).
The selection of the cloth binding the diet-filled glass tube for laying more viable eggs
by the rice moth is evidently a behavioural phenomenon exhibited by females whose
antennae were intact. It is likely that these head appendages might bear certain
chemoreceptors, as in Plodia interpunctella (Deseo 1976), guiding the females to arrive
at the site nearest to food odour source for greater oviposition. This aspect was also
considered in the present enquiry. Removal of antennae in females resulted in their
Rice moth reproductive behaviour
560
255
ID
Li.
CL
e>
UJ
UJ
80 -
500
LU
U-
340
CD
180
CD
O
UJ
- 20
A B C D
LARVAL DENSITY REGIMEN
Figure 2. Histogrammic representation of mean total and viable eggs laid by C. cephalonica
in relation to certain rearing regimes (data for each bar pooled from 5 females). A -Adults of
both sexes developed from single-reared larvae. B, C and D -Adults of both sexes developed
from larvae kept together in lots of 5, 20 and 40 members per group respectively for the first 25
days after which the caterpillars were always allowed to grow individually. For explanation of
other features pertaining to the histograms, see legend for figure 1.
Table 3. Estimates of oviposition and hatchability of eggs in C. cephalonica on muslin
cloth pieces covering separately the larger glass container and the diet-containing
(normal jowar supplemented with yeast) small glass tube kept within the former (data
pooled from five females).
Oviposition site
Mean number of total
eggs laid (±SE)
Mean number of viable
eggs laid (±SE)
On muslin cloth piece
covering the larger
glass container
On muslin cloth piece
covering the small glass
tube holding diet
18-8 ±9-81*
1244 ±20-58
11-6 ±6-36*
80-8 ± 15-68
* Significantly different at 1 % level (r-test) from the value just below in the column.
SE = standard error.
having no specific preference for deposition of total and fertile eggs on the cloth
covering the diet tube or on the top muslin closing the larger oviposition chamber
(P > 0-05) (table 5).
3.5 Effect of time of mating
Results concerning the effects of time-related copulations during the scotophase part of
the daily light-dark cycle on oviposition and egg hatchability are summarized in table 6.
S S Krishna and S N Mishra
Table 4. Estimates of oviposition and hatchability of eggs in C. cepha-
lonica on muslin cloth piece covering the small glass tube containing normal
or variously extracted jowar supplemented with yeast (data pooled from five
females)*.
Yeast-supplemen ted
diet within small
Mean number of total
Mean number of viable
glass tube
eggs laid
eggs deposited
Water-extracted jowar
168-2 ab
124-4 a
Normal jowar
124-4 b
80-8 b
Ether-extracted jowar
54-0 c
34-8 c
Mean
115-5
80-0
LSD(1%)
72-6
53-0
(5%)
51-8
37-8
* Means in the same vertical column followed by the same alphabet do not
differ significantly at the 1 % or 5% level by the LSD test.
Table 5. Estimates of oviposition and hatchability of eggs in anten-
nectomised females of Corcyra cephalonica on muslin cloth pieces covering
separately the larger glass container and the diet-containing (normal jowar
supplemented with yeast) small glass tube kept within the former (data
pooled from five females).
Mean number of total Mean number of viable
Oviposition site eggs laid (±SE) eggs laid (± SE)
On muslin cloth piece
covering the larger
glass container 59-8 ± 14-59 NS 35.4 + io-95 NS
On muslin cloth piece
covering the small
glass tube holding diet 123-6 ± 23-60 83-2 ± 16-56
NS — Mean values in a column not significant from one another.
SE = standard error.
Table 6. Estimates of oviposition and hatchability of eggs in C.
cephalonica subjected to different time-related mating schedules
(data pooled from five females).
Time period Mean number of total Mean number of viable
(hr) eggs laid eggs laid
1830-2230 119-4 63-6
2230-0230 175-2 132-2
0230-0630 143-6 79-2
Rice moth reproductive behaviour
257
Females which mated during the 4hr interval (2230 through 0230 hr) falling in the late
night period, interestingly, deposited the highest number of total and fertile eggs, while
those which completed this sexual function in the early scotophase (1830 through
2230 hr) were least productive with respect to both yield and viability of eggs.
A female was also often found capable of mating, within the 5-day tenure of the
experiment, twice with the same male paired with her and, occasionally, even thrice (as
in the case of one individual) (figure 3). The duration of an individual mating act was
quite brief and ranged between 2 and 8 min. For each quartet, there were varying
proportions of matings between pairs possessing different temporal lengths (table 7).
Majority of the coitus sessions lasted for 2 or for 5 min.
3.6 Effect of age
Males or females as old as 6 days, except in very few instances, courted and mated with
freshly emerged individuals of the opposite sex. Newborn mated females showed an
almost equal competency to unload eggs when copulated with 0, 3 or 6-day old males
although the proportion of fertile eggs released by them was largest when such
individuals were sexually united with freshly enclosed males (table 8).
u /
Q
O
cr
0
ijj
on
CL
CD
cr
I
o
n n
0
CO
CM
n n
n
U)
UJ
cr
O
X
ro
§—
(NJ
o
1
O
D
^
rn
Q
UJ
fvj
(M
n n
n n
O
5
CO
0
PO
n
cr
fN
o
CNJ
1
O
n
n
ro
00
00
n
n
UJ
*- 0 1 2 3 U
FREQUENCY OF COPULATION
Figure 3. Schematic diagram showing the number of matings during each of the three
quartets of a 12 hr period in 5 separately arranged pairs of males and females of C. cephalonica
in a 5-day test period. Each squarish symbol within a segment above the corresponding mating
number (given in abscissa) in the diagram represents the datum concerning frequency of
copulation of a female individual during the entire test period.
S S Krishna and S N Mishra
Table 7. Frequency distribution for length of mating period in total matings C, cephalonica*.
Experimental day
(counted from
pairing day)
Total
matings
Length of mating period (in min)
1
2
3
4
5
6
7
8
4
0
1
1
0
1
0
0
1
1
2
0
0
0
0
2
0
0
0
2
0
0
0
0
0
0
2
0
3
0
0
0
1
2
0
0
0
2
4
0
3
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
3
2
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
* Entire data based on observations collected from 15 mated pairs (5 tested for each quartet
separately), a single pair constituting an independent replicate.
Note: (i) Upper number of each entry for every experimental day refers to mating(s) in the 5
pairs tested during the 1st quartet (1 830-2230 hr).
(ii) Middle number of each entry for every experimental day refers to mating(s) in the 5
pairs tested during the 2nd quartet (2230-0230 hr).
(iii) Lower number of each entry for every experimental day refers to mating(s) in the 5
pairs tested during the 3rd quartet (0230-0630 hr).
Table 8. Estimates of oviposition and hatchability of eggs in C. cepha-
lonica subjected to different age-related mating schedules (data pooled from
five females).
Age (in days) at
the commencement
of mating Mean number of total Mean number of viable
M F eggs laid eggs deposited
263-4
23-2
7.fl
142-4
3-8
Rice moth reproductive behaviour
3.7 Effect of sex ratio
259
Total egg output and egg viability estimates reached highest levels when the sex ratio
between males and females was maintained at 1:5 and the mean values significantly
surpassed all others obtained in tests conducted with males and females held in varying
numbers (P < 0-01) (table 9).
3.8 Effect of nutrition
Females raised completely on normal jowar fortified with yeast laid significantly more
number of total and viable eggs than those whose postembryonic development took
place on yeast-mixed normal jowar up to the first 15 days and later on jowar extracted
with water, chloroform or 100% ethanol and then reinforced with yeast (P < 0-01)
(table 10). Egg hatchability further pronouncedly increased if these females reared
initially on normal jowar were continued to grow from the 16th day of their larval lives
on yeast-supplemented ether- instead of water-, chloroform- or 100% ethanol-
extracted jowar (P < 0-01 or < 0-05). If, on the other hand, rice of a particular strain m
20 in place of m 8 supplemented with yeast became the food instead of normal jowar in
the rearing medium of these moths since their birth, the productivity of females in terms
of egg output and egg viability became significantly poorer (P < 0-01 or < 0-05) (table
11).
4. Discussion
This investigation has brought to the fore several unexplored or insufficiently known
and, nonetheless, important environmental and physiological factors affecting mating,
oviposition and egg viability in the reproductive biology of C. cephalonica. They are
Table 9. Estimates of oviposition and hatchability of eggs in C.
cephalonica held on different sex ratio regimes (data pooled from
five sets of experiments for each sex ratio)*.
Sex ratio Mean number of total Mean number of viable
M F eggs laid eggs laid
1 : 5
1321-2 a
866-8 a
2 : 4
850-8 b
548-4 b
3 : 3
605-6 c
421-4 be
4 : 2
573-2 c
468-4 be
1 : 1
398-0 d
321-2 c
5 : 1
333-8 d
275-6 c
Mean
680-4
483-6
LSD (1 %)
196-2
299-6
(5%)
144-8
221-1
* Means in the same vertical column followed by the same
alphabet do not differ significantly at the 1 % or 5 % level by the
LSD test.
260 S S Krishna and S N Mishra
Table 10. Estimates of oviposition and hatchability of eggs in C. cephalonica
maintained on certain prescribed dietary schedules during the insect's postembryonic
development (data pooled from five females)* .
Mean number of Mean number of
Diet + 5 % yeast total eggs laid viable eggs laid
Normal jowar all through 410-8 a 364-0 a
Normal jowar for the first 1 5 days followed
by:
(i) ether-extracted jowar
369-0 ab
333-0 a
(ii) 100% ethanol-extracted jowar
309-0 bed
252-2 b
(iii) water-extracted jowar
291-6 c
175-6 c
(iv) chloroform-extracted jowar
269-8 cd
152-8 c
Mean
330-0
255-5
LSD (1 %)
96-5
95-6
(5%)
70-8
70-1
* Means in the same vertical column followed by the same alphabet do not differ
significantly at the 1 % or 5 % level by the LSD test.
Table 11. Estimates of oviposition and hatchability of eggs in C.
cephalonica raised completely on normal jowar or on one of two different
strains of rice fortified with yeast (data pooled from five females)*.
Mean number of total Mean number of viable
Diet 4- 5 % yeast eggs laid eggs laid
Rice IR 8 430-8 a 369-0 a
Normal jowar 410-8 a 364-0 a
Rice IR 20 330-8 b 284-4 b
Mean 390-8 339-1
LSD (1 %) 98-4 99-2
(5%) 70-2 70-8
*Means in the same vertical column followed by the same alphabet do not
differ significantly at the 1 % or 5 % level by the LSD test.
now considered at length here for a comprehensive appreciation of the problem in
relation to the establishment of this pest on stored materials, specially on jowar.
Oviposition and fertility of eggs were conspicuously greater in females experiencing
total scotophase or a complete photophase condition where red or yellow light
prevailed instead of blue. This fascinating finding, apart from motivating further
explorations into the role of photoperiod in regulating the intrinsically lodged
mechanisms involved in the behaviour and physiology associated with reproduction in
C. cephalonica, provides a basis to hint at the possibility of achieving appreciable
measure of success in reducing the evolution of fresh populations of this pest if stored
commodities harbouring a heavy infestation of these moths are exposed to continu-
ously illuminated environment lit by blue light.
Caterpillars raised for the first 25 days of their lives in groups of 5, 20 or 40
individuals per batch even under congenial nutritional conditions in glass containers
yielded adult females which, following copulation, were less fecund than counterpart
mateds reared individually all through. Presumably availability of relatively more space
Rice moth reproductive behaviour 261
for uninterrupted movement within and/or outside the diet medium in the glass
containers during the growth period of caterpillars reared singly was a positive factor
contributing to their development into healthy moths exhibiting higher reproductive
potential. This postulation, emphasizing the importance of the extent of space
availability during postembryonic development in relation to the reproductive
efficiency of these moths, is further strengthened by the record of greater egg yield and
egg viability values from females associated with such individually grown larvae in
bigger glass containers. However, imposition of limitation in the availability of space
for reproductive males and females, during their adult lives, within the oviposition
chamber by progressively lowering only its height did not severely affect the moth's egg
deposition or egg viability unlike in the case of another lepidopteran species, Earias
fabia (Mani and Krishna 1984).
There was pronounced increment in the number of eggs laid and in the hatchability
of these eggs when the females of C. cephalonica oviposited on a site nearest to the larval
food (normal jowar supplemented with yeast). Apparently, the odour of some volatile
compounds emanating from this diet seems to have exerted a stimulating influence on
the ovipositional behaviour of these lepidopterans — a point missed by Seshagiri Rao
(1954) in his observations — favouring them to select the substratum closest to the food.
The fact that egg output and egg fertility declined significantly on the same oviposition
site when the food comprised ether-extracted jowar enriched with yeast clearly shows
the ether-soluble chemical nature of these volatile compounds playing the role of
ovipositional stimulants for these insects. The inability of the mated females to
continue laying greater number of total and viable eggs, consequent to removal of both
antennae, on the cloth (oviposition site) proximal to the larval diet is enough evidence
to infer that the smell of these volatile constituents present in the food is olfactorily
perceived by chemoreceptors located on these cephalic appendages as in another stored
grain moth, Plodia interpunctella (Deseo 1976).
A male and a female individual were often successful in entering into sexual union
more than once during the 5-day experimental period. Nevertheless, within a prescribed
quartet on any single day they performed only one coition. The existence of this
multiple mating phenomenon in the reproductive behaviour of the rice moth, while
contradicting the observations of Chakravorty and Das (1983a), was, to some extent,
similar to that already reported for this species by Sehgal and Chand (1978) and to that
observed in certain other stored products insects like Tribolium confusum (Good 1933),
Tribolium destructor (Reynolds 1944), Trogoderma granarium (Karnavar 1972) and
Tribolium castaneum (Singh and Krishna 1980). But the fact that the duration of a single
mating act in C. cephalonica was short, though not as brief as that recorded in T.
castaneum (Singh and Krishna 1980), testifies the inherent incapability of these insects
to remain in coition for extended periods, notwithstanding provision of facility for
these moths to accomplish such a task associated with their reproductive activity.
Evidently, these findings raise interesting questions concerning the not yet elucidated
extrinsic and intrinsic conditions regulating frequency and duration of mating in this
insect.
Highest number of total and fertile eggs were laid by females which were paired with
males for mating between 2230 and 0230 hr daily during the 5-day test period.
Presumably copulations which occurred during this quartet of the nocturnal period
afforded the best opportunity for the females to get maximally stimulated for
impregnation upon receipt of the largest amount of viable sperms from the males and
262 5 S Krishna and S N Mishra
for oviposition. Admittedly, this calls for more detailed investigations in future to
examine into the relationship between time and spermatozoa content (quality as well as
quantity) in the males of the rice moth and its implication on the reproductive potential
of the females subjected to mating at different periods during scotophase of a 24 hr day-
m gAge°of male or female insect prolonged up to 6 days from eclosion did not prohibit
these moths from mating with freshly emerged individuals. Plausibly in such
combination of sexes the ability of the females to produce and release sex pheromones
and the competency of the males to perceive these chemical agents remain unaftected by
advancing age in the reproductive life of the rice moth. But the occurrence of high levels
of egg output by mated females when they were associated from the day of their
emergence with newborn or 3- or 6-day old males and the laying of maximum number
of viable eggs by these females coupled with just eclosed males suggest that oviposition
and egg viability in this lepidopterous pest are possibly regulated by endogenously-
based mechanism(s) possessing an interestingly intricate operational relationship with
age of males and females at the time of their mating.
Considering the effect of variation in sex ratio in the adult population on the
reproductive capacity of C. cephalonka females, it was found that a 1 :5 ratio between
males and females resulted in maximum egg output and egg hatchability by the mated
females. This characteristic feature in the reproductive behaviour of the rice moth is
quite unlike that reported in another, though taxonomically unrelated, stored grain
pestjribolium castaneum (Lee and Smith 1976) in which, however, egg deposition and
egg fertility values associated with the first oviposition period of more or less same
duration did not show great disparity among different sex ratios. Egg yield and
hatchability also pronouncedly shot up if 4 females were housed with 2 males rather
than a single female enjoying the company of a lone male or 5 individuals of this sex. All
these observations clearly indicate that the lower the proportion of males the greater
would be the reproductive potential of these moths which, in turn, would increase their
population levels during a given period of time. This relationship between the
population density of males and productivity in C. cephalonica could be of applied
significance from the standpoint of pest management if a pesticide was used that killed a
higher proportion of females than males — a hypothesis different from that put forth by
earlier workers (Shorey 1970; Otake and Sukuratani 1972; Otake and Oyama 1973;
Kehat and Gordon 1975, 1977) wherein the necessity of drastic reduction in male
population for achievement of economic control of the female's reproduction in
lepidopterous pests was emphasized.
With regard to the impact of varying larval nutrition on the reproductive potential of
the rice moth, it is clear from the results that normal jowar or rice of a particular strain
IR 8 enriched with yeast provided right from the first day during the caterpillar stage
facilitated the mated females to be more productive than all other prescribed dietary
schedules though females reared initially on normal jowar combined with yeast up to
the first 15 days and subsequently on yeast-supplemented ether-extracted instead of
water- or chloroform-extracted jowar also showed marked augmentation in their egg
output and egg hatchability. The precise nutritional factors resident in jowar and IR 8
rice strain and their involvement in regulating the physiological mechanisms (most
likely neuroendocrinal as in several other insects (Wigglesworth 1960; Engelmann,
1970)) connected with reproduction are worth examining in follow-up studies in this
insect.
Rice moth reproductive behaviour 263
Acknowledgement
This work was supported by funds obtained from the UGC, New Delhi.
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151-154
Otake A and Oyama M 1973 Influence of sex ratio and density on the mating success of Spodoptera litura F.
(Lepidoptera: Noctuidae); AppL Entomol. Zool. 8 246-247
Otake A and Sukuratani Y 1972 Egg production of the adult tobacco cutworm Spodoptera litura F.
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Paterson D D 1939 Statistical technique in agricultural research (New York: McGraw-Hill) pp. 263
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264 S S Krishna and S N Mishra
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© Printed in India.
Behavioural analysis of feeding and breeding in Orf hopteran insects
S Y PARANJAPE
Department of Zoology, Modern College, Shivajinagar, Pune 411 005, India
Abstract. Various aspects of the feeding and breeding behaviours in Orthoptera with special
reference to Acridoidea and Tetrigoidea are discussed. The changes in the incisor and molar
mandibular surfaces, laciniae and galeae of the maxillae, in relation to graminivory, herbivory
and omnivory are cited as specific manifestations of the feeding behaviour. Similarly, in sharp
contrast to Acridoids the rather poor foregut armature and small and compact feculae in
Tetrigoids is suggested as an evidence indicating the correlation between food and feeding
habits. While describing the breeding behaviour a generalized comparison of the utilization of
the acoustic sexual signals in crickets and grasshoppers causing attraction and copulation or
otherwise is made. Differences in the ovipositors, mode of egg-laying and the types of eggs in
Acridoids and Tetrigoids are stated as characteristic features of reproductive behaviour.
Factors influencing these behaviours in Orthoptera as well as the behaviours bringing about
succession and changes in the patterns of life-forms are mentioned.
Tools of behavioural investigations leading to the formulation of ethograms are briefly
stated. Methods and techniques generally adopted in studying these aspects of behaviours are
referred to as application of such ethological studies. The causative effect of feeding and
breeding behaviours is depicted by proposing the adaptive radiation diagrams for the order
Orthoptera.
The article, in conclusion, points out certain areas related to these behaviours on which,
work would seemingly be useful. For example, determination of the cues that bring about
mating in grouse-locusts in the absence of stridulatory and tympanal organs; the energy budget
on account of their peculiar diet; and diapause are few such areas. The possibility of these
forms turning out to be good models for experimental, lab-oriented studies is suggested. Since,
as compared to Acridoids very little studies in the areas of economic and ecological impact in
terms of population dynamics have been made on the Tettigonioids and Tetrigoids, it is further
suggested that these if undertaken, would also furnish valuable information.
Keywords. Ethology; ethogram; trophic and reproductive behaviour; methodology; adapt-
ive radiation.
1. Introduction
Feeding and breeding behaviours are of fundamental importance though highly
complex in nature. Feeding represents a form of maintenance activities and is,
therefore, of an individualistic nature, while breeding constitutes a type of com-
municatory activity. Together they represent ways of interaction with environment
through adjustment: feeding providing the energy source and breeding ensuring
survival and continuation of the species.
An attempt is made here to present an overview of the feeding and breeding
behaviour of a very large and economically important insect-order, Orthoptera. The
modo et forma of how this has been done is outlined below.
There are a number of related aspects, such as the role of visual, chemoreceptory,
olfactory, gustatory cues; that are essential in finding and recognition of the food before
the actual act of feeding. Similarly the nervous, hormonal and pheromonal factors are
known to particularly influence the insect breeding behaviour and also phase
265
266 S Y Paranjape
polymorphism in locusts. Together with abiotic factors, the hormones also probably
influence the phenomenon of diapause. The manifestation of feeding and breeding
behaviours in these Orthopteran insects is outlined through some representative
structural and functional adaptations. In doing so the term Orthoptera, sensu stricto
means Acridoids (grasshoppers and locusts) in general and the Tetrigoids (grouse-
locusts) in particular. While referring to Tetrigoids four species of family Tetrigidae;
namely, Euscelimena harpago Serville, Eucriotettix flavopictus Bolivar, (Subfam.
Scelimeninae); Euparatettix personatus Bolivar (Subfam. Tetriginae) and Potua sab-
ulosa Hancock (Subfam. Cladonotinae) are considered representative forms (figures
1 A-C). These none too studied but, very interesting forms allied to Acridoids, are being
investigated at this Centre. While describing the spatio-temporal manifestations of
these behavioural aspects through proposed adaptive radiation and interrelationships,
the term Orthoptera is used sensu law. The account of related methodology and
adaptive radiation has been presented in the form of schematic representations both for
brevity and clarity.
2. Review of literature
2.1 Food and feeding behaviour
Studies on food habits and biology of Acrididae by Gangwere et al (1976); on food
selection in Orthoptera together with feeding behaviour by Freeland (1975), Gangwere
(1961), Gangwere and Agacino (1973), Gangwere and Ronderos (1975) and Mulkern
(1967, 1969); on effect of specific food on growth by Bajoi and Knutson (1977); on food
preferences by Lambley et al (1972); on regulation of food intake by Bernays and
Chapman (1974); on host finding and food availability modifying the feeding
behaviour, as well as studies on succession in grasshoppers by Gangwere (1972); on host
related responses by Browne (1977) and Mitchell (1975); on chemosensory responses by
Schoonhoven (1977); on the observations of a monophagous grasshopper by Knutson
(1982); on the structural adaptations of the mouthparts by Gangwere (1965), Isely
(1944) and Muralirangan (1978); on foregut morphology, its armature as an adaptation
to food preference, as well as the taxonomic significance of foregut armature by
Muralirangan (1980) and by Muralirangan and Ananthakrishnan (1974, 1981); on
control through feeding with use of deterrents by Munakata (1977) and wheat bran bait
with chemical and biological agents by Onsager et al (1980, 1981); on population
ecology and energetics by Delvi and Pandian (1971, 1972, 1979), Hoekstra and
Beenakkers (1976), Khan and Aziz (1976), Muralirangan and Ananthakrishnan (1981),
Muthukrishnan and Delvi (1973y 1974) and Onsager (1983) are quite informative.
2.2 Different aspects of breeding behaviour
Studies on the life history strategies in insects by Dingle (1 974); on endocrine research in
Orthopteran insects by Penner (1983); on sequential analysis of insect behaviour by
Richard (1974); on hormones and insect behaviour by Riddiford and Truman (1974); on
acoustic and courtship behaviour by Otte (1972) and Loner and Chandrashekaxan
(1972); on chorusing flight by Willey (1979) are of interest. Observations by Cantrall
(1979), Gangwere (1964-65), Mores (1904) and Otte (1979) also appear useful. <
Feeding and breeding behaviour in Orthoptera
267
mr
*
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) .1,
.
v
1 H
J
K
Figure 1. A. Euscelimena harpago. B. Euparatettix personatus. C. Potua sabulosa.
D. Mandible of Euscelimena, with blunt incispr dentes (id) and the molar ridges (mr).
E. Details of molar ridges in Euscelimena. F. Mandible of Tetriginae showing sharp incisor
dentes (id). G. A typical Tetrigoid maxilla. H. Foregut armature in Euparatettix. I. Details of
the foregut armature. J. Feculae of an Acridoid (A), Euscelimena (B) and Potua (C). K. A
typical Tetrigoid ovipositor.
268 S Y Paranjape
2.4 Books
Those of topical interest as far as ethology is concerned are by Eibl-Eibesfeldt (1970),
Huntingford (1984), Grzimek's encyclopedia of Ethology (1977), Tavolga (1969) and
Wallace (1979). The books referring to the various aspects related to feeding and
breeding behaviour of insects in general and Acridoids in particular by Bei-Bienko and
Mischenko (1951), Chapman (1973), Cummins et al (1965), Friedlander (1978), Frost
(1959), Gillot (1980), Matthews and Matthews (1978), and the two volumes on
grasshoppers and locusts by Uvarov (1966, 1977) are invaluable.
2.5 Work on Tetrigoidea
Compared to grasshoppers and locusts there are relatively few studies carried out on
the different aspects of grouse-locusts, including feeding and breeding behaviour.
However, those referred herein are by Bhalerao and Paranjape (1982, 1984), Hodgson
(1963), Hancock (1898, 1902, 1906-1916), Hartley (1962), Karandikar and Paranjape
(1964), Kevan (1982), Nabours (1919), Nabours and Stebbins (1950), Paranjape (1976),
Paranjape and Bhalerao (1984), Poras (1979), Rehn and Grant (1961) and Sabrosky et
al (1933). Some, so far unpublished data of this Centre is also included in this article.
3. Material and methods
The feeding and breeding behavioural studies involve a variety of approaches. These in
turn contribute in ways more than one, to the development of an ethological profile or
ethogram for a particular species. A representative form of various steps is presented in
table 1.
4. Discussion
4.1 Feeding behaviour
The Orthopteran biting-chewing mouthparts in general and the mandibles and
maxillae in particular show structural adaptations in consonance to the feeding habit
and have been an object of study indicating the feeding behaviour of these insects (table
1). The degree of asymmetry allowing the left mandible to overlap the right one when
closed, the molar and incisor surfaces together with their teeth (dents or dentes) of the
mandibles and the laciniae together with their recurved teeth (dents or maxadentes) and
galeae of the maxillae are particularly important in this connection.
As the acridoids are known not to be polyphagous, but at the same time showing
considerable, species-related selection and variation in the range of phytophagy, these
tend to show some broad groups of mandibular patterns indicating adaptive radiation
in relation to the feeding behaviour (table 5). The nomenclature of the patterns is,
however, not unanimous and furthermore, while discussing Acridoid feeding be-
haviour the term omnivorous is used by some workers synonymous to polyphagous,
meaning thereby feeding on 'everything green'. However, as the Orthoptera is under
Feeding and breeding behaviour in Orthoptera
269
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discussion as a group, the terms herbivorous (feeding on plants) and omnivorous (i.e.
feeding on plants as well as animals) are used in a broader sense, unless specified
otherwise. Thus those feeding mainly on grasses and similar type of plants show
graminivorous type of mandibles (figure 2A). These overlap but slightly and have the
incisor regions represented typically by prominent parallel ridges (cusps or teeth) which
often tend to fuse to develop a cutting edge; while the molar regions show somewhat
flattened ridges and furrows useful for grinding the food. The forms that feed mainly on
broad-leaved dicotyledonous plants show herbivorous (= forbivorous according to
Brustia
Molar,
denies
Maxillary palp
lea
(F) (G)
10 -
Tetriginae
Figure 2. A. Graminivorous mandible. B. Herbivorous mandible. C. Carnivorous man-
dible. D. Maxilla of a marginal feeder. E. Partly exposed piece of a spongy (SP) egg-pod of an
Acridoid. F. Eggs (E) of Euscelimena arranged in tiers (T) Note prominent horns (H). G. Eggs
ofPotua arranged in a cluster. H. Relative acceptance of foods by subfamilies of Tetrigoidea.
Feeding and breeding behaviour in Orthoptera 271
some workers) type of mandibles (figure 2B). These overlap to a greater degree and
show dentes instead of ridges; those of the incisor regions are pointed while those of the
molar surfaces are subconical. The forms that are capable of feeding both on grasses
and herbaceous plants show a range of mandibular patterns of intermediate nature.
These are called ambivorous ( = herbivorous according to some) types of mandibles. In
some forms, the feeding is mainly on shrubs, woody shrubs and on foliage of trees. The
mandibles in such cases are stouter, with sharp dentes and each molar surface bearing
varying degree of concavity for grinding. Such arborivorous mandibles are variously
called forbivorous and dendrophagous types representing modified herbivorous
condition. The carnivorous Orthoptera, e.g. the mantis, possess mandibles that are
elongated, hook-like and bearing sharp dentes (figure 2C). On the other hand
omnivorous forms possess mandibles that are equipped with somewhat sharp dentes,
mostly uniform in length on the incisor surface, while the molar surface (figure ID) is
without dentes but bearing characteristic ridges. The ridges are near parallel, transverse,
closely placed, delicately denticulate, and alternating with grooves (figure IE). The
mandibles of grouse-locusts are of omnivorous type. Our observations on the three
subfamilies namely Scelimeninae, Cladonotinae and Tetriginae reveal interesting
variations when compared amongst each other on one hand and the Acridoids on the
other. For example, in Euscelimena the incisor dentes are not sharp but in Eucriotettix
they are sharp. Further the incisor dentes are more sharp and somewhat irregular in
Tetriginae (figure IF) than in Cladonotinae. However, in all the types, the molar region
bears the parallel 'ridge-groove' pattern mentioned earlier and is very unlike the 'ridges
and furrows or dentes' pattern of the Acridoids in general.
The maxillae also show adaptations in relation to the feeding habits. These features
are, however, less predominant and variable as compared to those of the mandibles. The
lacinial dentes are rather blunt in graminivorous and dendrophagous Acridoids. The
dentes are however long, sharp and somewhat curved in forbivorous, carnivorous and
omnivorous types. The galeae in 'margin-feeding' graminivorous forms are more
flattened than tubular (figure 2D). On the other hand, the galeae are of lobular type in
forms that are 'centre-feeders'. The Tetrigoid maxillae have somewhat long, curved,
pointed lacinial dentes giving the lacinia a dinner fork-like appearance and lobular
galeae helping while feeding on pulpy food and fluids (figure 1G).
The diet of Tetrigoids is basically different and varied as compared to that of
Acridoids. The grouse-locusts feed mainly on algae, mosses, molds, lichens, humus,
detritous material and ingest superficial soil too along with the food. The grouse-
locusts are observed to frequent marshy places, paddy fields and to feed on grass and
other cereal sprouts in the fields as well as in the laboratory. Tough-textured leaves of
monocot and dicot plants are, however, not preferred by the grouse-locusts.
Euscelimena is observed to feed even on tender Eichhornia leaves. Some species from
Scelimeninae and Cladonotinae also show scavenging tendencies and feed on soft parts
such as the abdomen of dead and decaying insects including their own species. Thus
some grouse-locusts show necrophagy but cannibalism has not been observed. Another
interesting peculiarity observable in grouse-locusts feeding on green carpets of moss is
that, the mode of feeding tends to be similar to grazing. This results into almost
complete wiping out and cleaning of the small patch of substratum on which the moss is
seen before feeding.
It is for these reasons that these insects have the peculiar well-developed denticulate
ridges and grooves on the molar region of the mandibles. The incisor dentes of the
272 S Y Paranjape
mandibles help in biting, cutting and nibbling whereas during feeding molar surfaces
of the two mandibles and the surface of the left overlapping on that of the right
mandible, help in cutting further the algal filaments together with such other food, into
smaller fragments. At the same time the mandibles presumably help in pressing,
packing and filtering the pulpy food, humus, and fluid matter rather than acting as jaws
that chew or grind the food, as in most Acridoids. The sharp, fork-like lacinial dentes
and lobular galeae help in feeding and centrally pushing the food into the pre-oral
cavity. Regarding the food and feeding behaviour of Tetrigidae, it can be said that, as
compared to Acridoidea, the grouse-locusts show herbivory through omnivory, in the
general sense of these terms as indicated earlier. The relative acceptance of the food in
the three subfamilies of Tetrigidae is given in figure 2H. Other methods of study of the
feeding behaviour (table 1) such as, the study of crop contents and feculae also support
these observations. The difference in the usual pattern of feculae in the adults of
graminivorous Acridoids and ominivorous-herbivorous Tetrigoids can be easily made
out from figure U.
The foregut armature which is widely studied in locusts and grasshoppers such as
Locusta, Schistocerca, Eyprepocnemis, Cyrtacanthacris is yet another feature that has
been considered mainly from taxonomic point of view and is also believed to be
mechanically helping the processing of food in Acridoidea. Our studies on grouse-
locusts indicate absence of spines as well as cuticular folds in Euscelimena but presence
of scanty longitudinal rows of sclerotized, spiny elements in the posterior region of the
crop in Eucriotettix and Euparatettix (figures 1H and I). But in all grouse-locusts the
proven ticular valve bears numerous, delicate and minute teeth. This poor development
of foregut armature observable in grouse-locusts is presumably because, the soft, pulpy,
food present in the shortened foregut might not be requiring the mechanical treatment
as required in locusts and grasshoppers.
There are a number of factors that influence the feeding behaviour of grasshoppers in
general. These can be briefly enumerated as under:
Abiotic factors such as light, humidity, wind, air, ground-level heat and seasonal
variations influence feeding behaviour. Similarly the type of habitat, the nature and
density of vegetation, the changes in pattern of vegetation due to topographic changes,
man's activities and such other factors can not only alter food habits on the basis of
availability but also lead to changes in grasshopper associations and succession of
which the feeding behaviour forms an important basis. The feeding behaviour is also
influenced by physical characteristics of food material such as toughness and turgidity
of the plant tissues and chemical constituents that cause the food to be a stimulant or
deterrent. The physiological state of the insect such as of hunger, thirst, satiety, injury,
disease as well as its state of development such as hatching, moulting, maturation of
gonads and the state of reproduction can also influence or modify the feeding
behaviour. As compared to the extensive observations and work carried out on
Acridoidea, much remains to be understood as far as the influence of these aspects on
the feeding behaviour in Tetrigoidea is concerned.
There are certain other aspects that are related to the food and feeding behaviour.
For example, the types of food-plants can have profound effect on the fecundity, the
rate of completion of the life-cycle, number of instars and also on the growth of wings as
has been indicated by studies on Locusta, Schistocerca, Melanoplus species. Generally, a
mixed diet, rather than a single plant diet, has a better effect leading to faster
development and lower mortality. The feeding behaviour can lead to diet-based
Feeding and breeding behaviour in Orthoptera 273
variation in phytophagous forms resulting into the development of either polyphagous,
steno- or oligophagous or even monophagous species. The latter condition is, however,
very rare in Orthoptera. Another interesting aspect is the quantification of feeding in
the different instars and/or adults as well as in the somatic growth period or
gonotrophic cycle of a particular species. For these studies, various parameters such as
the food consumed during a certain period or on per day basis, weight of a single meal,
ratio of food per body weight and the increase or the decrease in daily food
consumption depending upon the sexual maturation in the male or in the female, are
used. Similarly the amount of green food consumed by an individual Acridoid in its life-
time, its economic and ecological impact, importance and effect of biomass and energy
transfer, population dynamics and energetics are also ultimately related to the feeding
behaviour. The literature, reviewed indicates that much remains to be done in this area
as far as the Acridological studies in India are concerned and that practically no related
information is available on the Tetrigoids.
4.2 Breeding behaviour
As compared to feeding, the breeding or reproductive behaviour is still complex a
phenomenon influenced by a variety of internal and external factors. The breeding
behaviour works internally through the maturation of gonads, while it manifests
externally, mainly through the mating and oviposition behaviour.
The maturation of genital products depends on the nervous, hormonal and also to a
certain extent on the trophic factors. The gonidial state of the reproductive system in
the male and female Orthopteran insects, in turn, triggers the development and visible
expressions of the breeding behaviour. In most of the Orthoptera, especially the
crickets, long and short-horned grasshoppers, acoustic sexual signals mark the
beginning of the mating behaviour. The devices to produce songs differ in the crickets
and the grasshoppers (table 3). The patterns of songs essentially differ in 'time'
distribution of the pulses of sound; the songs also differ in their meanings as per the
changing conditions. As far as the mating behaviour is concerned, it can be said that the
songs are sung mostly by the males to -attract the receptive females of the same species
and to disuade indulgence, territorial intrusion, to indicate dominance and so on,
between members of similar sexes. The songs, based on the purpose they serve are
variously called as calling songs, courtship songs, post-copulatory songs, or rival's song
suggesting threatening. The courtship songs aid the copulatory behaviour while the
post-copulatory songs are mainly meant to ward off further attempts by other males
and thus prevent any interference with the physiological events to follow in the
inseminated female. The rival's song in crickets invariably ensues a fight, while in
grasshoppers it is not so much for threatening but a. suggestion to 'keep away' or avoid,
a result that is generally achieved. It is known at least in the crickets, that the songs are
innate means of communication and that they have genetical basis. The various songs
are also species specific. There are no stridulatory and tympanal organs in the grouse-
locusts and may be that certain other sensory cues initiate the mating behaviour in
Tetrigoids.
The next stage in mating behaviour leads to act of copulation through the courtship
phase. The copulatory behaviour is also dependent on variety of stimuli and mutual
reactions. This culminates into copulation, an act which differs in duration and posture,
274 5 Y Paranjape
the latter depending upon the difference in the relative sizes of the rather small male and
the larger female of the copulating pair and the type of concealed copulatory apparatus
in the male. The completion of copulation tends the now non-receptive female to
undertake the last of the phases in the breeding behaviour, namely, the opposition
behaviour culminating into the act of egg-laying. The oviposition behaviour is both
complex and highly specific. Many factors such as habitat, vegetation, soil conditions,
temperature, humidity, crowding, etc. play an important role in the oviposition
behaviour.
The act of oviposition also depends upon the structure of the ovipositor, the site of
oviposition etc. (tables 3, 4). The eggs in Acridoidea are laid mostly in the soil, in the
form of an egg-pod (figure 2E) and are either arranged regularly as in Acrtda or
irregularly as in Nomadacris. The egg-pod is formed of frothy secretion. The depth at
which the eggs are laid is dependent on the soil humidity; drier the habitat deeper are
the eggs laid. On the other hand in progressively humid to wet habitat the eggs are very
near or even on the surface. In some Acridoids eggs are laid at peculiar sites such as in a
leaf-stalk or under a floating leaf of an aquatic plant, while some show group-laying, e.g.
Dociostaurus. The ovipositor, oviposition and the eggs of Tetrigoids markedly differ
from those of the Acridoids in general. The ovipositor has serrated valves ending in a
rather straight or slightly curved spine-like structures (figure IK). It is unlike that of
Acridoids, wherein the valves are generally smooth (except the serrate ones in Oxya)
and terminally bearing a hook-like structure. In the grouse-locusts, the eggs are laid in
richly humid soil or in marshy habitat about 1-3 cm deep and the valves therefore are
presumably more useful in separating the vegetation and digging a superficial rather
than a deeper hole. Unlike Acridoids such as Locusta, Melanoplus, Gomphocerippus, the
Tetrigoid females do not cover the hole after oviposition. However, as observed in
Euscelimena and Potua since the holes are dug in moss carpets or such other low
vegetation, these many a times are concealed. Our observations on Euscelimena under
experimental conditions also indicate that the development gets completed and the eggs
can hatch even if kept completely under water. The structural modification of the
ovipositor in grouse-locusts is thus in accordance with their oviposition behaviour. The
eggs in Tetrigoids are laid not in egg-pods but in clusters that may or may not be
showing a tier-like arrangement (figures 2F and G). Furthermore, the Tetrigoid eggs are
loosely glued together and each carries a chorionic horn or filament at the anterior end.
In a cluster all the horns point upward. At the posterior end, the egg has a hydropyle
that facilitates water-uptake and the resulting increase in size is partly accommodated
because of the horn mentioned above. The horn of the egg is longer in the species that
are semiquatic or inhabiting marshy habitat while it is shorter or even knob-like in the
species that are more terricoles in nature. Tetrigoids show nymphal or adult diapause,
tendency to hibernate and also show univoltine or biannual cycles. Our studies on
grouse-locusts indicate some interesting features of the breeding behaviour of the
tropical forms. For example, the widely distributed semiaquatic Euscelimena breeds in
natural conditions practically throughout the year, except the summer months as
evidenced by the presence of all nymphal instars and gravid females throughout the
year. On the other hand the pigmy locusts, Potua shows an altitude limited distribution
inhabits cooler places and feeds predominantly on moss (Funaria sps.) The pigmy
locusts are therefore observed to be aestivating as adults by remaining buried 5-6 cm
below thesoil surface along the fence-walls, without feeding, to tide over the summer
months. The aestivation period terminates with the onset of rains when these adults
Feeding and breeding behaviour in Orthoptera 275
come out, copulate and lay eggs in, by then newly developed moss carpets. Some
Tetrigoids are reported to show parthenogenesis.
Both the feeding and breeding behavioural aspects are capable of influencing each
other. Furthermore, although dependent on many factors, both possess some
instinctive basis and exhibit some stereotype, sequential pattern. An attempt has been
made in table 2 to analyze these events in the reproductive behaviour with reference to
the food and feeding behaviour in particular.
4.3 Ecological features and adaptive radiation
These studies in Orthoptera in general and Acridoidea in particular form the basis of
various important aspects. For example, the phase polymorphism is now known to be
due to changes both in the behaviour and in the endocrines. The studies also throw
more light on the economic aspects and also help understand the possible trends in the
adaptive radiation and phylogeny. Acridoid families show some broad grouping based
on food predominance and preference. For example, subfamilies Acridinae, Oxyinae of
the family Acrididae are graminivorous; Eyprepocnemidinae is herbivorous and
ambivorous; while families Pamphagidae and Pyrgomorphidae are herbivorous.
Secondly herbivorous, dendrophagous and forbivorous types are generally considered
as less advanced as compared to the graminivorous types that are believed to be more
progressive ones. The habitats such as terricoles (geophilous/geophiles/geobionts by
some workers), planticoles (phytophilous/phytophiles/phytobionts by some) and its
modifications, e.g. the herbicoles, graminicoles etc. and these behaviours have also
given rise to different life-forms. For example, herbicoles represent the typical body-
shape of the grasshoppers, while terricoles being restricted for feeding and breeding to
the ground only have strongly depressed body form, and graminicoles have laterally
compressed body and more oblique face. The feeding and breeding behaviours through
course of time have established patterns that indicate different lines of adaptive
radiation shown by Orthoptera in general (tables 3-4) and Caelifera in particular (table
5).
5. Conclusions
(i) As compared to Acridoids, more such studies need to be undertaken on
Tettigonioids and Tetrigoids. The bio-ecological studies related to these aspects of
behaviour could further contribute in the designing of control measures on the
economically important order Orthoptera.
(ii) As far as the grouse-locusts are concerned, the detailed study of the mouthparts,
quantification of feeding and energy budget studies, the role of sensillae in the feeding
and breeding behaviours (this important area in relation to the feeding behaviour of
Acridoids is being studied at Entomology Research Institute, Loyola College, Madras),
determination of the cues that bring about mating, in the absence of stridulatory and
tympanal organs generating and receiving the acoustic signals respectively, the life-
cycle and diapause would furnish valuable information.
(iii) Euscelimena, Eucriotettix species of the Tetrigoids seemingly have the potential
as good models for experimental, lab-oriented test-studies.
276
S Y Paranjape
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Feeding and breeding behaviour in Orthoptera
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280 5 Y Paranjape
Acknowledgements
It was very kind of Prof. T N Ananthakrishnan to suggest the author to undertake this
work and later enlighten him on the different aspects of the article. The encouragement
received from Principal S Y Gambhir and the financial support by ICAR, New Delhi was
extremely valuable. Immense help through discussions, photography, critical readings
of the typescript was extended by colleagues Dr H V Ghate and Mr A M Bhalerao.
Valued assistance in the preparation of the article by Uma Mahajani and Narendra
Naidu, and help towards typing the script including the adaptive radiation charts, by
Suvarna Kale need special mention. The author is deeply grateful to all of them for their
help.
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SSSR 23 pp. 83
Not cited in original.
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 283-294.
© Printed in India.
Physico-chemical factors in the acridid feeding behaviour
(Orthoptera: Acrididae)
M C MURALIRANGAN* and MEERA MURALIRANGAN**
Entomology Research Institute, Loyola College, Madras 600034, India
'Department of Zoology, Guru Nanak College, Madras 600032, India
"'Department of Natural Science, S.D.N.B. Vaishnav College for Women, Madras 600044,
Abstract. Though acridids are generally polyphagous, they are not indiscriminate feeders as
is observed, on the basis of extensive studies on several species of grasshoppers like
Eyprepocnemis alacris alacris (Serv.), Oxya nitidula (Walker), etc. The feeding behaviour of
such herbivorous insects in general is of immense importance because of their direct relevance
to applied ecological problems. These insects live in environments with abundance of food, but
their suitability is differently related to each available plant species in the environment. Hence
the feeding behaviour patterns are seen to be generally influenced by several factors such as the
morphological correlates of the insect as well as the physico-chemical factors of the host plant.
In this context, consideration is being given to, (a) physical factors of the host plant such as
the thickness of the leaf lamina, the presence of trichomes, the position of the leaf blade, the
general colour pattern and the effect of blinding, (b) morphological correlates of the insect
such as mandibular modifications in relation to the host, its changes during post-embryonic
development and their role in the shift of the host, the foregut armature and its influence on
host selection, (c) the influence of chemicals like silica, phagostimulants and deterrents of the
hosts on the insect feeding behaviour and (d) the role of sensory structures of the insect in the
detection and discrimination of the host.
In addition to the discussion on the general pattern of feeding, the factors responsible for the
initiation, continuation and termination of feeding are also analysed.
Keywords. Phagostimulant; deterrent; sensilla; palpation; acridids.
1. Introduction
The feeding behaviour and the pattern of host selection in acridids are known to be
conditioned not only by their ecological requirements but also by their general
behaviour. Though they are generally polyphagous, they are not indiscriminate feeders
and the range of host plants is often correlated with natural taxonomic plant groupings,
but the host plants may be randomly distributed among different plant families as
evident in several species of acridids. As such the effective allocation of grazing
privilages to herbivorous insectslike acridids therefore tends to lead to diversity of host
selection patterns. Generally the distribution of grasshoppers is related to the
composition of the vegetation, as the movement of grasshoppers is much less in areas of
favourable vegetation, which accounts for the concentration of several species of
acridids in a particular habitat. However, the existence of many plant species and
grasshoppers in a given habitat may not indicate the host specificity of the insects as the
general behaviour of the insect is also involved. Feeding behaviour in acridids therefore
revolves around specific feeding patterns, proximate cues influencing the plant choice.
This paper highlights some aspects of the feeding behaviour on the basis of the studies
on Eyprepocnemis alacris alacris (Serv.) and Oxya nitidula (Walker) in relation to
283
284 M C Muralirangan and Meera Muralirangan
accrued infonnation on other acridid species, both endemic and exotic, focusing on the
physico-chemical factors which influence the pattern of feeding and feeding behaviour.
The following aspects of the feeding behaviour are discussed:
Orientation behaviour of acridids towards the food source; Feeding behaviour of
acridids in the host selection; General feeding patterns in acridids.
2. Orientation behaviour of acridids
Visual attraction, mechano- and chemoreception involving gustatory and olfactory
receptors are associated with the orientation behaviour of acridids towards the food
source.
2.1 Visual attraction and light
Attraction of an activated insect towards the host plant, involves a number of factors
such as visual and olfactory cues and continuation of the feeding activity, an essentially
light controlled reaction (Chapman 1954) mainly rests on the receipt of favourable
stimuli from the host. The movement of grasshoppers towards the food source in
reaction to light involves visual cues. Though feeding and other activities also take place
in the dark, most species are diurnal. Pielou (1948) showed in Nomadacris sep-
temfasciata (Serville) that they reacted positively to light. Kaufmann (1968) in
Melanoplus differentialis Uhler showed that it preferred Taraxacum to Poa grass in an
uniformly lighted environment, but if Poa was kept in the light and Taraxacum in the
shade, the insect preferred Poa to Taraxacum. The importance of visual attraction and
the role of vision in perception and identification of the hosts have also been
investigated in experiments involving the painting of eyes; total blinding results in
random feeding as observed in several grasshoppers (Mulkern and Mongolkiti 1977;
Meera 1982).
2.2 Position of the leaf blade
Many of the acridids tested in the laboratory showed a preference for upright blades
over those lying on the floor (Williams 1954; Hjelle and Mulkern 1964; Ba-Angood
1977; Meera 1982). In Zonocerus variegatus (Linn.), Chapman (1955) and Kaufmann
(1 965) demonstrated that the insect was attracted to the vertical objects within 7 cm and
could identify the leaf form from a distance of 10-15 cm. Hjelle and Mulkern (1964)
projected the leaf pattern on ground glass and found that Melanoplus femur-rubrum
(De Geer) was attracted to the vertical patterns, readily ascending the projected pattern.
Both in the laboratory as well as in the field, Meera (1982) observed a similar behaviour
in Oxya nitidula. Hence the upright leaf blades are always appreciated by the climbing
forms of acridids, although the leaf on the floor may be selected when there is no choice.
2.3 The colour of the host plant
The colour of the host plants also influences the food selection behaviour in
grasshoppers, though each grasshopper is believed to be attracted only to green colour,
Acridid feeding behaviour 285
grasshoppers like M. differentialis (Cresitelli and Jahn 1939) have been found to be
more sensitive to blue/green wave-lengths, though generally showing a preference to
the bright lush-green blades of the host. Most of the North Dakota grasshoppers
studied by Mulkern are responsive to a range of wave-lengths including green, and
hence it may be suggested that colour might also play a role in the feeding behaviour,
especially in the location of the host.
3. Tactile and chemoreception
Chemical stimuli may play the most important role in the detection, discrimination and
selection of host plants. It is likely that these insects may use combined visual and
chemical information to locate the potential host. In this connection, the structure
and functions of the receptors of the antennae and mouth parts are of considerable
importance in the various behavioural aspects connected with feeding by acridids.
The sense of smell plays a significant role in host selection and in feeding behaviour as
each insect responds only to smell which is relevant to it. It has been shown in all
acridids studied (mostly Locust sp.) that the surface of the antennae is crowded with
sensory hairs and tiny pits concerned with olfaction, useful in testing the host plant
during feeding (Goodhue 1963). In Melanoplus differentialis differentialis, M. mexicanus
mexicanus and Romalea microptera (Beauvois), Slifer et al (1957) has demonstrated that
the thin- walled basiconic pegs of the antennae are the major olfactory sensilla used in
testing the host for its palatability. Similarly, the maxillary palps also have a group of
sensilla which are responsive to volatile chemicals.
Many tactile and olfactory receptors on the tips of the maxillary and labial palps also
provide the insect with considerable amount of information, much of it being related to
feeding behaviour (Blaney and Chapman 1969a,b). Thurm (1965) and Nicklaus et al
(1967) associate the palp- tip sensilla with the mechano receptive function. On the basis
of his study on Locusta migratoria L., La Berre et al (1967) concluded that these sensilla
are capable of functioning as mechano- and chemoreceptors while the structure of
other? suggest a purely mechanoreceptor function. In Locusta migratoria and
Schistocerca gregaria (Forsk.) it has been shown that they have a circle of mechanore-
ceptors surrounding about 400 chemoreceptors. The chemoreceptors are of three types
viz (a) short-peg sensilla with pores and olfactory in function, (b) another group of
sensilla that reacts to solutions and volatile chemicals and (c) crested sensillae with a
single pore at the tip which are contact chemoreceptive and mechanoreceptive in
function. Such sensilla are also identified on the inner surfaces of Poekilocerus
hieroglyphicus (Klug) (Abushama 1968) and M. differentialis (Frings and Frings 1949).
Haskell and Schoonhoven (1969) proved the domes of the maxillary palps of L.
migratoria to be mechanoreceptor for testing the hardness of the grass, or contact
chemoreceptors which play an important role in food selection when the insect is not
starved for long (Blaney and Chapman 1970; Blaney et al 1973). Blaney (1974) has
shown that the individual neurons respond to a wide range of chemicals and are capable
of responding to more than one type of chemicals. The property of mechanoreception
was observed only in 47% of the sensilla tested. Blaney and Chapman (1969) and
Blaney et al (1971) found similar sensilla in S. gregaria.
In addition, to the sensilla of the palps, the laciniae and the galeae also carry
campaniform and trichoid- sensilla on their surfaces (Louveaux 1973; Chapman and
286 M C Muralirangan and Meera Muralirangan
Thomas 1978). These sensilla are believed to be directly concerned with the feeding
behaviour as they are found to be fewer in graminivorous species than herbivorous
species.
On the inner surface of the epipharynx lining the cibarial cavity is a closely-packed
group of sensilla which are regarded as chemoreceptors because of their characteristic
position (Viscuso 1974; Chapman and Thomas 1978). In S. gregaria and L. migratoria,
the inner surfaces of the clypeo-labrum contains very fine sensilla. Simple behavioural
experiments conducted by Cook (1972, 1976) with several chemicals have established
that the hexose sugars are the most powerful phagostimulants, stimulating these
sensilla. In Zonocerus variegatus, Chapman and Thomas (1 978) have identified several
types of scattered chemoreceptors.
Uvarov (1977) had postulated that the sensilla on the surface of the mandibles may
play an important role. Though it has been identified in certain species, it appears that
they are useful as secondary structures acting as chemo- or mechanoreceptors and they
may not play as important a role in host-selection as the palp sensilla. In addition,
before the actual testing of the host is initiated, acridids are found to tap the host with
their tarsi. Kendall (1971) showed that there are some chemoreceptors in the tarsi of S.
gregaria with which the insect, to begin with, is able to perceive and discriminate the
hosts.
4. Physical factors of the host and the morphological correlates of the insect
Many physical factors of the host have been observed as influencing the feeding
behaviour and host selection in acridids.
4.1 Leaf thickness
The leaf thickness has been shown to decide the nymphal feeding pattern with regard to
the gap of the mandibles and has been correlated with the inability of the early ins tars to
open their mandibles wide. Bernays and Chapman (1970) and Meera (1982) have
attributed the failure of the early nymphs of Chorthippus parallelus (Zett.) and Oxya
nitidula to feed on Festuca sp. and the crop plants like Oryza sativa, Panicum maximum
respectively to this factor. In 0. nitidula the adults feed on the mid-lamellar region while
the young ones prefer the apices of the leaves of Cyperus rotundus and Cynodon
dactylon.
4.2 Trichomes
In addition, the trichomes on the leaf lamina have also been found to influence the
nymphal feeding behaviour and food selection in 0. nitidula and C. parallelus. In 0.
nitidula, the early instars are unable to feed on the crop plants like Oryza saliva and
Panicum maximum because of their long trichomes which may hinder feeding of the
early mstars either physically or through their secretion and modify the general
behaviour of the insect.
Acridid feeding behaviour 287
4.3 Morphological correlates of the insect
The morphological correlates of the insect are also important as the physical factors of
the host. Observations of Eyprepocnemis alacris alacris (Serv.) and 0. nitidula
(Muralirangan and Ananthakrishnan 1978; Meera 1982) have shown that there seems
to be a difference in the feeding behaviour of the early instars and the late instars and
adults. The post embryonic development of the mandibles, especially the molar region,
if analysed, clearly exhibits a transformation of the morphological structure. As a result
of the transformation, the molar ridges becomes more complex during the post
embryonic development. If proper studies are undertaken, such observations could be
made even in other species as well.
Similarly, difference in the pattern and complexity of the foregut armature could also
be correlated with the feeding behaviour and the food habits of the grasshoppers and
this could be considered an ecological adaptation of the grasshopper concerned.
(Uvarov 1966). Muralirangan and Ananthakrishnan (1974, 1978) have analysed the
foregut armature pattern of 30 species of south Indian acridids and of the five zones
recognised in the foregut region, Z II and Z IV exhibit a marked developmental
differentiation during post embryonic development. Such a differentiation in the
foregut morphology has been observed from the very early instars, thus accounting for
the variation in the food habits. Based on the observations of E. alacris alacris and 0.
nitidula the morphological adaptation in the foregut structure is manifested in an
increase in the number of ridges, the complexity of teeth arrangements and the basal
chitinisation of the Z IV teeth from the III instar onwards, an adaptation that enables
the insect to process the tougher leaves of the crop plants with high silica content.
All these factors, viz. the physical factors of the host as well as the morphological
correlates of the insect are responsible for the absence of certain hosts in their diet,
especially in the I and II instars. Greater complexity of the grinding surfaces of the
mandibles with well-differentiated and well-chitinised foregut armature is the morpho-
logical adaptation not only to the silica content of the host but also to the thickness of
the leaves, when the nymphs are not able to open their mandibles wide. As a result of
these factors, a change in the feeding behaviour occurs, resulting in the shift of the host
from III instar, thereby increasing the total amount of food ingestion. Perhaps this may
be the reason for a sudden increase in the body weight of nymphs gained from III to IV
instar.
5. Chemical factors of the host influencing feeding
Though the feeding activity could be initiated by the sensory receptors of the insect, the
activation of these receptors requires a stimulatory factor to evoke normal feeding
behaviour. The leaf surface chemicals play an important role in determining the feeding
activity and acridids have been known to identify the phagostimulatory ones from the
deterrents by palpation and recognise the leaf form from the leaf surface attributes
(Chapman 1977).
Many substances stimulate feeding and such phagostimulants have been identified
and established for Schistocerca sp. and Locusta sp. (Bernays and Chapman 1977;
Cook 1977; Uvarov 1977). The substances which may stimulate feeding are specific and
the occurrence of suitable phagostimulants may, in part, effect feeding. Several such
288 M C Muralirangan and Meera Muralirangan
phagostimulants have been found to evoke a striking feeding activity in the older
nymphs and adults of Melanoplus bivittatus Say and Camnula pellucida (Scudder)
(Thorsteinson and Nayar 1963); S. gregaria (Goodhue 1963) and L. migratoria
(Mehrotra and Rao 1 966). Some aminoacids (like L. proline and L. serine), hexose sugar
and disaccharides are found to be phagostimulatory for L. migratoria (Cook 1977), and
Mulkern el al (1978) have analysed the experimental results on the attractants and
phagostimulants used for the control and estimation of grasshopper populations.
Some hosts are rejected because of the presence of certain chemicals that are
deterrents to feeding and such deterrent chemicals seem to play an important role in the
feeding behaviour of acridids. Combinations of feeding deterrents have also been
shown to be additive in their effects on the feeding behaviour of Locusta migratoria
(Adams and Bernays 1978). The release of HCN from the non-deterrent cyanogenic
glycosides has been shown quantitatively to play an important role with the increasing
maturity in the unpalatability of Sorghum bicolor to L. migratoria (Woodhead and
Bernays 1978). Probably all the plant species contain a combination of nutritional
substances, some of which tend to promote feeding activity while others inhibit it.
Some secondary compounds, deterrents for Locusta are ineffective as deterrent for
Schistocerca and some which are deterrent at high concentrations stimulate feeding at
lower concentrations (Bernays and Chapman 1978). Thus the sensory system of
forbivorous Schistocerca must receive more information on the nature and concen-
tration of secondary compounds than by an oligophagous Locusta. Rowell (1978),
while discussing the feeding strategy in relation to other aspects of life cycle, presumed
that the high diversity of secondary plant chemicals makes a general strategy
impractical. This is because acridid species have evolved a diversity to recognise only a
small number of phagostimulants rather than a larger number of feeding inhibitors.
Hence feeding depends on the balance between the phagostimulants and the feeding
deterrents and the response varies in different species so that a plant may be acceptable
to some but not to other species, or the rejection of hosts by these insects may be owing
to the presence of one or more chemicals in amounts which inhibit feeding; Poaceae and
few other plants are readily accepted only because of the absence of deterrent chemicals
in sufficient quantities to limit feeding (Bernays 1978; Bernays and Chapman 1977).
In order to differentiate these chemicals, a number of sensilla located on the mouth
parts, initiate the feeding behaviour on being favourably excited. Evidence suggests that
each of these sense cells in the chemoreceptors are particularly sensitive to one class of
chemical substances, perhaps because of the form of its receptor membrane (Chapman
1974a). With the excitation of different cells, it is able to differentiate between some
classes of chemical compounds, as between inorganic salts and sugars, and to identify
certain 'key' chemicals to which the cells are specially sensitive. Chemically similar
complex substances are differentiated, possibly by producing responses in several
sensory cells in each sensillum, the pattern of response of each cell varying in a
particular way so that the overall pattern from all the cells combined produces a
characteristic effect which could be interpreted within the central nervous system. As
the sensory inputs of some cells sensitive to feeding deterrents is not qualitatively
different from that of positively stimulating cells, differentiation between positive
stimulation and inhibition is presumably a function of the central nervous system
(Chapman 1974a,b). Only after elaborate exploratory behaviour of the insect— first by
the sensilla of the antenna, then by those of the maxillary and labial palps, test-biting
takes place. As a result, the group of sensillae i.e. contact chemoreceptors found on the
Acridid feeding behaviour 289
inner surface of the labrum are excited and they decide the palatability of the food
before it is engulfed; Chapman (1977) has estimated that at least 4000 sense cells and
12000 neurons are involved in the feeding behaviour of acridids.
6. General feeding patterns in acridids
6.1 Basic pattern of feeding
Feeding does not start until 6-1 2 hr after eclosion and no feeding occurs for the first few
hours after ecdysis. But the first one or two feeds are much longer. In the acridids so far
studied, as in several Locust sp., Oxya nitidula, Eyprepocnemis alacris alacris etc. the
first two feeds last over 20-30 min followed by a short resting period of 5-7 min. The
first instar generally feed on the apices of the leaf lamina while the late instars and the
adults are marginal feeders. In 0. nitidula, the leaves, leaf bases and the stems of C.
rotundus and 0. saliva, and occasionally those of P. maximum are consumed entirely
during shortage of food, while the stem and leaf bases of C. dactylon are never
consumed, even during shortage of food. Feeding commences after the test bite from
the -margins of the leaves (both in Oxya and Eyprepocnemis), continuing downward
towards the mid-rib in a semi-circle. Further cutting is inner to the first site, starting
from an anterior region and proceeding downwards. On the basis of 50 such
observations we have concluded that a semi-circle is formed either inward or above the
previous one. Such a definite pattern of feeding behaviour has been observed in most of
the graminivorous acridids so far studied.
With different types of food habits, non-graminivorous acridids deviate from the
graminivorous pattern though the feeding sequence remains the same. The forbivorous
species generally eat ovoid, net- veined leaves rather than the linear and parallel veined
leaves. Because of the venation, it is unable to remove morsels by a combination of
incision and splitting between veins. The cut made — by incision only — is necessarily
escalloped and irregular.
The classification of acridids into herbivorous, forbivorous and graminivorous types,
is associated closely with the structure and the shape of the mandibles. Mandibles of the
forbivores (Catantopinae) have an armature of irregular and sharp incisors; those of the
graminivores (Truxalinae and Acridinae) have incisors in parallel ridges often fused or
worn into a semi-continuous cutting edge; the herbivores have incisors intermediate
between the above two types. The three kinds of cutting patterns of the leaves exist as a
result of the three types of mandibles; frilled margin of leaf by forbivores; even
margined cutting by graminivores and intermediate one by herbivores (Gangwere
1972).
The initial choice of the host by adults is made by visual stimulus followed by tapping
of the leaves by their antennae and tarsi. The over-all picture of the feeding behaviour
seems to be similar in all acridids (Gangwere 1972; Mordue (Luntz) 1979; Meera 1982).
While selecting a host plant, both maxillary and labial palps are repeatedly moved. This
"palpation" (Blaney and Chapman 1970) continues, when the animal is in search of
food as well as during feeding; after the interfeed period, again palps are projected. On
coming into contact with the host plant, the head is lowered with the hypognathous
mouth parts pushed outward touching the food. Upto this point, the behavioural
pattern is considered as 'exploratory behaviour' (Mordue (Luntz) 1979). The head first
290 M C Murcdirangan and Meera Muralirangan
moves in a backward and downward path to bring the incisor cusps of the mandible in
contact with the food from its margin. The continuous palpation during feeding is also
attributed to their mandibular movement. Depending upon the acceptance of the test-
bite the host plant is either well consumed, nibbled or rejected altogether, rejection
taking place while probing, palpating or test-biting.
Thus, palpation is a step towards the selection of the host plant, the final choice or
rejection being made only after the test-bite by which the sensory inputs are produced
from the sensilla; and if the reaction is favourable, then the initiation of feeding begins.
The role of maxillary palps in feeding has been assessed in 0. nitidula through
palpectomy and antennectomy and these experiments have shown that (i) without both
the maxillary palps, the adults are able to identify their hosts with their antennae to a
certain extent, but even an inert crape paper strip is bitten immediately; (ii) with only
one palp, O. nitidula palpates and feed normally, but the rate of feeding is slower than in
normal-control insects. In the absence of the palps, the labrum is brought into contact
with the food often and in S. gregaria exploratory behaviour is significantly reduced in
insects without palps. Mordue (Luntz) (1 979) has shown in S. gregaria that the ability to
perceive gustatory stimuli is reduced by removal of maxillary and labial palps, because
single chemical like sucrose is not recognised easily in the absence of palps, (iii) without
both antennae but with both the palps intact, the insects are much slower in the location
of the food than the palpectomised insects, the adults palpating slowly on the food
plants as well as on the inert media (Meera 1982). Antennectomised grasshoppers do
not behave normally in feeding as they fed even on the non-preferred host. This is more
pronounced when both antennae and palps are removed.
6.2 Initiation and continuation of feeding
If the grasshoppers are not deprived of food for a long time, feeding is normally
initiated by chemical stimulation. If starved, they bite indiscriminately and only after a
full meal, they revert to the normal feeding behaviour as reported in several locust sp.,
0. nitidula and Eyprepocnemis a. alacris. In acridids no specific chemical has been
identified initiating biting and swallowing (Goodhue 1963). In acridids, feeding is
generally initiated by sugars, by sucrose in particular (Bernays and Chapman 1978),
which are perceived by the sensilla of the palps, the epipharynx and the hypopharynx,
and the stimulation of any of these sensilla initiate feeding. Immediately after a full
meal, the grasshopper generally exhibits little or no feeding and shows a post-prandial
rest for about 40-70 min, after which feeding once again commences but this process of
starting again is sudden. The increase in potential reponsiveness after the post-prandial
rest can be envisaged as a result of a progressive decrease in the various inhibitory
inputs which lead to the cessation of feeding. As a result, the insect starts to feed again.
The mechanism which causes the feeding behaviour even in the absence of any changing
external conditions is unknown (Chapman 1982) but Simpson (1981) indicates the
rhythmic changes occurring in the central nervous system as a possible factor.
For continuation of feeding, a continuous and sustained sensory input is necessary.
Feeding stops in Locusta, if they are provided with an inert substrate (Chapman 1982).
So in a normal meal the chemosensory inputs are continuous, but the effects of sensory
inputs are not limited to the immediate response; but according to Barton Browne
(1975) there is also a sustained preservation effect, which has been demonstrated in
Acridid feeding behaviour 291
acridids like Locusta migratoria (Bernays and Chapman 1974) and Chonhippus
terminifera (Barton Browne et al 1975). It is probable, therefore, that these inputs
influence the duration of the meal and continue to have their effects on the sensilla.
Perhaps the initial stimulation sets the level of the central excitory stage (see Barton
Browne 1975; Dethier 1966) which then persists in the presence of chemosensory inputs
signalling the existence of a suitable host even after the original stimuli have ceased to
be effective.
6.3 What causes them to terminate feedingl
According to Chapman (1982) two types of phenomena are involved in the termination
of feeding, viz volumetric feed-back and chemosensory inputs. The feeding stops when
the foregut gets filled with food. The last part to get filled is the anterior-most end of the
foregut. If the post-pharyngeal nerve which runs from this region to the frontal
ganglion is cut, it results in an excessively large and prolonged meal (Rowell 1963;
Bernays and Chapman 1973). If the inner oesophageal nerve is cut, the food is retained
in the front part of the foregut distending the crop, and the feeding stops although
relatively little food has been ingested. So it seems certain that the inputs from the
stretch receptors from the pharyngeal region may have an inhibitory effect on feeding.
The implication in such cases, as suggested by Sinoir (1968) and Bernays and Chapman
(1974), is that the distension of the foregut is the major importance in the termination of
the feeding behaviour.
While the volumetric feed-back possibly imposes an absolute limit in the meal size,
there is evidence for its interaction with other inputs from the peripheral chemorecep-
tors. For example, there is a difference in the meal size of nymphs of L. migratoria when
fed on mature grasses. If they are fed on Agropyrun sp. previously, they consume more
of this grass in one meal than Poa grass but after pre-feeding or habituation on Poa the
converse is true (Bernays and Chapman 1972). A similar observation has been made in
0. nitidula (Meera 1982) and S. gregana (Azzi 1975) as well. Chemical stimulation of
receptors of the mouth parts before feeding leads the insect to take a larger meal.
The importance of chemosensory inputs and the termination of feeding have been
clearly demonstrated by the experiments of Blaney and Duckett (1975); continuous
stimulation of the sensilla of the maxillary palps of L. migratoria with feeding
deterrents have been observed to reduce the length of the meal even though the other
sensilla are exposed to the whole meal in the normal way. In contrast, a sucrose extract
on the palp lengthens the duration of the meal. Cook (1977) and Bernays and Chapman
(1978) have shown in L. migratoria and S. gregaria respectively that the meal size is
dependent on the concentration of sucrose. Hence it may be concluded that the effect of
stretch receptors is modulated by the chemical inputs.
Normally the duration of the feeding may be longer, often lasting more than 10 min.
During this period, the adaptation of the sensilla on the tips of the palps is offset to
some extent by the rapid vibration of the palps, which makes sure that they are brought
in physical contact with the host ten times for a very brief period of about a second
(Bernays and Chapman 1970). As a result of this, the information is transmitted to the
central nervous system (Blaney and Duckett 1975). But Mordue (Luntz) (1974) has
observed that even in palpectomised nymphs of S. gregaria, duration of feeding is
292 M C Muralirangan and Meera Muralirangan
normal. In such circumstances it is suggested that the cibarial receptors as well as the
antennal sensilla might overtake this function.
The work of Bernays and Chapman (1974) indicated that the sensory adaptation is of
little importance in the regulation of meal size in acridids feeding on normal food. Then
how does the chemical input influence the meal size? The experiments of Blaney and
Duckett (1975) have shown that the inputs from the sensilla have some lasting effect,
resulting in a decrease in the meal size. Since the palps provide information on the meal
size intermittently during feeding, it is likely that the closure of the sensilla, which
deprives the insect of the stimulatory inputs, may lead to the cessation of feeding. But the
meal lengths do not differ in controls as well as in palpectomised insects (Chapman 1982).
Bernays and Chapman (1973) have reported that the distension of the crop by a full
meal leads to the release of a hormone from the storage cells of corpora cardiaca.
Bernays and Mordue (1973) have suggested that the hormone effects the tips of the
palps by closing the pores of the terminal palp sensilla which then become non-
ifunctional. This hormone also decreases the locomotory activity after feeding (Bernays
1980). It is suggested (Cazel 1969) that they might enhance the movement of the foregut
so that the food moves out and the volumetric feedback from the crop to stop feeding is
removed.
It may be concluded that the first information from the stretch receptors of the
foregut is relayed to the brain so that feeding behaviour is continued or switched off.
More long term effect is brought about by the release of one or more hormone from the
carpora cardiaca. This effect persists for an hour or more during the post-prandial rest,
but after 2hr or more they become fully functional (Bernays et al 1972; Bernays and
Chapman 1973). This closure of the terminal sensilla decides the end of the meal and
prevents further feeding.
Acknowledgements
The authors express their deep sense of gratitude to Prof. T N Ananthakrishnan for
encouragement and valuable suggestions, and also for critically going through the
manuscript. One of the authors (MCM) thanks the UGC for financial assistance.
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© Printed in India.
Behavioural response (feeding preference and dispersal posture) of
Aphis gossypii Glover on brinjal crop
T K BANERJEE and D RAYCHAUDHURI
Department of Zoology, Calcutta University, 35 Ballygunge Circular Road, Calcutta 700019,
India
Abstract. During late July to early August (kharif) and late November to late December
(rabi), alates of Aphis gossypii Glover appear on the old leaves of the tender brinjal plants.
Population build up is quite gradual with a sigmoid growth and the pest reaches peaks during
late December (kharif) and late February (rabi) when plants become mature. In course of
incidence they distribute from old to young to tender leaves and the aphid incidence on these
leaves is always followed by the migration of alate immigrants. Thereafter, the aphid prefers
mature tissues of the crop and in course of crop maturity its dispersing trend follows from old
to the other leaves as well as plants in a sigmoid posture in a latero-angular spectrum.
Keywords. Brinjal; Aphis gossypii; sigmoid growth rate; matured tissue preference; latero-
angular sigmoid dispersal contour.
1. Introduction
Aphids appear to have evolved the ability to profit from the environmental cues (e.g.
mutual tactile stimulation, photoperiod, properties of different parts or ages of the host
plant or of different plant species) to develop the morphs most suited to successful
exploitation. They are found to disperse to the host plant as immigrant alates to exploit
it if it is suitable and if not they tend to be away from there as emigrants. Thus the
dispersive behaviour of alate migrants for better niche facilities is directly dependent on
crop infestation. However, several factors for the development of the said form have
been studied by several authors (Wadley 1923; Evans 1938; Dickson and Laird 1962;
Johnson 1966; Lees 1967; Dadd 1968; Mittlcr and Kunkel 1971; Raccah et al 1971;
Schaefers and Judge 1971; Ghosh and Mitra 1979; Rajagopal and Kareem 1979) in
different species. Kennedy and Booth (1951) categorised the factors and flavour stimuli
and nutrient stimuli to enhance alate migration. Muller and Unger (1951) also
proposed a funnel-like locomotory path of alate immigrant of Aphis fabae Scop, in
intercrop ( Viciafabd) migration. The present study aims at gathering knowledge about
the feeding preference and intracrop-dispersal posture of Aphis gossypii Glover on
brinjal.
2.. Material and methods
In a plot (20 m x 20 in) in Hooghly district (W.B.) twenty plants of brinjal
(Krishnanagar cluster in kharif and Pusha purple long in rabi seasons) were planted.
Observations were made on randomly selected three leaves (old, young and tender) per
plant at 15 days interval. During observations adults and nymphs (3rd and 4th instars)
295
296 T K Barter jee and D Raychaudhuri
of both forms (alate and apterous) were separately counted along with the counting of
colonies. Simultaneously the rate of infestation of plant and leaves were also recorded.
Observations on kharif (1980-82) and rabi (1981-83) crops were recorded separately.
3. Results
3.1 Incidence pattern
The data on mean population (table 1) shows that after the initiation of colony aphids
(A. gossypii Gl.) on brinjal plant followed a gradual increasing trend to reach the climax
during late December (kharif) and late February (rabi). In both the seasons the aphid
retained colony over the crop up to its harvest. Further, A. gossypii Gl. appeared on 2
month old hosts and the individuals in the population pyramid of the same increased
with the rise of the host age (figures 1 C and F). Again, initiation of population was
Table 1. Kharif and rabi incidence of aphids and correlation coefficient between the
increasing change of plant infestation with the decrease of alate adult incidence from old leaves.
Month
Incidence
of aphid (%)
Incidence
of alate
immigrants
(%)
Increasing change
of infested
plant (%)(*)
Decreasing change
of alate adults (%)
from old leaves (y)
Correlation
coefficient
(r) between
x and y
Time
Kharif
J12
18-33
13-66
0
0
A
57-77
18-94
8-35
8-33
*2
68-33
13-86
12-20
9-96
Si
113-88
10-22
3-35
+ 2-00
*2
98-33
4-16
-5-00
6-79
o,
126-66
4-11
5-00
+ 5-74
*
02
156-10
6-15
5-00
8-88
0-605
*1
123-33
6-47
5-00
8-60
*2
199-99
10-87
1-65
3-98
01
168-88
15-30
-3-30
3-52
02
199-99
17-06
0-80
5-58
Rabi
*2
13-88
4-00
0
0
01
71-11
13-98
10-00
35-77
02
80-22
10-33
1-70
12.63
Jl
108*33
20-54
1-30
+ 7-20
J2
151-66
11-40
7-00
+ 1-50
fl
188-88
9-21
3-35
7-63
**
F2
247-22
8-90
3-35
15-19
0-689
M,
244-99
9-75
1-65
0-05
M2
152-77
13-79
-10-00
4-79
*i
118-88
19-11
-5-55
4-95
AI
175-55
26-06
-0-55
2-91
Data plotted here, mean of three years' observation.
* Significant at 1 %.
Significant at 5%.
Behaviour response of Aphis gossypii 297
mostly restricted to the old leaves and within a short period, aphid incidence could be
traced on old, young and tender leaves. In course of aphid incidence on the old leaves it
was marked with a gradual decrease whereas it was reverse on the young leaves. But on
the tender leaves no such trend could be traced though there was a relatively higher
incidence on it just prior to the harvest. Interestingly, the old, young and tender leaves
had higher incidence values during early, middle and late sessions of the crops
respectively in both the seasons but in no case incidence on old leaves could be
surpassed by the others.
Another interesting feature was the incidence of the alate adults which could be
marked with a higher percentage during early and late sessions of the crop seasons. The
incidence of the said morph was relatively lower during the mid session. Like the trend
of aphid incidence, the higher values of alate adults were marked successionally on old,
young and tender leaves during early, middle and late sessions of the crop respectively
(figures 1A and D; figures 2 B and E).
3.2 Growth rate
The growth curves (figures 1 B and E) show a modified sigmoid form where the upper
asymptote level was marked during late November (kharif ) and late February (rabi)
when the plants were fully mature (5-7 months old).
3.3 Infestation percentage
Maximum incidence of A. gossypii Gl. infested plants and leaves in maximum
frequencies (figures 2 A and D). Furthermore, the leaf infestational trend was
distributed from old to young to tender leaves in course of its incidence (figures 2 C and
F). Again it could be seen (table 1) that decrease of alate immigrant incidence from old
leaves positively correlated (r = 0-605/kharif and 0-689/rabi) with the increase of plant
infestation.
4. Discussion
It has often been shown that the aphids (A. gossypii Gl.) favour winter with low
temperature and optimum rh (Bodenheimer and Swirski 1957; Agarwala and
Raychaudhuri 1979; Roy and Behura 1979). Here too the aphids showed abundance on
Figures 1 A-F. Preferential behaviour of aphid to the host tissues. A, D. Population
distribution on different leaves B, E. sigmoid (s-shaped) population growth form and
C, F. population pyramid of Aphis gossypii Gl in kharif and rabi crops of brinjal. These show
that the aphids always favour the mature tissues of the brinjal crop.
Figures 2 A-G. Intra crop dispersal-trend of aphid (Aphis gossypii Gl.) on brinjal.
A, B. Incidence of aphid on the crop in relation to plant and leaf infestation.
C, D. Distribution of alate adult at the different levels of the leaves. E, F. Rate of infestation
of different leaves during pest incidence. These suggest that the aphids tend to be dispersed
from old to young to tender leaves. G. Model showing intra crop dispersal contour of aphid
(Aphis gossypii Gl.) in brinjal.
T K Banerjee and D Raychaudhuri
c
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( %) 30U3ppUl
Behaviour response of Aphis gossypii
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mature brinjal plants. Nevertheless, the forms of population pyramids diverged
towards old age, as they were active during that period. Again, the trends of aphid
incidence (colony as well as alate immigrants) from old to young to the tender leaves
and the retention of the same over old leaves during harvest reflect a behavioural
affinity of the aphids towards the older hosts. Furthermore, the higher growth rate with
sigmoid form on the old plants during late session, indicates the host status where the
aphids find normal situation in which population size deviates less from the asymptote
(Odum 1971). This could be placed as a supplementary factor for the former reflection.
Again to pay emphasis over aphid-host interaction in relation to matured tissues several
factors including emergence of largest veins (Gibson 1972), intermittent water stress
(Wearing 1972), nitrogen saturation (van Emden et al 1969) have been referred.
Moreover, Turner (1971) found that the growth of A. gossypii Gl. was haltered in the
absence of methionine. Though the present study does not deal with any of these
factors, it is obvious that the aphid prefer to feed on older tissues rather than the young
or tender.
Based chiefly on alate incidence and the relative infestation of the plants by them the
speculation has often been advanced that the initiation of aphid colony is being made
by the alate adults. Subsequently, factors like feeding competitiveness (Ghosh and
Mitra 1979), crowding effect (Mittler 1973) and reduced moisture content of the host
(Ratanlal 1951) enhance aphid incidence thereby increasing the rate of host infestation.
But it is obvious that throughout the season alate persisted in all the leaves though the
relative percentage varied during different periods. It is noteworthy that in the
observation, the preabundance of alate adults led the abundance of aphids in general
along with the peak percentage of infestation. It is quite clear from the correlation
between the decrease of alate immigrant incidence and increase of plant infestation,
that the aphids tried to avail latero-angular dispersal than vertical or irregular. Thus the
projectile of alate percentage on different leaf level, a sigmoid dispersal contour
(Wolfenbarger 1946; Odum 1971) appears (figures 2 B, E and G).
Acknowledgements
The authors thank Dr M R Ghosh and B C K V V Kalyani for constructive suggestions
and Mr S N Basu for the drawings.
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(D Printed in India.
Behavioural analysis of feeding and breeding in LaiBellicorn beetles
G K VEERESH and K VEENA KUMARI
Department of Entomology, University of Agricultural Sciences, Bangalore 560 024, India
Abstract. Social behaviour is recognised in nine families of Coleoptera. The Lamellicorn
beetles, in the families Passalidae and Scarabaeidae exhibit varying types of social behaviour.
Sound production by stridulation in both the larvae and adult passalids is attributed as a
social behaviour to hold the families together.
Some South American scarabs live very close to the anus of sloths and monkeys in order to
oviposit on their dung. Many have association with ant nests either for food, shelter or
breeding.
The dung beetles present a whole sequence of bisexual cooperation in the nesting behaviour,
excavation and ball-rolling. Parental care is exhibited to a varying degree.
An attempt has been made to review the feeding and breeding behaviour of Lamellicorn
beetles in the light of available Indian literature including studies made by the authors.
Keywords. Behavioural analysis; Lamellicorn beetles; feeding and breeding behaviour.
Feeding and breeding habits seen in many Lamellicorn beetles are fundamental
features of their biology which determines the characteristics of their behaviour,
distribution, morphology and development. Their food habits are varied but most of
the free living scarab adults and larvae are saprophagous or phytophagous. In addition
many members belonging to the six subfamilies, the Scarabaeinae, Aphodiinae,
Ceratocanthinae, Melolonthinae, Dynastinae and Cetoniinae are found in the nests of
ants, termites, honey-bees, wasps etc.
Although "Truly Social" or "Eusocial" species which meet three criteria like living in
groups as adults of different generations, with co-operative activity and different
individuals performing different roles for the success of the colony, are not found in
scarabs, yet varying types of social behaviour are encountered especially in Passalidae
and Scarabaeinae.
The hind legs of larvae of the scarabaeoid families Lucanidae and Passalidae and the
subfamily Geotrupinae often have stridulatory organs on the coxae working against
those of the middle legs. In Passalidae the great reduction and modification of the hind
legs has little effect on locomotion but are believed to be of great help in communication
by stridulation to hold the family together which is attributed as a social behaviour.
The Passalids are considered to have a primitive society. They are gregarious living in
the same tunnel system but during reproduction, each beetle maintains its own tunnel,
lays eggs. The developing young feed on the material prepared by the adults. The wood
consuming species lack digestive symbionts and mix fecal pellets and frass which act as
a substrate for bacterial and fungal development, as was found by Matthews and
Matthews (1978) in Odontotaenius disjunctus whose larvae cooperate with the adults in
the construction of the pupal chamber. Parental care is also exhibited in passalids.
Two species of passalids are commonly found in the decaying, wet logs in the
evergreen forests of the Western ghats in Karnataka. These two species Episphenus
indicus (Stol.) and Plaurarius brachuphyllus Stol. are often in the same log, side by side
303
304 G K Veeresh and K Veena Kumari
but will have distinct tunnels in which their own larvae are lodged separately, thus
exhibiting a niche behaviour. The larvae, like adults, are gregarious, 3 cm long, with
well developed first two pairs of legs, the hind legs being greatly reduced and stubby.
These larvae live very close to each other in the tunnel and move quickly to come
together when separated.
In Cetoniinae, the adults are usually phytophagous and the larvae feed on dung,
humus or decaying wood. However some species become adapted to the nests of ants
and termites. Potosida cuprea spends the larval and pupal stage in the nests of Formica
rufa in Europe (Wheeler 1910). Larval cases are generally ignored by the ants as they
look like lumps of earth. Other cetoniids like Potosia cuprea (Fab.) and P. lumgarica
Herbst feed as adults upon honey in bee hives while the larvae feed on decaying organic
matter (Caron 1978).
In South Africa the larvae of the green protea beetle (Tnchostethafascicularis) live on
termite droppings for two years in the mound of the termite Amitermes hastatus,
whereas the adults are flower feeders (Skaife 1955).
A cetoniid beetle Coenochilus taprobanicus Westwood and a valgine scarab
Oreoderus argillaceus(HopQ) are commonly found in the nest of Odontotermes
wallonensis in South India. They feed on the fungus reared by the termites (Rajagopal
and Veeresh 1981). According to Kistner(1982) the principle adaptation here seems to
be the conditioning of the wood by the termites rather than social interactions with
similar eating habits.
The Cetoniiae beetles having predaceous food habit is reported from North America
and India. Adult Cremastocheilus stathamae Cazier (Cetoniinae) are obligate predators
of the ant larvae in the nests (Cazier and Mortenson 1965). Another interesting feed-
ing behaviour of a cetoniid beetle Spilophorus maculatus (Gory and Percheron) has
been reported by Ghorpade (1975) from Southern India. These beetles feed on the
nymphs of the treehopper Oxyrhachis tarandus Fab. occurring on Acacia concinna.
Cremastocheilini feed on a variety of insects but it is not known whether they became
predaceous before or after their invasion of ant nests.
The two genera Chaetopisthes and Corythoderus of Aphodiinae, are found with
Odontotermes sp. in India. Chaetopisthes assmuthi Wasmann is quite common in the nests
of Odontotermes obesws(Rambur) (Wasmann 1903). Although these beetles are nor-
mally found in the fungus gardens, they may also occur in the royal cells. The termite
workers find the trichomes attractive and carry the beetles from place to place and the
beetles feed on the fungus (Kistner 1982) but how this help the termites is not known.
Melolonthines are rarely reported from ant and termite nests. A species ofDiplotaxis
is reported from the nest of Pogonomyrmex occidentalis (Idaho, USA) and a species of
Maechidias in ants and termites nest is reported from Australia (Lea 1910).
The Dynastid, Coelosis bilobata Linn, found with Ana sexdens in Brazil is supposed
to be the largest of all the myrmecophilous arthropods (Eidmann 1931). The adult
beetles lay eggs in the leaf mulch which are carried to the fungus gardens where they live
in oval earthen holes and feed on fungus.
Nest making reaches its epitome among dung and carrion feeding Scarabaeinae
(Eickwort 1981). Since the food source, dung or carrion, is ephemeral and randomly
scattered it should be removed and protected from desiccation and has to be buried
before egg laying. Both sexes frequently participate in food provisioning, defence and
prevention of fungus contamination resulting in parental care and true subsocial
behaviour.
Feeding and breeding behaviour in Larnellicorn beetles 305
The peak of subsocial behaviour is seen in Cephalodesmius and Necrophorus in which
larvae are provided food by regurgitation (Wilson 1971). In Cephalodesmius the male is
responsible for foraging and the female molds the food into 'cake' and allow it to
ferment for two weeks adding adult feces to it. This "home made dung" is thus
partitioned by the female into six to ten brood balls. The male and female remain in the
nest till their offspring emerge as adults (Halffter 1977).
According to Halffter and Matthews (1966). "Scarabaeine beetles live in a world of
smell and touch almost exclusively and that a suitable ambient temperature is the first
requisite for activity". Olfaction seems to be the dominant sense of scarabaeines, image
perception not being that dominant due to poor vision. Light is used perhaps only for
orientation. Sound production although exists in most scarabaeinae, auditory stimuli is
very less. Tactile perception seems to be highly developed particularly in ball-rolling
beetles.
Considering the Scarabaeinae as a whole, the food used by the majority of the species
both for the larvae and for adult is the excrement of large animals, particularly of
mammals and man, suggesting that these beetles are coprophagous. However other
types of food habits, among the scarabaeines are not very rare.
Necrophagy is found in one genus Onthophagus in South America and India. A
number of species of Onthophagus are known to be carrion feeders in India.
Onthophagus igneus, 0. unifasciatus, 0. pygmaeus and O. kchatriya were found in the
carcasses of crows and frogs in Bangalore (Veena Kumari 1984).
Saprophagous scarabaeids are not uncommon. There are different types of
saprophagous Scarabaeinae feeding on leaf litter, vegetable debris, decaying fruits,
fungi etc.
Predatory habits among the scarabaeines are rare except a Brazilian species of
Canthon which attacks ants of the genus Alia (Navajas 1950).
In addition, there are several special ecological niches where the scarabaeines are
found, although their food habits are not well defined.
There are reports of scarabaeines on ectocommensals of mammals like monkeys in
Brazil, sloths in America, kangaroos and wallabies in Australia.
As endoparasites of mammals there are reports of scarabs causing 'Scarabiasis' in
India among human beings resulting in recurrent intestinal illness accompanied by
bloody diarrhoea due to Onthophagus bifasciatus (Fab.) and Caccobius vulcanus (Fab.)
(Senior-white 1920; lyengar 1923).
In recent years there are reports of Scarabaeinae, mainly Onthophagus, occurring in
nests and burrows of vertebrates, particularly in rat burrows.
Examples of termitophily and myrmecophily in Scarabaeinae are many.
Arrow (1931) has reported presence of Sisyphus longipes in the nest of Pheidole
rhombinoda in Madras.
Largest number of Scarabeinae are known from Grassland biomes and Forest
ecosystems. High mountain colonization of these beetles is known from the Himalayas.
Various species of Copris are known to climb high mountains between 2000 and
2500m. Caccobius himalayanus Jekel has been collected frequently at 3000m and
Onthophagus tibetanus Arrow lives between 3000-4200 m in Sikkim and Tibet. The
highest locality known for any scarabaeid is that of 0. cupreiceps which is found
at 5200 m and the same has not yet been collected below 4000 m (Arrow 193 1; Balthasar
1963). Feeding and breeding behaviour of these high altitude scarabs are not known
clearly.
306 6 K Veeresh and K Veena Kumari
Fossilized scarab brood balls have been described from various tertiary deposits in
South America, which demonstrate nidification behaviour at a fairly advanced level in
Scarabaeinae, as early -as in the lower Oligocene (Balthasar 1963).
Detection of food and approach behaviour like search flight, altitude of flight,
distance at which the smell of food is first perceived in the Scarabaeinae are not fully
understood. These behaviours differ from species to species and place to place
depending on the source of food.
Most of the dung beetles land a little away from the pat and crawl towards the food.
Some, like most Coprini come to semiliquid cow dung immediately after deposition,
and utilise it in that state. In the case of human excrement many species come a few
minutes after deposition of the feces. The Eucraniina dung beetles habitually go to dry
excrement under semi desert conditions. 0. tritinctus is found attracted to dry dung in
Bangalore (Veena Kumari 1984).
Feeding behaviour differ from group to group. Adult Scarabaeinae and Geotrupinae
nearly always bury the food both for themselves and for their larvae directly beneath or
beside the food source. In the genus Gymnopleurus most of the species feed 'at the
surface. G. miliaris and G. spilotus recorded from Bangalore fall under this category.
Overland transportation of food without formation of ball, is done in three
ways: (i) carry food with forelegs and walk backward towards the burrow e.g. Copris
spp. and Onthophagus spp. (ii) pieces of food rolled away from the source without
making balls and walking forward and pushing with its head and forelegs. This
"butting" technique is seen in Onthophagus tritinctus (iii) the beetle grasps the food
with the forelegs and head, and elevating the fore body it runs rapidly forward on the
remaining four legs as found in Argentine subtribe Eucraniina (Kolbe 1905).
Overland transportation with formation of ball is common to the tribe Scarabaeini
with a few exceptions. Ball rolling behaviour of dung beetles has been studied in detail
in various parts of the world (Scarabaeus spp., Gymnopleurus spp., Sisyphus spp. and
Canthon spp.)
The biological advantage of ball rolling is that the ephemeral food source, scattered
randomly has to be protected from competition from other insects and desiccation.
The ball rolling behaviour seems to have originated with the habit of carrying more
or less spherical pellets such as those of rodents, lagomorphs and caprines and later they
might have developed other techniques for rolling (Halffter and Matthews 1966).
Ball making behaviour, initiation of ball rolling, the role of sexes in ball rolling, the
direction in which balls are rolled, distance rolled and burial of the ball have all been
well documented for several species from many parts of the world and the same is
beautifully summarised by Halffter and Matthews (1966). The above behaviours are
well exhibited in Gymnopleurus miliaris, and Gymnopleurus geoffroyi.
Nidification behaviour in Scarabaeinae may be classified into four groups:
(i) Egg laid directly in the food mass packed into the blind end or branch or a burrow
dug near or under food source e.g. Onthophagus spp., Onitis spp.
(ii) Egg laid in a pear shaped shell covered with soil, constructed under the food
source e.g. Catharsius spp.
(iii) Spacious underground chambers are constructed near or under the food source,
a large mass of dung is compacted and then divided into several brood ovoids
containing one egg each not enveloped in a clay shell. Male and female remain in the
nest till the larvae develop, e.g. Copris, Synapsis, Catharsius spp.
(iv) Formation of ball of food on the surface and rolling away on the surface and
Feeding and breeding behaviour in Lamellicorn beetles 307
laying an egg in it. e.g. Gymnopleurus, Scarabaeus, Sisyphus etc.
Male and female cooperation in ball formation and burying the dung, combat and
parental care have been reported from a number of species like Copris hispanus, Copris
lunaris, Heliocopris dilloni etc.
The larval behaviour of some of the Scarabaeinae are interesting. In Copris repertus
the larva repairs the breech in the brood ball if it is damaged. Also the larvae make a
scratching noise when the ball is touched. This noise is a result of scratching the inner
wall of the ball with its mandible. Melolonthine beetles emerge at a particular intensity
of light (foot candle) in the evening after the first summer rains and get back to the soil
early morning at the same intensity of light. Although rain is a must for adult emergence
yet unless the gonads are well developed, the young ones will not emerge inspite of the
rains. Holotrichia serrata needs a minimum of 23°C soil temperature for its gonads to
mature (Veeresh 1983).
Among the phytophagous lamellicorn beetles some species show strong tendencies
towards a particular host plant, the presence of which decides the distribution of the
pest. Adults of Holotrichia serrata F. goes only to neem plants Azadirecta indica in the
midst of several of its host plants. Likewise adults of Holotrichia nilgiria Arrow has
decided preference to Ficus racemose and the pest distribution is confined around the
adult beetle's, host plants, in coffee plantations. Similarly Holotrichia reynondi Bl.
concentrates around Moringa oleifera to which it is highly attracted.
Concentration of Holotrichia to a particular patch of field seems to be guided
by the adults egg laying behaviour, which in turn is influenced by the first few adults
going to a particular side of a host tree or a particular patch of a field for egg laying.
There is a strong tendency of the beetles following a pheromone trial (Veeresh 1983).
The Leucopholis spp. have no attractive adult host plants. Leucopholis coneophora
female attracts the male while it is still in the process of emergence and more often it gets
back into the soil from where it has emerged, for egg-laying.
These are but a few scattered reports of behavioural analysis of feeding and breeding
in this vast and widely distributed group of Lamellicorn beetles. Except for a few
reports, nothing is available from India on the behaviour of Lamellicorn beetles. Many
more fascinating accounts of behaviour particularly of Scarabaeinae will come to light
if more and more attempts are made on these abundantly available beetles.
References
Arrow G J 1931 The fauna of British India including Ceylon and Burma. Coleoptera Lamellicornia HI
(Coprinae) (London: Taylor and Francis) V-XII 428 pp. 28 pp.
Balthasar V 1 963 Monographic der Scarabaeidae und Aphodiidae der palaearktischen und orientalischen
Region. Tschechoslowakishe Akad. Wissenschaften. 1 391 pp.
Caron D M 1 978 Other insects. In Honey bee pests predators and diseases (ed) R A Morse (Ithaca New York:
Cornell Univ. Press) Chap. 10 pp. 158-185
Cazier M A and Mortenson M A 1965 The behaviour and habits of all myrimecophilous scarabs
Cremastocheilus (Coleoptera: Scarabaeidae); J. Kans. Entomoi Soc. 38 19-44
Eickwort G E 1981 Presocial insects in Social insects (ed.) H R Hermann (New York: Academic Press) Vol. 2
pp. 199-280
Eidmann H 1931 Die Gaste and Gastverhaltmisse der Blattschneidermeise Ana sexdeus L; Z. Morphol.
Oekoi Tiere 32 391-462
Ghorpade K D 1975 A remarkable predaceous cetoniid Spilophorus maculatus (Gory and Percheron) from
Southern India (Coleoptera: Scarabaeidae); Coleopt. Bull 29 226-330
308 G K Veeresh and K Veena Kumari
Halffter G and Matthews E G 1966 The natural history of dung beetles of the subfamily Scarabaeinae
(Coleoptera: Scarabaeinae); Folia Entomol Mex. 12-14 312
Halffter G 1977 Evolution of nidification in the Scarabaeinae (Coleoptera: Scarabaeidae); Quaest. Entomol.
13 231-253
lyengar MOT 1923 Occurrence of a coprid beetle Caccobius mutans Sharp in human intestines; Proc. fifth
Entomol Meeting Pusa pp. 201-201
Kistner D H 1982 The social insects (ed.) H R Hermann (New York: Academic Press) Vol. Ill 459 p.
Kolbe H J 1905 Uber die Lebensweise und die geographische Verbreitung der coprophagen Lamellicornier;
Zool Jahr. SuppL 8 475-594
Lea AM 1910 Australian and Tasmanian Coleoptera inhabiting or resorting to the nest of rats, bees and
termites; Proc. R. Soc. Victoria 23 116-230
Matthews R W and Matthews J R 1978 Insect behaviour A Willey Interscience Publication 507 pp.
Navajas E 1 950 Manifestacoes de predatismo em Scarabaeidae do Brasile alguns datos bionomics de Canthon
vireus; Ciencia cultura 2 284-285
Rajagopal D and Veeresh G K 1982 Termitophiles and Termitariophiles of Odontotermes wallonensis
(Isoptera: Termitidae) in Karnataka, India; Colemania 1 129-130
Senior-White R A 1920 On the occurrence of Coleoptera in the human intestine; Indian J. Med. Res. 1
568-569
Skaife S H 1955 Dwellers in darkness (New York: Longmanis Green)
Veena Kumari K 1984 Studies on dung beetle communities with special reference to the biology and ethology of
some coprise dung beetles (Coleoptera: Scarabaeidae) Ph.D. thesis, UAS, Bangalore, India pp. 235
Veeresh G K 1983 White grubs In Applied soil biology and ecology (eds) G K Veeresh and D Rajagopal
(Bangalore: Sharada Publications) pp. 252-301
Wasmann E 1903 Zur Lebensweise Eimiger in-und auslandischen ameiseugaste; Z. Wiss. Insektenbiol 10
329-336
Wheeler W M 1910 Ants their structure, development and behaviour (New York: Columbia Univ. Press)
pp. 663
Wilson E O 1971 The insect societies (Cambridge, Mass: Belknap Press of Harvard Univ. Press) pp. 548
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 309-324.
© Printed in India.
Evolution of insect sociality — A review of some attempts to test
modern theories
RAGHAVENDRA GADAGKAR
Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560012, India
Abstract. An important feature of insect societies is the presence of a sterile worker caste that
makes it possible for the fertile queens to produce a large number of offsprings. The
mechanism of evolution by natural selection of such sterility and similar, though less extreme,
forms of altruism has long been considered as a paradox. In recent years a large body of
theoretical ideas has accumulated that purports to explain altruistic behaviour within the
framework of the theory of natural selection. With special reference to insect sociality three
theories namely kin selection, parental manipulation and mutualism have been suggested.
Some attempts have now been made to empirically test the mutually exclusive predictions
arising out of these alternative theories. A somewhat different approach to empirically
distinguishing between kin selection and parental manipulation is to measure sex-investment
ratios. This approach was at one time believed to have provided overwhelming support in
favour of the theory of kin selection. It has now been realised that several complicating factors
such as local mate competition and multiple mating have to be considered before arriving at
appropriate theoretical predictions of the two rival theories. I argue in this paper that rigorous
quantitative studies on< inter-individual variations in behavioural strategies in primitively
eusocial insects constitutes yet another approach that is likely to help in understanding the
forces that mould the evolution of insect societies.
Keywords. Social insects; kin selection; parental manipulation; mutualism; altruism; quanti-
tative ethology; sex-investment ratios.
1. Introduction
Living organisms present a fantastic diversity %of structure, function and behaviour
unparalleled by anything in man's experience. This diversity has kept generations of
biologists so busy in describing and cataloguing life phenomena that biology has
sometimes been compared to stamp collecting. It is perhaps no exaggeration to say that
Darwin along with Wallace changed this scene and converted biology into a 'Real
Science' by his unifying theory of evolution by natural selection. Darwin set the
tradition of asking such questions as why do peacocks have those incredible feathers?,
why is sickle cell anemia more common in certain parts of Africa?, why do Hanuman
Langurs commit infanticide?
2. The theory of evolution
The theory of evolution by natural selection is as pretty as it is simple. Living organisms
normally produce many more offsprings than can be supported by the environment.
This results in intense competition for survival. The individuals in each generation are
not all identical but show a whole range of variation in their structure, function and
behaviour that leads to differences in efficiencies of survival and reproduction;
differences both from one variant to another as well as from one environment to
310 Raghavenara Gaaagkar
another. If these features are inherited, as they commonly are, it follows that in any
given environment some kinds of organisms survive and reproduce better than others
and thereby come to dominate the population. The variants that dominate in any given
environment are often described by biologists as being 'adapted' to or being 'fittest' in
that environment and the efficiency of survival and reproduction is called the fitness.
Darwin put together a lifetime's experience in natural history to amass evidence for his
theory (Darwin 1 859). The result was overwhelming but there was one odd fact that did
not fall in place. It is a testimony to Darwin's perceptive mind that he did not fail to
notice this anamoly.
3. The puzzle
Many animals ranging from slime molds to man live in societies of varying degrees of
organisation. Individuals in these societies sometimes behave as if they are not
maximising their own fitness but lowering it in order to maximise somebody else's
fitness. At the approach of a predator a squirrel gives an alarm call that warns off its
neighbours but the individual that gives the alarm call itself attracts the attention of the
predator and increases its chances of becoming prey: The grand finale in the evolution
of such altruistic behaviour is the case of the worker honey bee that never reproduces on
its own but rather spends its entire life-time working for its colony. Nor does this
superaltruist hesitate to sting an approaching predator notwithstanding the fact that
stinging is suicide. Every honey bee that stings dies within the next few minutes because
its barbed sting as well as a part of its intestines are pulled out as it tries to fly away. It
was this example of sterile workers in social insects that attracted the attention of
Darwin.
4. The social insects
Understanding the forces that mould the evolution of social behaviour is one of the
most challenging areas of modern biology. A whole new field of sociobiology has
developed to meet this challenge (Wilson 1975; Barash 1982). Social insects, especially
the ants, bees and wasps have been the focus of special attention in this context (Wilson
1971). The reasons for this have been three fold. Firstly, social insects show the most
extreme forms of altruistic behaviour such as the case of the sterile worker bee.
Secondly these insects exemplify a series of stages in the course of evolution from the
solitary to the highly eusocial Thirdly the ants, bees and wasps are characterized by a
peculiar kind of genetics known as haplodiploidy that introduces asymmetries in
genetic relatedness between siblings on the one hand and between parents and
offsprings on the other thereby predisposing them towards sociality, (see below).
5. The theory of kin selection
5.1 Statement of the theory
Hamilton (1964a, b) proposed what has now come to be known as the theory of kin
selection. Hamilton argued that social or altruistic traits are selected for by natural
Evolution of insect sociality 311
selection because, although they decrease the classical individual fitness of an animal,
they serve to increase the number of copies of the genes coding for such behaviour. This
is because altruism is often directed towards genetic relatives who are also likely to
carry the same genes. Thus there is really no altruism from the point of view of the
genes. Altruism at the level of an individual animal is simply the genes' way of making
more copies of itself. Thus animals that behave altruistically may still be maximising
their 'inclusive fitness' which is the sum of their direct contribution through their
offspring to the gene pool and their indirect contribution through their relatives. This
argument can be stated precisely as follows:
As altruistic act will be favoured if
b 1
- >-
c r
where b is the benefit to the recipient, c the cost to the donor and r the coefficient of
genetic relatedness between donor and recepient, benefit and cost being measured in
fitness units. In other words an altruistic act will be favoured if the benefit to the
recipient devalued by the probability that he carried the gene in question is greater than
the cost to the donor. Altruistic traits will therefore spread rather easily either if the
benefit to cost ratio is high or if the recipient and donor are very closely related.
5.2 Haplodiploidy
Consider a hypothetical diploid organism that gives up producing its own offspring and
instead helps its parents to produce more of its siblings. This behaviour, if genetically
coded, will spread in the population only if our hypothetical organism can raise more
siblings than it gives up offspring. This is because both siblings and offsprings are
equally related to it (r = 0-5). On the other hand if the siblings were more closely related
to it than its own offspring, then the behaviour would be selected even if less siblings
were produced than offspring given up. This is the kind of situation that occurs in many
social insects. The only truly social (with sterile castes) animals are among ants, bees,
wasps, termites and a single example from higher animals, the naked mole rat. Of these
ants, bees and wasps belong to the insect order Hymenoptera a group characterised by
haplodiploidy. Males develop from unfertilized eggs and are consequently haploid.
Females develop from fertilized eggs and are diploid. This introduces asymmetries in
genetic relatedness (table 1). For example a female hymenopteran is more closely
related to her sister (r = 0*75) than her own daughter (r = 0-5).
Table 1. Coefficients of relatedness under haplodiploidy assuming complete outbreeding.
Daughter Son
Sister Brother Mother
Father
Fpmni* n-s n-s rvs n.^
Av = 0-5
1-0 0-0 1-0 OO
0-5 0-5
Av = 0-5 Av = 0-5
312 Raghavendra Gadagkar
5.3 Implications of haplodiploidy
The concept of inclusive fitness together with a knowledge of the asymmetries in genetic
relatedness lead to a number of predictions summarised by Wilson (1971) as follows:
(i) True sociality should be more common in haplodiploid organisms than in diploid
ones.
(ii) Queens should not be mated by more than one unrelated male.
(iii) Males should be more selfish than females.
(iv) Females should be more altruistic towards their sisters than towards their
brothers or neices.
(v) Workers should prefer their own sons over their brothers.
Wilson (1971) also summarised the evidence supporting these predictions and
showed that most of the evidence is in qualitative agreement with the theory of kin
selection. As pointed out by him this qualitative agreement means that 4the factor of
haplodiploid bias should be taken into account in future evolutionary interpretations
and as a guideline in planning some further empirical research' but not as proof of the
correctness of the theory. What then should we do in order to ascertain the validity of
kin selection theory? First we ought to put the theory to a critical test by generating
quantitative predictions that can potentially falsify the theory and perform the
appropriate experiments to see if the predictions are borne out. This alone is sometimes
considered inadequate and what we need therefore is also a comparison of the
predictions of two or more competing theories (see Lakatos and Musgrave 1970 for a
detailed discussion of the methodology of scientific research). In recent years some
attempts have been made in providing both these requirements and in the remaining
pages some of these studies are reviewed.
6. Alternative theories
6.1 Parental manipulation
The theory of parental manipulation advanced by Alexander (1 974) states that altruistic
behaviour could evolve even if it does not increase the inclusive fitness of the altruistic
animal because selection can act on the parent to manipulate some of its offspring to be
altruistic towards the rest. Ecological conditions could be imagined under which, an
animal that produces a certain fraction of sterile offspring which in turn help the
remaining fraction of fertile offspring to survive and reproduce better, could leave
behind more grandchildren than an animal that produces all fertile, selfish offspring.
6.2 Mutualism
Lin and Michener (1972) emphasizing the ecological factors involved in the evolution
of altruistic behaviour argue that at least in the early stages of the evolution of sociality,
when complete sterility had not yet evolved, mutual advantage in defence against
predation for example, might have been an important force. This idea might be more
important in present day primitively eusocial insects than has been hitherto suspected
(see below).
Evolution of insect sociality 313
7. Testing the theories
In this section three major approaches that are being pursued in making quantitative
tests of the theories are discussed. The first is the direct measurement of inclusive fitness,
the second tests predictions of optimal sex investment strategies and the third involves
quantitative studies on the ethology of primitively eusocial insects. I will discuss the
logic behind each of these approaches and then very briefly review illustrative examples
of empirical investigations representing these approaches, making however no attempt
to provide a comprehensive review of the literature. The interested reader may consult
several excellent reviews dealing both with theoretical ideas as well as empirical studies
on social insects (Evans and West-Eberhard 1970; Wilson 1971; Hamilton 1972;
Spradbery 1973; Michener 1974; West-Eberhard 1975; Hermann 1979-82; Starr 1979;
Edwards 1980; Jeanne 1980; Barash 1982; Charnov 1982; Michod 1982; Brian 1983).
7.1 Computation of inclusive fitness
This approach has mainly been applied to primitively eusocial wasps of the genus
Polistes. Typically these wasps follow one of two strategies. Sometimes a single female
initiates a colony on her own and raises the first brood of offsprings unaided by any
other wasps and later the daughters from her first brood become workers and help her
to raise reproductive male and female offsprings. Alternatively a group of females of the
same generation, typically sisters, jointly found a nest and again produce a first brood of
workers which help them raise reproductive offspring. Here all the cofoundresses do
not contribute equally to the production of reproductive offspring. Sometimes only a
single dominant (a) female lays all the eggs while the other cofoundresses remain
subordinate and behave like workers. Even if the subordinate (ft) cofoundresses lay
some eggs this number is usually less than that laid by the a foundress. In such a
situation it is possible to calculate the productivities of the single foundress colonies
and the multiple foundress colonies. With a knowledge of the genetic relatedness
between cofoundresses and, the exact number of offsprings produced by each
cofoundress one can compute the fitness of the solitary foundress as well as the inclusive
fitness of the dominant cofoundress and each subordinate foundress in multiple
foundress colonies.
For the sake of simplicity let us consider two full sisters a and ft, each having mated
with any one male, in a diploid population (table 2). When nesting solitarily (table 2A)
let them produce 10 offspring each thus having an individual fitness of 5 (10 offspring
x 0-5, the coefficient of genetic relatedness to each offspring) and an inclusive fitness of
7-5 each (individual fitness of 5 + indirect contribution of 2-5 as a result of 10 neices or
nephews x 0-25, the coefficient of relatedness to each nephew or neice). Note that the
term individual fitness is used for the direct contribution to the gene pool and the term
inclusive fitness for the sum of the direct and indirect contributions. When they nest
together (table 2, B) and if their summed productivity does not increase but merely a
produces all the 20 possible offspring and ft produces none of her own but helps her
sister, a has a fitness as well as inclusive fitness of 10 while ft has a fitness of 0 but an
inclusive fitness of 5. This situation is advantageous to a but not to ft. ft will therefore not
be selected to accept this subordinate role. Nor will selection acting on the parents of a
and ft favour their manipulating ft into remaining subordinate and helping a.
314 Raghavendra Gadagkar
Table 2. Conditions for the evolution of sterility in a diploid population where a and j9 are
full sisters.
a 8 Remarks
Number of offspring
10
10
A
Classical individual fitness
5
5
Solitary
Inclusive fitness
7-5
7-5
Nesting
Number of offspring
20
0
Sterility
B
Classical individual fitness
10
0
Will not be selected
Inclusive fitness
10
5
Number of offspring
21
0
Parental
C
Classical individual fitness
10-5
0
manipulation
Inclusive fitness
10-5
5-25
Number of offspring
31
0
Kin selection
D
Classical individual fitness
15-5
0
Inclusive fitness
15-5
7-75
Number of offspring
21 orO
Oor 21
Classical
E
Classical individual fitness
10-5 or 0
0 or 10-5
Individual selection
Inclusive fitness
10-5 or 5-25
5-25 or 10-5
Sterility on the part of ft will thus not spread by any of the mechanisms under
consideration, parental manipulation, kin selection or individual selection. On the other
hand, when nesting jointly (table 2, C), if there is even a slight increase in the total
productivity such as a being able to produce 21 offspring then the situation is quite
different. Although ft still has a lower inclusive fitness than when she nested solitarily, a
and ft together have done better than when they were nesting solitarily. Thus it pays the
parents of a and ft to manipulate ft into being subordinate and sterile because now the
parents of a and ft have more grandchildren. Parental manipulation can thus promote
the spread of sterility on the part of/? although ft loses in the process. If the productivity
increases substantially (table 2, D) such that a produces, say, 31 offspring then ft gets an
inclusive fitness of 7-75 although she does not produce a single offspring. Now kin
selection acting on ft will promote the spread of subordinate behaviour as ft now
contributes more to the gene pool of the population than when she was nesting solitarily
and producing 10 of her own offspring. The exact amount by which productivity under
joint nesting should increase would depend upon the degree of relatedness between the
altruist and those she individual raises. Under haplodiploidy, if a female worker raises
sisters in place of daughters then the productivity need not even increase because the
workers are more closely related to their sisters than to their daughters (0-75 vs 0-5). If
they raise equal numbers of brothers and sisters of course they gain no more fitness than
they would, had they raised sons and daughters instead. The asymmetries in genetic
relatedness created by haplodiploidy could however be capitalised on if the workers
produce more sisters and less brothers. This by itself is a prediction of kin selection
theory whose verification has also been attempted (see below).
It is of course possible that both a and ft produce offspring when nesting jointly and
Evolution of insect sociality 315
that both produce more offsprings than when they were nesting solitarily. This will lead
to the evolution of joint nesting by classical individual selection but there will be no
sterility. Such a situation is envisaged by Lin and Michener (1972) in the early stages of
the evolution of sociality. Ecological conditions can be sufficiently harsh and it may for
example be impossible for a single female to guard her nest against predators as well as
forage for food. Thus by nesting jointly both a and ft may increase their offspring
production. Once such joint nesting gets established in the population it could give
scope for the evolution of parental manipulation or kin selection. Another interesting
case of evolution of sterility is possible through classical individual selection
(mutualism) which may be more important in present day primitively eusocial insects.
For example, let a and ft nest jointly but who will lay the eggs and who will remain
sterile be decided by chance (table 2, E). If a and ft each have on the average an equal
chance of egg laying then, even if there is a slight increase in productivity due to joint
nesting, classical individual selection will promote such sterility. When nesting,
solitarily a and ft each get an inclusive fitness of 7-5. Here each gets on the average
10-5 + 5-25 = 15-75/2 = 7-875. In Polistes exclamans the ft foundress remains subordi-
nate and waits for the a foundress to produce a batch of workers. When it is time to lay
eggs that will mature into reproductives the ft foundress suddenly becomes very
aggressive and challenges the a foundress. In a substantial number of cases the ft
foundress succeeds in driving away the a foundress and then lays all the eggs. (Alan
MacCormac personal communication). This would be a situation analogous to the
example described above and here subordinate behaviour and sterility with a certain
probability could be brought about by classical individual selection.
Perhaps the most complete study attempting to distinguish between different
theories by the computation of inclusive fitness is that of Metcalf and Whitt (1977) on
Polistes metricus. They used enzyme electrophoresis to determine the genetic relatedness
between different individuals in the colony and knowing the productivity of single and
multiple foundress colonies they were able to calculate the inclusive fitnesses of a
solitary foundress, the dominant a foundresses and the subordinate ft foundresses of
joint nesting colonies. Their results are as follows:
In the population studied by Metcalf and Whitt, 83 % of the colonies were solitary
foundress colonies. In multiple foundress colonies, a foundresses had a relative inclusive
fitness 1-83 ± 0-57 times that of a solitary foundress while the ft foundress had a relative
inclusive fitness 1-39 ±0-44 times that of a solitary foundress. A female's expected
number of grandchildren from two of her daughters jointly founding* nest is 1-55 times
what she would achieve by the two daughters acting as two solitary foundresses.
Since the ft foundress does not have an inclusive fitness significantly greater than that
of the solitary foundress, neither kin selection theory nor mutualism (classical
individual selection; table 2, E) can be accepted as the selective force responsible for the
altruistic behaviour of ft although the authors themselves conclude that their results are
in accordance with the predictions of kin selection theory. Parental manipulation could
explain this behaviour on the part of ft provided the figure of *l-55 times more
grandchildren' is statistically significant (no standard deviation is provided by the
authors). Besides one does not know how to interpret the fact that 83 % of the colonies
were single foundress colonies (see below). A very similar study was conducted on a
related species P. fuscatus (Noonan 1981). Not having used electrophoresis Noonan did
not know the exact values of genetic relatedness but made her computations of inclusive
fitness for different possible values of relatedness between the dominant and
316 Raghavendra Gadagkar
subordinate females on a nest (full sisters, half sisters etc.). These results showed that
subordinate females lay some eggs (and gain some individual fitness) but not enough to
make subordinate roles better than solitary nesting. Because foundresses are often
sisters the subordinates have inclusive fitness values greater than their individual fitness
values. Noonan's data however do not permit distinction between parental manipu-
lation and kin selection models because of the high variances associated with her mean
values of inclusive fitness. Thus we find that the best empirical studies using the
approach of computing inclusive fitness does not permit us to draw any definite
conclusions.
I would also like to argue that there are difficulties with this approach itself. Firstly
the kind of analysis described above compared wasps which are naturally nesting either
solitarily or jointly. Implicit in such a comparison is the assumption that the two females
being compared are reproductively equivalent and that any difference in productivity is
only because of solitary versus joint nesting. We have no information that might help
decide whether this assumption is valid or not. However, if solitary and joint nesting
females have different reproductive potentialities, then the effects of the intrinsic
differences in productivity and the effects of nesting strategy will be confounded in the
analysis. The second problem with this approach is that the frequency of solitary versus
joint nesting varies widely in different situations. No attempt has so far been made to
take this into consideration. Ideally, the theory should be able to predict the frequency
distribution of foundress size associations. Even if we find that the inclusive fitness of
the subordinate foundress is much higher than a solitary foundress, there would still
remain a puzzle if it turns out that most of the females prefer to nest solitarily.
7.2 Sex investment ratios
We argued earlier that haplodiploidy predisposes the hymenopterans to the path of
sociality because a female is more closely related to her sister than to her daughter. But
this asymmetry exactly cancels out because a female is less closely related to her brother
than to her son. Her average relationship to her offspring of 0-5 is exactly the same as her
average relationship to her siblings ( ~ \ Trivers and Hare (1976) argued
therefore that workers in hymenopteran societies should be capitalizing on the
asymmetries created by haplodiploidy by investing differentially in their sisters and
brothers, in fact in the ratio 3 : 1, which is the ratio of their genetic relatedness to their
sisters and brothers respectively. Since it is the workers who feed the larvae, it should be
easy enough for them to feed their sisters and brothers differently and achieve the
maximum possible inclusive fitness. Notice however that on the Fisherian argument
(Fisher 1930) the mother queen who is equally related to her sons and daughters would
prefer an equal investment in brood of the two sexes (assuming on outbreeding, random
mating population). In other words there is a conflict of interests between the queen
(who prefers a 3:1 ratio of investment) and the workers (who prefer a 3:1 ratio of
investment). Notice that this approach too makes a precise quantitative prediction and
also contrasts kin selection theory with the parental manipulation theory. If the par-
ental manipulation theory is correct, then it means that the mother queen should be able
to have her way and manipulate the workers to invest equally in the two sexes although
it is not the optimum strategy for them. If kin selection theory on the other hand is
Evolution of insect sociality
317
o
S 6
10
O.Or-
3:1
INVESTMENT
t RATIO
1:1 *
, INVESTMENT
\RATIO
5 10 15 20 25
WEIGHT RATIO
30
.0 1.5
SEX RATIO
2.5
Figure 1. A. Relationship between the sex ratio and weight ratio of reproductive caste ants.
The two lines show 1 : 1 and 3 : 1 investment ratios. B. Relationship between sex and weight
ratios for solitary bees (open circles) and solitary wasps (closed circles) compared for 1 : 1 and
3:1 investment ratios (modified slightly from Trivers and Hare 1976, after Barash 1982.
Reprinted with permission).
correct, then workers behave altruistically only because this is the strategy that
maximises their inclusive fitness. But it would not maximise their inclusive fitness if they
invested equally in their brothers and sisters. Workers should therefore have their way
and invest in the ratio 3:1.
Trivers and Hare (1976) weighed the total male and female reproductive brood in a
large number of species and appeared to provide overwhelming support for kin
selection theory. In a large number of monogynous ant species the weight ratio of the
reproductive brood was significantly close to the 3 : 1 prediction. Besides, in solitary bees
and wasps, and termites the investment ratio as expected was close to 1:1.
Soon after the publication of this study, Alexander and Shermann (1977) pointed out
that there are serious problems with the interpretations of Trivers and Hare. The
predictions used by Trivers and Hare are valid only if the queens have mated only once
and only if the populations are completely outbreeding. If the queens mate more than
once then the workers are not necessarily full sisters of the reproductive siblings and
therefore they would not be expected to invest in the ratio 3:1. If the population
inbreeds then according to the theory of local mate competition proposed by Hamilton
(1967) even the queens would prefer a female biased sex investment ratio. According to
Alexander and Shermann, Trivers and Hare's data thus may not represent the triumph
of kin selection theory over parental manipulation theory but may simply be a response
to local mate competition. Supporting this argument is a 1:1 ratio of investment
demonstrated in Polistes fuscatus in a situation where local mate competition was
known to be absent (Noonan 1978). One obvious difficulty here is the lack of good
empirical data on multiple mating and local mate competition. Multiple mating has
long been realised to be very common in social insects (Wilson 1971) but it has often
been ignored because the sperms from different males have been assumed not to mix in
the spermatheca (Orlove 1975, for example). Page and Metcalf (1982) have recently
demonstrated that at least in the honey bee, sperms from different males do mix and
that the average genetic relatedness between workers and the reproductive sisters they
rear can be quite low. In any case it is clear that multiple mating and local mate
competition both influence the predictions of optimum sex investment ratios in the
framework of kin selection theory and parental manipulation theory. Information on
the extent of multiple mating and local mate competition in social insects is thus
urgently needed.
318
Raghavendra Gadagkar
theory would be different from those used hvT°ry ^ Parental ma^P»lation
Gadagkar (1985) have modd^^^^™""1 "^ ****** Joshi and
investment ratios under different let s of ir, ^ C°mpUted the °Ptimum s«
(ngure2). This has been done b con? P "^^ and loc*l mate competition
S^cotonieswhereaoerSn^S'rft; haplodiPloid Population with
outbreed while the remaining fraction H K reproductive offspring disperse
be summarised as follows (n±T?n^f brothei"sister Bating. Our results
e '
ma
can be summarised as follows
mate competition, parental
investment ratio while
mate competition biases
both the theories. For any
investment ratio predicted b
that predicted by kin selectio
the parental manipulation theory a
km selection theory more male
exact predictions of sex inves ment
provided by us will be used in a fre"h
future studies that should atte
^ levels of multiple mating
ng. ur resuts
th? T' °f multiPle ^ting and local
, y (qU6en contro1) Pred^ts a 1:1
(fem^™H Local
in the frame work of
mate comPetitio" however, the
^ le biased than
the prediction of
tmui" investment ratio under
adng ^ We h°Pe ^ the
framework of these two theories
theSe the0ries i
i
QUEEN CONTROL
[WORKER CONTROL
2 INSEMINATIONS
I (DOMINANT)
[WORKER CONTROL /
SINGLE
( INSEMINATION /
teo PtonM- w s Contro te ratio
Evolution of insect sociality 319
7.3 Quantitative ethology
In the last few years I have been studying the behaviour of adults on colonies of
primitively eusocial wasps such as Ropalidia in India and Polistes in America with the
hope that these studies will in the long run provide a third approach to understanding
the forces that mould the evolution of social behaviour (Gadagkar 1980; Gadagkar and
Joshi 1982a,b, 1983, 1984, 1985). Taking Ropalidia marginata in India as an example I
shall now illustrate this approach. Colonies of Ropalidia marginata are initiated by one
or a group of females (foundresses) at any time of the year (Gadagkar et al 1982a,b).
One of these foundresses assumes the role of the queen while the others remain
subordinate to the queen and assume the role of workers. Of the female offspring
produced in such a colony many remain at the parent colony and become workers
although some leave it to found or join other colonies. Males disappear from the colony
within a few days after their emergence. The lack of a very severe winter in peninsular
India permits these colonies to be perennial. A single colony can therefore survive for
many years. This makes it possible for queens to be replaced (Gadagkar unpublished
observations). When an existing queen dies or is driven away, one of the other females
takes over and begins to lay eggs. In addition, females sometimes leave their parent
colonies to found their own colonies either alone or in small groups where one of them
again becomes the queen. These events can occur any time in the year and therefore
every female must have a fair chance of becoming a queen.
I argue that in such a situation these colonies cannot simply consist of a queen and a
bunch of willing workers but each colony must be a highly competitive association of
female wasps each trying to maximise its chances of becoming a queen either by leaving
the colony at the appropriate time and perhaps with the appropriate company or to
challenge the existing queen at the appropriate moment and inherit the nest, its brood
and the workers. Using two monogynous colonies of JR. marginata, I sought to discern
the competitive strategies of these wasps by carefully studying and quantifying their
behaviour. Instead of following the classical method of concentrating either on species
specific behaviour patterns or on certain kinds of behaviours that appear to be
important from our point of view, I decided to study the patterns in which the wasps
allocated their time between different behaviours. Since time must be a very limiting
resource, I considered those behaviours important in which the wasps spent more time.
Besides, my emphasis was on the differences between different individuals within a
colony. Sitting, sitting with raised antennae (alert to external disturbance), sitting with
raised antennae and raised wings (a state of alarm), walking on the nest, being in the
cells, and being away from the nest turned out to be. the activities in which the wasps
spent most of their daylight hours. For these six behaviours I constructed time-activity
budgets for a number of individually identified animals. As it turns out, all animals
studied spent about 95 % (95-9 ± 44) of their daylight hours in these six behaviours but
the manner in which they allocated their time between these behaviours was highly
variable (figure 3).
These time-activity budgets were analysed by multivariate statistical techniques such
as principal components analysis and hierarchical cluster analysis with the aim of
understanding the difference between individuals. The wasps could be classified into
three distinct behavioural castes (figure 4) with sitting, sitting with raised antennae and
being absent from the nest as the main attributes of the three clusters respectively
(figure 5). I had also collected data on the frequencies of certain rare behaviours such as
320
Raghavendra Gadagkar
60r
SIT & GROOM
ANIMALS
Figure 3. Time-activity budgets of 20 individually identified animals from 2 monogynous
nests for 6 behaviours. Animals 1 and 14 are the queens of nest 1 and 2 respectively.
+ 40
LU
8
g
O
5
OL
20
-20L
II (FIGHTERS)
13
III (FORAGERS)
-20 0 +20
PRINCIPAL COMPONENT I
.60
Figure 4. Behavioural castes of R. marginata. Twenty wasps from 2 different nests are shown
as points in the coordinate space of the amplitudes associated with the first two principal
components. The points fall into three clusters (or castes) by the criterion of nearest centroid.
Circled dot = centroid. (From Gadagkar and Joshi 1983a. Reprinted with permission).
Evolution of insect sociality
321
n2
Figure 5. Mean behavioural profiles of the clusters obtained in figure 4A, mean percentages
of time spent in each of the six activities that were used in obtaining the clusters are shown for
sitters, fighters and foragers. B. mean frequencies per hour of the five activities that were not
used to obtain the clusters are shown for sitters, fighters and foragers. (From Gadagkar and
Joshi I983a. Reprinted with permission).
dominance behaviour, egg laying etc. which were not used in the analysis. Sitting with
raised antennae is positively correlated with dominance behaviour (P < 0-01). Wasps
absent from the nest often return with food loads (figure 5B). The three clusters were
thus named Sitters, Fighters and Foragers (although it is possible that wasps absent
from their nests spent some of their time looking for other nests to join or new nest sites
to start their own nests).
We can now begin to interpret the biological significance of this behavioural caste
differentiation. Note that the two queens (individuals 1 and 14) are among sitters
(queens are recognised by their egg laying behaviour). Thus the queens do little other
than sitting and grooming because it is perhaps the best strategy to conserve their
322 Raghavendra Gadagkar
energy and maximise their egg laying capacity. But this also means that the queens face
little competition from their nestmates who can therefore be 'trusted' to do all the work
for the colony. This seemed to be the situation in the colonies of R. marginata that were
studied. Significantly when some polygynous colonies of another species R. cyathifor-
mis were studied, the queens were not sitters but fighters. In the latter case the queens do
not just sit but they were the most active individuals in the colony. In R. marginata
however, there were many sitters who were not queens. These we hypothesise are
'hopeful queens' who still have some chances of becoming queens. On the other hand
the fighters could also be hopeful queens who are following an alternative strategy of
maximising their chances of becoming queens. These hypotheses assume significance
because they can be readily tested. Experiments are in progress where I am studying a
number of colonies, classifying the wasps and then removing the queens to see who
takes over. Take over somebody does, but preliminary results indicate that either a
Sitter or a Fighter could be the replacement queen (Gadagkar unpublished observa-
tions). The foragers are interpreted as having the least chance of becoming replacement
queens. This interpretation holds whether the foragers really spent all their time away
from the nest in foraging or attempted to join or initiate new nests. The fighters very
frequently behave dominantly towards other members of the colony. Apart from
establishing their claim to the position of the next queen this fighting could have other
functions. For example it may serve to keep the foragers active. I have often seen
foragers leave the colony after a series of attacks by a dominant wasp. That the queens
could be sitters or fighters depending on the extent of reproductive competition they
face from their nest mates seems to be supported by our results. As mentioned above
queens of monogynous colonies of R. marginata were sitters while queens of
polygynous colonies of R. cyathiformis were fighters. Even in R. cyathiformis when a
single foundress was attending a colony and thus had no one to compete with, she was a
Sitter i.e. she did not spend more time with raised antennae. Furthermore in Polistes
versicolor in Panama, I had an opportunity to study both pre-emergence colonies as
well as post emergence colonies. Pre-emergence colonies are associations of females of
the same generation. In such a situation, the closest possible relationship between the
workers and the brood they rear is 3/8 (neices or nephews). These colonies are therefore
likely to be much more competitive than post-emergence colonies consisting of a
mother queen and her daughter workers; the latter caring for their siblings. The queens
in two pre-emergence colonies studied were Fighters while the queen in the post-
emergence colony studied was a Sitter (Gadagkar and Joshi in preparation).
The behaviour of the wasps is very highly variable and seemingly unpredictable. But I
believe that in this variability lies the clue to the understanding of the evolution of
sociality. Wasps that all look morphologically similar may behave as sitters, fighters or
foragers, they may be queens or subordinate workers, they may challenge an existing
queen and chase her away to take her position, they may leave the colony alone or with a
submissive group to start new colonies. Kin selection theory predicts that the wasps
should behave so as to maximise their inclusive fitness. Parental manipulation would
predict that the animals should at least sometimes behave not according to 'their own
optimum criteria' but according to what is best for their parents to produce the largest
number of grandchildren. If we know what factors influence the chances of a wasp
becoming an egg layer in a given circumstance it should be possible to predict the
behaviour of a given animal within the framework of a particular theory. With this long
term aim in mind experiments are in progress in my laboratory to determine the relative
Evolution of insect sociality 323
contributions of body size, age, prior social experience, hormone levels etc. to the egg
laying capacity and the capacity to win in an encounter with a conspecific.
It is in the context of such present day primitive insect societies that I have been
discussing above that mutualism or classical individual selection of the kind considered
in table 2E might be important. For instance a wasp may stay on at the parent colony
and work as a subordinate individual if it has a fair chance of becoming the next queen.
There may be a certain probability of not succeeding but if more inclusive fitness is
gained on the average by taking the risk than by solitary nesting, classical individual
selection will favour this behaviour and we will see certain individuals remaining sterile
even if that particular individual does not have higher inclusive fitness than its solitary
counterpart.
8. Conclusions
In conclusion it may be said that today we have a very attractive body of theoretical
ideas concerning the forces that might be responsible for the origin and maintenance of
social behaviour especially in social insects. Several simple minded attempts have been
made to test these theoretical ideas but none has yielded unambiguous results. This is
primarily because the social insects live in rather complex societies and pursue complex
strategies. We need much more information on the details of the lives of these insects,
especially in areas such as breeding structure of populations and the factors that
influence the chances of an animal's success in an encounter with a conspecific before we
can launch more sophisticated empirical tests of the theories.
Acknowledgements
It is a pleasure to thank Madhav Gadgil, N V Joshi, Mary Jane West-Eberhard, Charles
D Michener and V Nanjundiah for reviewing an earlier draft of this paper and W D
Hamilton with whom many of the ideas presented in this paper were discussed. My own
research discussed here has been supported in part by a grant from the DST,
Government of India.
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© Printed in India.
An analysis of the superparasitic behaviour and host discrimination
of chalcid wasps (Hymenoptera: Chakidoidea)
T C NARENDRAN
Department of Zoology, University of Calicut, Kerala 673635, India
Abstract. Superparasitism is frequently met with in chalcids. The actual mechanism of
suppression of the supernumerary individuals is by mutual combat though exceptions to this
general rule may also be seen rarely. Many chalcids are known to discriminate between
parasitised and healthy hosts. It is an interesting phenomenon that superparasitism occurs
even when a female is capable of discriminating parasitised and unparasitised hosts. Several
factors play prominent roles in causing superparasitism and the avoidance of superparasitism
by a chalcid is the result of maximisation of its reproductive success.
Keywords. Superparasitism; host discrimination; analysis; chalcids.
1. Introduction
The chalcid wasps are well known for various salient features of their ethology. The
majority of chalcids are solitary, developing singly upon their hosts. Superparasitic
behaviour is frequently exhibited by many species of chalcids. Superparasitism is the
parasitisation of an individual host by more larvae of a single parasitic species than can
mature in that host. In superparasitism usually a single parasite individual survives or
all may die or the brood may produce undersized weaker adults. When a parasite
superparasitises a host it usually condemns its own progeny to death thus resulting in a
wastage of its own eggs. To avoid such a contigency it must be able to discriminate
between parasitised hosts and unparasitised hosts. Such avoidance of superparasitism
is an interesting aspect of insect behaviour. This paper presents an analysis of some of
these interesting aspects of the ethology of chalcids.
2. Ethology and analysis
Supernumerary individuals are usually suppressed by destruction by mutual combat
between the first instar larvae. They attack each other with their mandibles and finally
only one survives. However if an egg is laid in a host that already contained an advanced
larva then the younger of the two dies due to oxygen starvation and apparently no
fighting occurs in most cases, though autoparasitism and hyperparasitism are
oLsionally met with in chalcids. A different method of suppression of super-
numenme.^
of caterpillars of Artona. In this case when more than one egg is laid on a host, the
eclos^n of one egg causes immediate cessation of development of the remaining ones
finds a host a sequence of behavioural patterns follow (figure 1).
325
326
T C Narendran
Figure 1. Behavioural patterns of chalcids when they come across different types of hosts
(suitable and unsuitable hosts).
Fiske (1910) recognised that "the prevalence of superparasitism depends entirely upon
whether or not the female parasite is gifted with a prescience which will enable her to
select healthy hosts for her offspring". The same author pointed out that the total
absence of such an instinct would make the prevalence of superparasitism wholly
dependent upon the laws of probability. Fiske found that a vast majority of parasites
studied by him showed no such instinct and oviposition therefore, occurred at random
with a consequent incidence of superparasitism. Thompson (1924) accepted Fiske's
theory of 'random distribution' and gave a brief mathematical interpretation to the
problem by providing the formula:
Y=N
1-e-x
~N
where N is the number of hosts, x is the number of parasite eggs distributed, Y is the
number of hosts parasitised and e is the Napierian logarithmic base. Stoy (in Salt 1932)
also believed in the random distribution of eggs and explained that the probable
number of hosts that will receive a given number of parasites can be calculated by using
the formula:
Z = NxCp(l/N)p (1 - l/N)x~p
where N is the number of hosts, x is the number of parasites distributed, Z is the
number of hosts containing p parasites. Explaining this formula Askew (1971) pointed
out that "A binomial distribution would be approached when several eggs are
distributed over a limited number of hosts, but the rate of parasitism is not too high a
Poisson series, which is probably more easily calculated, may be used to obtain values
for random distribution. The probability of occurrence of 0, 1 , 2, 3 etc parasites per host
is then
ze~
21
3!
etc
Superparasitism and host discrimination of chalcid wasps 327
where e = 2-72 (natural logarithm base), and
_ total number of parasites
total number of hosts
If the actual distribution of parasite eggs differs significantly from the calculated
random distribution in the direction of more hosts than expected supporting only one
parasite, and fewer than expected remaining unparasitised then it can be said that the
parasite exercises discrimination." There are several reports which show that many
chalcids have the ability to discriminate parasitised and healthy hosts. Melittobia acasta
Walker (Eulophidae) will not oviposit in puparia of Diptera which contain either their
own larvae or pupae or those of Pteromalus (Pteromalidae) or Dibrachys
(Pteromalidae) (Thompson and Parker 1927). The female Trichogramma evanescens
Westwood (Eulophidae) is capable of discriminating parasitised hosts from un-
parasitised hosts (Salt 1934, 1937; Flanders 1937). With her antennae the female can
recognise the residual odour of the tarsal gland's secretion left on the eggs that have
been walked on by another female of the same species. This was subsequently termed by
Flanders (1951) as the 'spoor effect". Parasitised hosts may be thus detected by the
female Trichogramma evanescens initially by the antennal 'drumming' on the surface of
the host. If this initial examination of the host with the antennae fails to indicate
parasitism due to the washing away of the odour by rain or by other means, the female
tested the hosts by inserting its ovipositor into the host and withdrawing it immediately
(Salt 1934, 1937). The present author in his observations has noted that several species
of Brachymeria (Chalcididae) were unable to discriminate between parasitised hosts
and healthy hosts in the beginning stages and superparasitism was a common
occurrence. However the females were found taking a longer time than usual for
'drumming' if they happened to meet hosts which were parasitised for the first time by
another female of the same species or by the same female, 4 to 6 days earlier. In such
instances the female either abandoned the hosts after a thorough antennal 'drumming'
or pierced the host with its ovipositor just to withdraw it immediately and then left the
host. It is suggested that changes in the physical or chemical condition of the parasitised
hosts might be responsible for providing the stimuli for discrimination between
potential hosts (Salt 1938; Wylie 1965; Fisher 1971; Narendran 1975; Narendran and
Joseph 1977). In those cases where a parasitised host is detected only after the
penetration of the ovipositor, it is evidently the sense organs of the ovipositor that are
believed to be responsible for detecting the hosts. Such sense organs especially
chemosensory "pores" are seen in several species of chalcids such as Eurytoma tibialis
Boheman (Eurytomidae), Brachymeria lasus Walker (Chalcididae), Tetrastichus rapo
(Walker) (Eulophidae), Nasonia vitripennis (Walker) (Pteromalidae), Microterysflavus
(Howard) (Encyrtidae), Aphytis sp. (Aphelinidae) and in several other species of
chalcids (Fulton 1933; Varley 1941; Copland and King 1971a, b, 1972a, b, c; Fisher
1971; King and Rafai 1970; Jackson 1966, 1969; Edwards 1954; Wylie 1958; King and
Fordy 1970; Bartlett and Lagace 1961; Quendnau and Hubsch 1964; Weseloh 1969;
Narendran 1975; Askew 1971). It is suggested by Fisher (1971) that biochemical
changes of the host's haemolymph are likely to act as sign stimuli for discrimination
between parasitised and unparasitised hosts. Mouthparts and tarsi are also reported to
play a role (probably a minor one when compared to antenna and ovipositor) in the
host detection behaviour of certain chalcids of the family Aphelinidae (Viggiani 1984).
328 T C Narendran
In Spalangia drosophilae Ashmead, either the smell of the host's haemolymph clotted
around the oviposition puncture or the odour of the fluid left by the ovipositor
provided the stimulus for the detection of parasitised hosts from healthy hosts
(Simmonds 1954). The discharge of a "venom' presumably from the poison apparatus of
the ovipositing female was suggested (Jackson 1 966) to be responsible for providing the
stimulus for discrimination between parasitised and unparasitised hosts by
Caraphractus cinctus Walker (Mymaridae). The vibrations produced by the palpation
of the host provided the stimulus for discrimination between parasitised host and
healthy host in the case of Microplectronfuscipennis Zett (Eulophidae) (Ullyett 1936).
Similarly movement or lack of movement by the host might contribute part of the
stimulus for detecting healthy and parasitised hosts by Spalangia drosophilae, and
Nasonia vitripennis (Simmonds 1954; Wylie 1965). In certain chalcids like
Lasiochalcidia igiliensis Steffan, (Chalcididae), active movement of the host is an
essential requisite for oviposition. This interest ing chalcid parasitises the larvae of ant-
lion. The female provokes its host to come out from its burrow and to seize the leg of the
chalcid whereupon the chalcid inserts its ovipositor into the membrane between the
head and thorax of the host. Among the chalcid parasites of the knapweed (Centaur ea
nigra L.) gall fly (Urophorajaceana Hering) only the endoparasitic Eurytoma tibialis is
able to discriminate parasitised and healthy hosts by avoiding superparasitism whilst
the four ectoparasitic chalcids species either distribute their eggs randomly or even in an
aggregated manner (Varley 1941). In perilampids and eucharitids the planidium or the
first instar larva upon emergence undergoes a freeliving period during which it must
find its hosts. In these cases this is "more exactly a waiting period rather than a
searching period; for relatively little movement takes place and the greater portion of
the time is passed in the erect position awaiting the arrival of a host or carrier" (Clausen
1940). In these cases, the instincts of the planidium are not sufficiently developed to
enable it to discriminate parasitised and unparasitised hosts and in several instances the
planidium responds to virtually any moving object that approaches its immediate
vicinity.
In chalcids, superparasitism is not always caused by the failure of the discriminative
ability. In Trichogramma evan&scens, Encarsia formosa Gahan (Aphelinidae),
Pachycrepoides vindemmiae Rondani (Pteromalidae) and in several other species,
superparasitism occurs although the females were capable of discriminating parasitised
and unparasitised hosts. One of the possible reasons for this is the breakdown of the
'restraint' of the ovipositing females when there is a scarcity of healthy hosts. Another
possible suggested explanation is that the female has to learn to discriminate between
parasitised and unparasitised hosts if it is an inexperienced one (Salt 1934; Van
Lenteren and Bakker 1975; Van Lenteren et al 1978). It is known that the 'restraint'
exercised by the chalcid Caraphractus cinctus Walker in avoiding superparasitism of
water-beetle eggs was best developed in old females and inexperienced young females
would superparasitise the hosts (Jackson 1966). In the case of Ooencyrtus kuwanae
(Howard) (Encyrtidae) the female tends to retain her eggs rather than deposit them if
she finds only parasitised hosts and the exercise of this 'restraint* in this case is shown to
be related to the developmental stage of the parasite in the parasitised hosts, the age and
condition of the ovary of the female and the number and nature of the hosts available
(Lloyd 1940). There are several other possible causes for superparasitisation such as,
when a female lays more than one egg after the first oviposition within the period which
is needed for building up the factor which causes avoidance of superparasitisation and
a* te. discrmirnli,,* of CUcU
in one host (Van Lenteten and
whe» two « more fences oviposit
Bakkerl975). mv,™™icr,arasitism are also met with in the case of
GKgarious parasitism and "*%%%*££** .__ ^ h()st bu, th
r^'i^.ws
.
including chalcids especially when the e is a blished how exactly the
scarcity of healthy hosts. However ins ^not undouD y ^ ^ ^
parasite recognise the hosts Wlthj?f ^ some species. Hence the
known clearly is that such an abl1
-s Wlth different nun^er rf
eggs-
3. Conclusion
Superparasitism and avoidance of
spetTef to species. It may be based 01
used by certain species.
In chalcids superparasmsm s
healthy hosts, inability to learn to ch
hosts by inexperienced P^'
females on a host, la
causes for
also by discriminating
the
and chemical
the
lack of ability to discriminate
e 'restraint' due to scarcity of
itised and unparasit1Sed
more than one or two
, aie some rftte
by chalclds, „
from unparasitised hosts bu
^ ^.^^ Q
a of its reproductive success an<
of an effective bioiogical control agent.
Acknowledgements
T.eautho.than.sDrRRAskewofUnive.sityofManchesterfo.permissionto^
from one of his books.
330 T C Narendran
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Lloyd D C 1940 Host selection by hymenopterous parasites of the moth Plutella maculipennis Curtis; Proc. R.
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Narendran T C 1975 Studies on biology, morphology and host-parasite relationships of Brachymeria lasus
(Walker) (Hymenoptera: Chalcididae) Ph.D. thesis, Calicut University, India
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specificity (ed.) T N Ananthakrishnan, (Delhi: The Macmillan Company of India Ltd.) 127 pp.
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Superparasitism and host discrimination of chalcid wasps 331
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© Printed in India.
Application of sex pheromones in sugarcane pest management
H DAVID, B F NESBITT*, S EASWARAMOORTHY and
V NANDAGOPAL
Abst,.c, Seven species of
SSSeT^^^^^^
Sfield population. The « »» £ «£ S^recent years and among these the
stalk borer, shoot borer and top borer have oeen ^ attractants.
nternode borer and stalk **%^ internode borer while daruptw*
Keywords. Sex pheromones; sugarcane; moth borers.
1. Introduction
estimated to be 158 to 4 18 ou 2Q
t«
Dv/lCl. V^1-* Jr rt^«/"*ACl \(*Cl WLLlllll tUV' v A vj >-'•-•«'•'
Chaudhary 1964). These borers «e conowco w«« ^^ ^ for contact
not exposed for parasitisation or Pr^at^yltionbecomes difficult. This warrants
insecticides. Hence the suppression ol t n^ P"?.^ ^ tQ ^ integrated harmoniously
different methods of suppression strateg ^ injuring level. Among the modern
in order to manage them below the eco _ Qmones offers SCOpe for monitoring
technologies for pest suppression, use o F ^ fertility of Wud females thereby
the activity of these borers and also in _reau ^ ^^ This method Can also be
bringing down the fertile egg • PJP"™"! . in the pest management programme,
conveniently integrated with °tne' "^ have been camed out in four species of moth
Preliminaryinvestigationsonthisstrategyna
borers and these are summarised in this paper.
2 Sexual activity of the borer moths
it
^ta and 0200 hr in stalk borer
Mating has been observed to take place .be^fnthe niht in shoot borer (Usman ,t al
(Kalraand David V^d^.^^^odrtoL (David and Kalra 1965) and
1957) during the late hours of the night minternoaeo
334 H David et al
throughout the night in top borer (Kalra et al 1978). Prior to mating the virgin female
moths assume a 'calling position' by raising their abdominal tips and during this
process the pheromone is released. The male moths downwind take up the scent and fly
towards the female. A large number of male moths are attracted simultaneously
towards the virgin female and they fly excitedly around it and then mating takes place.
This chemical communication between the two sexes in these moths can be taken
advantage of for either mass trapping the male moths or for disruption of the
communication by suitable contrivances so that mating and fertility of wild females can
be reduced.
3. Studies with virgin females
Studies have been conducted on the potency of attraction of male moths by virgin
females of stalk borer (Kalra and David 1971), top borer (Kalra et al 1978), internode
borer (David and Chandra 1972) and shoot borer (David et al unpublished). The virgin
females collected from pupae which are separated and kept in isolation from the male
pupae, when placed in sticky/water traps attracted male moths. The number of male
moths attracted ranged from 0 to 107 in stalk borer, 0 to 5 in top borer, 0 to 10 in
internode borer and 0 to 33 in shoot borer. In stalk borer, the number of male moths
trapped was more during the first 24 hr and it declined subsequently. It was also
observed that four-day old virgin females or mated females did not attract male moths.
Abdominal extracts of virgin females taken in solvents like ether also attracted males of
stalk borer and the number trapped ranged from 0-1 to 4-2 moths/day/trap. These
experiments have indicated the presence of potent sex pheromones in the females of
stalk borer, internode borer and shoot borer.
4. Identification of sex pheromones
The fresh pupae of stalk borer and internode borer were air freighted to UK and the
pheromones were isolated and identified by Dr B F Nesbitt at the Tropical
Development and Research Institute. The pheromone of stalk borer consists of the
following four components, viz.,
(i) (Z)-7-dodecynyl acetate,
(ii) (Z)-S-tridecenyl acetate,
(iii) (Z)-9-tetradecenyl acetate and
(iv) (Z)-lO-pentadecenyl acetate,
while that of the internode borer consists of two components, viz.,
(i) (Z)-13-Octodecenyl acetate, and
(ii) (Z)-13-Octodecenyl alcohol.
The synthetic products of these pheromones have been prepared in different
combinations and were evaluated in the field. The sex pheromones of shoot and top
borers are yet to be chemically characterised.
5. Comparative performance of synthetic pheromone with virgin females
The comparative efficacy of the virgin female and synthetic pheromone in water traps
was assessed for 42 days in the stalk borer. The number of moths trapped/trap/day was
Sugarcane pest management 335
significantly more in synthetic pheromone traps (7-95) than in virgin female traps (0-39).
In the internode borer, the comparative efficacy was evaluated for 17 days. The mean
number of moths trapped was significantly high in water traps baited with synthetic
pheromone (5-35) compared to .virgin females (2-35).
6. Mass trapping
6.1 Stalk borer
Two combinations viz., 1:1:1:1 and 4:8:4:1 of the components (Z)-7-dodecenyl
acetate, (Z)-S-tridecenyl acetate, (Z)-9-tetradecenyl acetate and (Z)-lO-pentadecenyl
acetate at two different doses, viz., 0-1 and 1 mg were tried at Shamli in 1982 and the
results are given in table 1 . The combination of 4 : 8 : 4 : 1 at the dose of 1 mg was found
to be more potent than the three other treatments. During 1983, eight more
combinations (table 2) were tried and it was observed that the moth catch was more in
traps baited with vials containing a 2:1 ratio of (Z)-S-tridecenyl acetate and (Z)-7-
dodecenyi acetate and an 8 : 1 ratio of (Z)-8-tridecenyl acetate and (Z)-lO-pentadecenyl
acetate.
Between 25.2.1983 and 8.4.1983, mass trapping was attempted with 20 water
traps baited with 1 mg of 4:8:4: 1 combination. A total of 2863 moths were trapped
with a mean of 7-95 moths/trap/day. The maximum number of moths trapped was
259/trap/day.
6.2 Internode borer
The efficacy of different combinations of acetate and alcohol viz., 1:1;3:1;5:1;7:1 and
9 : 1 was assessed by conducting field experiments at Sakthi Sugars Ltd., Sakthi Nagar
and Deccan Sugars Ltd., Pugalur. The experiment had five treatments and each
treatment was replicated six times. The distance between the traps was 30m.
Observations on moth catch was recorded at weekly intervals for 1 1 weeks from the
Table 1. Evaluation of different components in the mass trapping of C.
auricilius.
Total
male
Dosage moths Mean moths/
Components (mg) caught trap/day
1:1:1:1
Z7:Z8:Z9:Z10
component
1
31
0.70B
1:1:1:1
Z7:Z8:Z9:Z10
component
0-1
25
060B
4:8:4:1
^7 • ^8 • ^9 " ZIQ
component
1
1643
16-5A
4:8:4:1
Z,:Z8:29:Z10
component
0-1
54
121B
Figures followed by the same letters are not statistically different.
336
H David et al
start of the experiment. It was found that the average moth catch was more at Pugalur
(6-23 moths/trap/day) than at Sakthi Nagar (3-44) and the difference was statistically
significant. Among the combinations tested, though the catch was more in 5:1
(acetate : alcohol) combination, the differences between the different combinations
were not statistically significant (table 3). The catch was more and on par during fourth
to eleventh weeks indicating the persistence of the material under field conditions for
longer duration.
Another experiment was conducted to find out the optimum height of the water trap
for maximum moth catch. The water traps were set at 60, 90, 1 20, 1 50 and 1 80 cm height
and the treatments were replicated five times. The mean moths trapped/trap/day was
more in traps set at 60 cm height (12-88) compared to 7-10 to 8-72 moths/trap/day in
other treatments.
After fixing up the optimum combination and height of traps, the synthetic
pheromone of the borer was used for mass trapping of male moths under field
conditions in two centres viz., Coimbatore and Pugalur during 1982. The area trapped
was about three hectares at Coimbatore and seven hectares at Pugalur. In both the
centres suitable control of about one hectare at a distance of 0-5 km was earmarked for
observations. The distance between the traps was 33 m. The traps were set up in about
five to six months old crop and two changes of the pheromone vials were given after two
Table 2. Evaluation of different components in the mass
trapping of C. auricilius.
Mean
Total
moths/
moth
trap/
Components
caught
day
4:8:4:1 mixture CA 83/35
Z7:Z8:Z9:Z10 component
751
1O80BC
Z7 component CA 83/86
21
0-31C
Z8 component CA 83/37
38
055C
Z9 component CA 83/38
39
0-56C
Z1Q component CA 83/39
275
3-94c
Z8:Z7 mixture 2: 1) CA 83/40
536
12-705
Z8:Z7 mixture 2:1) CA 83/41
1585
22-75*
Z8 + Z10 mixture 8: 1) CA 83/42
1524
21-78*
The dosage used was 1 mg for all the cases.
Table 3. Evaluation of different components on the mass
trapping of C. sacchariphagus indicus (K).
Components
Mean moths trapped
Sakthinagar Pugalur
1 : 1 Acetate : alcohol
3-28*
5-87*s
3:1 Acetate: alcohol
3-58*
5-80B
5:1 Acetate: alcohol
3.57/1
6-57*
7:1 Acetate : alcohol
3-39*
6-41*
9:1 Acetate: alcohol
3-38*
5.49/1
Sugarcane pest management 337
months. Observations on the moth catches were made weekly. Observations on the
progressive incidence and intensity of infestation of the borer, both cumulative and
fresh attack, were made at periodic intervals. Detailed observations were made at
harvest on incidence and intensity of the infestation, larval population, yield and quality
of canes at Pugalur.
A total of 9822 male moths were collected at Pugalur with a mean catch of
0-83 moths/trap/day. The mean borer incidence was 93-63 and 87-17% in the treated
and control plots and the difference was not statistically significant. There was no
significant difference in the intensity of borer infestation and larval population, yield of
cane and ccs % between the treated and control plots.
At Coimbatore, a total of 1122 male moths were collected with a mean of
0-59 moth/trap/day. The details of observation on incidence and intensity of borer
infestation are given in table 4. It was observed that there was significant difference in
the incidence of borer at harvest but not with respect to the intensity of borer
infestation.
During 1983, mass trapping was attempted in an area of five hectares at Pugalur.
Suitable control of one hectare at a distance of 0-5 km with the same variety and age of
the crop was earmarked for observations. The distance between the traps was 16-5 m in
one set and 24-8 m in another. The traps were set up in about four months old crop and
three changes of pheromone vials were done.
A total of 74697 male moths were trapped with an average catch of 3-4 moths/trap
/day. The mean moth catch was 2-95/trap/day in the traps set up at 24-8 m distance and
it was 3-8 /trap/day in the traps set up at 16-5 m distance and the difference was not
significant. The data collected at harvest revealed that there was significant difference in
incidence (table 5). The mean incidence in treated plots was 68-36 % and in control plots
Table 4. Internode borer incidence and intensity of attack at Coimbatore.
Treated plots Control plots
Per cent Per cent Per cent Per cent
Period incidence intensity incidence intensity
Before setting up of traps 8-73 4-07 13-65 3-39
30 days after setting up of
traps 13-44 5-27 19-18 5-08
60 days after setting up of
traps 17-16 ' 4-90 31-36 5-57
Table 5. Yield data from the pheromone treated and control
plots.
Parameters Treated Control
Per cent incidence (plot basis) 68-36* 76-85^
Per cent intensity— Old 2-86^ 3-26^
Fresh l-49A \-29A
Plot weight (kg) 564-41* 473-26*
Per cent purity 84-38^ 82-73*
ccs per cent* 9-63^ 9-00*
* Commercial cane sugar per cent
338 H David et al
it was 76-85 %. But the differences observed in the intensity of the borer infestation were
not significant. The cane yield was significantly more in treated plots and also there was
improvement in purity and ccs % due to mass trapping of internode borer male moths.
7. Communication disruption
7.1 Stalk borer
The scope of disruption of the pheromone communication system in stalk borer was
assessed by saturating the atmosphere near the pheromone trap with the optimum
combination of 4:8:4: 1 at the dose of 1 mg/vial during 1983. The surround vials used
for saturating the atmosphere contained either (Z)-7-dodecenyl acetate, (Z)-8-
tridecenyl acetate, (Z)-9-tetradecenyl acetate, (Z)-10-pentadecenyl acetate or 4:8:4:1
combination of the above components at the dose of 1 mg. Maximum reduction
(85-02%) in moth catch was recorded when (Z)-7-dodecenyl acetate was used in the
surround vials. The reduction in other treatments was 1-63, 0-0, 0-0 and 54-07%
respectively.
During 1984, two more experiments were conducted with the most effective
component viz, (Z)-7-dodecenyl acetate. The result obtained confirmed the earlier
finding and the per cent reduction observed was 76-18 % in one experiment and 94-96 %
in another.
7.2 Internode borer
The experiments conducted on the same lines as in stalk borer, did not give much
encouraging results. The reduction observed ranged only from 24-87 to 38-58 % when
different combinations were used in the surround vials.
8. Discussion
The observations on the sexual attraction in these species of moths showed that there is
potent sex pheromone in the females of stalk borer, internode borer and shoot borer
while it is apparently feeble in the case of top borer. Intensive studies on the former two
species indicate that these two vary with regard to the population density. The
population density is low in internode borer and high in stalk borer, especially in the
later broods and this is reflected in the number of moths trapped in the water traps with
a maximum of 259. Moreover, in the stalk borer, the emergence of moths is apparently
not continuous, being restricted to specific broods under subtropical conditions
whereas in internode borer, it is continuous throughout the year under tropical
conditions. Because of the continuous emergence, male moths of internode borer can be
trapped throughout the year using water traps. The traps are to be set up with the
commencement of internode formation with only two changes of the synthetic
pheromone vials at trimonthly intervals. In the case of stalk borer, the initial two broods
have low population and the setting up of the traps will be highly useful for identifying
the initial foci of infestation which is otherwise normally difficult to identify in the field.
Sugarcane pest management 339
The results of the communication disruption experiment have shown clearly that it may
be highly useful for field application in the case of stalk borer while it may not be much
effective for the check of internode borer.
These studies have indicated promise for field application of mass trapping and
disruption techniques using synthetic pheromones and hence further detailed infor-
mation on cheap and durable dispensers, simple traps for field use, optimum number of
traps/unit area and scope of combining this method with other methods in the
integrated pest management of these highly destructive borers may be obtained.
Acknowledgement
The authors thank Dr K Mohan Naidu, for facilities provided and also M/s. Deccan
Sugars Ltd., Pugalur; Sakthi Sugars Ltd., Sakthi Nagar and Upper Doab Sugar Mills
Ltd., Shamli for the excellent field facilities extended throughout the investigations.
References
David H and Chandra J 1972 Sex attraction in sugarcane internode borer, Chilo (Proceras) indicus Kapur;
Labdev. J. Sci. Technol BIO 159-160
David H and Kalra A N 1965 A sturdy of sugarcane internode borer, Proceras indicus Kapur; Proc. Cong. Int.
Soc. Sugarcane Technol 12 1444-1453
David H, Sithanantham S and Velayutham B 1979 Some aspects oflosses due to internode borer in sugarcane
in Tamil Nadu; Proc. Annu. Conv. Deccan Sugar Technol. Assoc. 29 27-40
Gupta B D and Avasthy P N 1954 A resume of the behaviour of varieties towards the incidence of major pests
of sugarcane in UP and other states in India; Proc. Con]. Sugarcane Res. Dev. Wkrs. India, 2 229-241
Kalra A N and Chaudhary J P 1964 Assessment oflosses by sugarcane pests — loss caused by top borer of
sugarcane; Indian J. Sugarcane Res. Dev. 8 261-264
Kalra A N and David H 1971 Studies on sex attraction in the sugarcane stalk borer, Chilo auricilius Ddgn;
Indian J. Agric. Sci. 41 21-24
Kalra A N, Mehrotra A K, David H and Chandra J 1978 Sexual response and mating behaviour in moths of
top borer, Tryporyza nivella Fabr. (Pyralidae: Lepidoptera), a major pest of sugarcane; Proc. Annu. Conv.
Deccan Sugar Technol. Assoc. 28 65-70
Parthasarathy S V, Krishnamurthy C S, Prakasam P and Ramachandrachari C 1953 Loss in yield due to pests
and diseases of sugarcane; Proc. Annu. Conv. Sug. Technol. Assoc., 22 1 3-22
Usman S, Sastry K S S and Puttarudriah M 1957 Report of the work done on the control of the sugarcane
borers Dept. Agric. Mysore State pp. 69.
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 341-350.
© Printed in India.
Role of behavioural studies in the development of management
strategies for forest insect pests
K S S NAIR
Division of Entomology, Kerala Forest Research Institute, Peechi 680 653, India
Abstract. Under forestry conditions, management techniques aimed at maintenance of pest
populations at moderate levels have greater chance of success than conventional methods of
pest control. Simple behavioural observations can sometimes be used to great advantage in the
development of such methods, some examples of which are given. Although there has been
considerable excitement over the past two decades on the possibility of using behaviour
modifying chemicals for control of pests through mass trapping or disruption of the insect's
normal communication systems, no significant practical achievement has so far been reported.
Difficulties in the use of these chemicals include inadequate information on the biological
responses of natural populations of insects; utilization by most insects of a complex
pheromone system involving several chemical components; non-reproducibility of laboratory
results under natural conditions due to several modifying factors; high cost of the development
and deployment of pheromonal control systems, particularly for low value forestry crops;
inadequacy of pheromonal control methods for coping with the high epidemic densities of
most forest pests; and the possibility of development of pheromone resistance. Behaviour-
modifying chemicals, such as food lures, sex pheromones and population aggregating
pheromones, however, are useful in pest management as tools for survey and ecological
research. Populations generally exhibit properties that cannot be understood by studying
individual insects; study of the behaviour of populations is therefore more important than
study of the behaviour of individuals for developing management strategies.
Keywords Forest pest management; pheromones in insect control; insect behaviour; food
plant chemicals; Hyblaea puera.
1. Introduction
The major insect pest problems of natural and man-made forests (forest plantations)
have been summarised by several authors (Pant 1974; UNESCO 1978; Mathur 1964;
Chatterjee and Sen-Sarma 1968; Sen-Sarma and Thapa 1981; Nair 1980). In the tropics,
most pest problems are noticed in man-made, as against, natural forests. Experience in
Kerala has shown (Nair 1980) that all forest tree species grown in a sufficiently large
area for at least one tree generation have brought up with them at least one or two
serious insect pests. Although the economic damage caused by most forest pests have
not been adequately assessed, many of them are considered serious enough to merit
application of control measures. The present paper will examine the unique problems
of forest pest control in the tropics and the relevance of behavioural studies in
developing control measures. Some examples of successful use of behavioural
observations for practical pest management will be discussed and a critical evaluation
made of the prospects of using behaviour-modifying chemicals for direct control of
pests.
342 K S S Nair
2. Unique problems of forest pest control
Although forests have fewer pest problems than agricultural crops, the problems
entomologists must face in devising control measures for them are much greater.
Conventional methods developed primarily for agricultural crops often turn out to be
unsuitable for forest crops for several reasons -(i) The extensive area under forest
plantations, often in inaccessible hilly terrain, impose serious restrictions on the
practical implementation of surveillance and control operations, in spite of theoretical
feasibility, (ii) Again because of the extensive land area covered, the risk of adverse
environmental impact of control operations, particularly the use of insecticides, is large
(iii) The large size of the trees makes special demands on techniques of applications of
control measures -whether it be spraying of a chemical or microbial insecticide or
hand-picking of insect larvae, (iv) The greater permanence of the forest crop (for
example, the harvesting period of teak is 60 years even in a good quality site) preclude
certain types of control measures like crop rotation, (v) Above all, the lower economic
returns from forest crops (compared to agricultural crops) limits the expenditure that
can be incurred to prevent insect damage.
The appropriateness of control measures for forest pests must be judged in the light
of the above considerations. Obviously, methods acceptable for use in forests must be
effective, cheap and environmentally "safe", a combination of qualities that is difficult
to accomplish in practice although it sounds simple and ideal. What is usually achieved
is something less than ideal: compromise usually involves tolerance of a certain degree
of damage (i.e., no control or partial control), acceptance of a high cost of treatment, or
neglect of minor environmental damage. Methods which make use of silvicultural
manipulations, natural enemies (including predators, parasitoids and pathogens) and
other population management techniques based on behavioural peculiarities of pests
and their natural enemies to keep the pest populations at reasonable levels are the most
suitable for dealing with forest pests.
If there are unique difficulties in dealing with forest pests, there is also at least one
unique advantage. Because the forests are owned by governments, indiscriminate use of
insecticides can be prevented by policy decisions of the governments, even if a certain
degree of damage is sustained. In agriculture, on the other hand, the profit motive of
individual farmers often works against the larger interests of society. The chances of
practising pest management, as distinct from direct kill of insects, is therefore greater in
forestry than in agriculture. Behavioural studies that could turn out useful in the
development of management strategies may be considered under two major heads -
simple behavioural observations and modification of insect behaviour by chemicals.
3. Simple behavioural observations for insect control
Simple behavioural observations can sometimes be used to great advantage for the
management of forest insect pests. Some examples follow.
The caterpillar of the moth, Hyblaea puera is a serious pest of teak plantations
throughout India. Experimental aerial application of insecticides has been tried in the
past (see Sen-Sarma and Thapa 1981) to control this insect. A 4-year field study
conducted in Kerala (Nair and Sudheendrakumar, unpublished) showed that in spite of
the insect's potentiality to pass through at least one generation per month, serious
Behavioural studies and forest pest management 343
outbreaks occurred only once or twice a year in most plantations, usually in late April to
August. Unpublished recent observations on the egg-laying behaviour of natural
populations of H. puera moths, on the behaviour of mature larvae, and on the
movement pattern of newly emerged moths have revolutionised our concepts of the
population dynamics of this important pest. The current working hypothesis is that
the greater proportion of the moth population, resulting from a larval build-up in one
area, migrate to another area about 5 to 10 km away, to start a new infestation. H. puera
moths lay eggs only on tender leaves. Although teak is a deciduous species, observations
have shown that there exists enough phenological variation among individual trees
within an area and among populations of trees in different areas within a larger
geographical region to make tender leaves available continuously, though not at the
same place, to sustain a residual population of larvae. With the onset of the general
flushing season, the insect population builds up, step by step, colonising newer and
newer areas, moving in the general direction of late flushing areas from early flushing
areas. Later, populations decline when parasite populations build up or large quantities
of tender leaves are no longer available. Existence of a residual population during the
off-season (non-flushing period of teak) and gypsy-like migration of adult moths, are
new concepts prompted by simple behavioural observations (unpublished), which if
proved, can lead to simple methods to control the outbreak. In plantations, initial
build-up of larval populations can be located by surveillance during the critical period.
As mature larvae descend to the ground for pupation, application of a contact
insecticide or appropriate microbial agent can prevent emergence of the moth
population and their subsequent spread to other areas. This method is much simpler,
practicable and environmentally less hazardous than aerial application of insecticides
that had been experimented with in the past.
Increasing evidence is now accumulating to suggest that migration may be more
common than hitherto suspected in many pests, particularly the noctuid moths
(Barfield and Stimac 1981; Oku 1983; Stinner et al 1983; Riley et al 1983). Management
strategies for such highly mobile pests must necessarily be based on observations on the
behaviour of natural populations.
Several examples can be cited to show how careful observations on the behaviour of
pests can lead to improvements in their control. Subterranean termites damage young
transplants of eucalypts within a few months of planting out. Field observations
revealed that characteristically the termites (mostly Odontotermes spp.) begin their
attack in the root collar region a few centimeters below groundlevel (Nair and Varma
1982). It is therefore possible to control the attack by treating the soil core immediately
surrounding the tap root, instead of the entire planting area. This can be accomplished
by drenching the polythene bag containing the seedling with an insecticide before
transplanting it in the field, thus effecting considerable reduction in environmental
contamination as well as cost of treatment. In African countries, on the other hand,
different species of termites, notably Macrotermes spp. cause most damage to
eucalypts; they forage at the soil surface level and attack the stem of saplings at or
above ground level. This behaviour of termites calls for a different approach to control.
Control of the sapling borer, Sahyadrassus malabaricus Moore (Hepialidae) by spot
treatment of the tunnel mouth is another example. This borer attacks saplings of forest
plantations and lives inside a large tunnel along the pith. The tunnel mouth is covered
by a mat-work of wood particles and faecal pellets. Observations revealed that if the
tunnel mouth cover is removed, the larva comes out at night and makes a new cover
344 K S S Nair
with wood particles gathered from the vicinity of the tunnel mouth. Control of insects
which live inside wood is generally difficult, but the above behavioural observation was
made use of to control this insect by pulling off the cover and applying a contact
insecticide at the tunnel mouth region (Nair 1982).
Behavioural observations also find application in the control of cerambycid wood
borers. Observations have revealed that they lay eggs only on logs with bark.
Debarking is therefore a standard practice for protection of stored logs from
cerambycid borers.
4. Modification of insect behaviour by chemicals
Over the last 15-20 years there has been considerable excitement among entomologists
on the possibility of using behaviour-modifying chemicals for control of pests. A large
number of chemicals have been isolated from a variety of insects and other sources,
including host plants or animals, and their exact chemical structures have been
established; many have also been synthesized outside the animal or plant system. These
are elegant achievements made possible by the active involvement of expert chemists
and general advances in instrumental analysis. Many such chemicals have been tested in
the laboratory for behavioural responses and found promising. Pilot-scale field tests
have also been carried out for some of them. We shall, consider them under two
heads -food plant chemicals and pheromones.
4.1 Food plant chemicals
Since Fraenkel's (1959) classic paper on the 'raison d'etre of secondary plant
substances', the role of plant chemicals in influencing the orientation of insects to food
plants for feeding or oviposition has been well recognized. Since then we have learned
much about the mechanisms of host plant selection of various phytophagous insects.
Several excellent reviews of the topic are available (Kennedy 1965; Dethier 1970 a, b;
Schoonhoven 1972, etc.) and several national and international symposia have also
been conducted on the topic (Ananthakrishnan 1977; Visser and Minks 1982; etc.).
Except in a few instances, however, it has not been possible to use this new knowledge
for developing control measures against pests. Nonspecific food lures like sugar
solutions or protein hydrolysates mixed with toxic compounds have often been used to
attract and kill insects like ants, cockroaches and flies. A toxic bait containing aldrin,
soya bean oil and dried citrus meal applied aerially at the rate of 2-2 kg/ha was reported
to be collected by leaf-cutting ants (Atta sp.), a pest of forest trees in the neotropics, in
preference to fresh leaves and to kill about 91 % of the ants in some experiments (Lewis
1972). The best known example in Indian forestry is the use of trap logs to attract the sal
borer, Hoplocerambyx spinicornis. This cerambycid borer is a serious pest of natural and
man-made forests of Shorea robusta (sal), an important timber tree in the northern
states of India (Beeson 1941) bad epidemics of which have occurred in the past in UP,
MP, Bengal and Assam. Normally a borer of sickly trees, at times of epidemics, even
healthy trees are attacked and killed. Adult beetles of both sexes are attracted to newly
exposed inner bark and sapwood of trees over distances upto 400m. It is therefore
standard practice to fell unhealthy or injured trees, cut them into 3m logs, beat up the
cut ends to expose fresh sapwood and distribute them in small heaps in the affected area
Behavioural studies and forest pest management 345
to attract the beetles. Trap trees at the rate of 3 to 5 per hectare are used; at intervals, the
logs are again cut into smaller billets to expose fresh sap. The beetles are collected and
destroyed daily. Collections of upto 1000 beetles per trap tree per day have been
reported (Beeson 1941). The nature of attractant has not been determined.
In general, although there has been initial optimism on the use of behaviour-
modifying host plant chemicals, particularly the secondary plant substances, for insect
control, this has not been borne out by experience. The main handicap is the
involvement of several chemicals, each having some influence on the chain of
behavioural responses of the insect leading to host plant recognition and acceptance.
The problems of using them for control can be illustrated using the example of the
cabbage root fly, Delia radicum (Anthomyiidae) a pest of cruciferous crops in Europe
and North America which has been studied in some detail (Traynier 1965, 1967a, b;
Coaker 1969; Coaker and Finch 1971; Nair et al 1974, 1976; Nair and McEwen 1976;
Finch 1978; Stadler 1978; Hawkes and Coaker 1979; Ellis et al 1982). In this insect, the
first step in host selection is taken by the adult female when it lays eggs in soil close to
the host plant. The newly hatched larvae bore into the roots where they feed and grow.
Highly volatile mustard oils, break-down products of glucosinolates (mustard oil
glucosides) present in cruciferous plants, stimulate the flies into greater activity and
attract them to the plant. The parent glucosinolates then induce oviposition. Allyl
isothiocyanate (AITC), one of the mustard oils tested in the laboratory, did not induce
oviposition by itself, but in the presence of a glucosinolate, very low concentrations of it
caused an increase in the number of eggs laid (Nair and McEwen 1976). Many of the
nutrients tested did not influence oviposition, but a protein hydrolysate inhibited it.
Flies also arrive and land on non-host plants by random movements. The gravid female
which lands on a plant probes the leaves with the proboscis (other sense organs may
also be involved) and if the plant is judged suitable, walks down to the bottom and lays
eggs in soil, close to the plant stem. A large number of factors, chemical, visual and
physical, apart from the physiological state of the insect, are involved in the sequence of
behavioural events leading to oviposition. In the next major step, similar factors affect
the establishment of the neonate larvae on the root. All crucifers are believed to contain
one or more glucosinolates, usually a mixture of several, but some crucifers did not
elicit oviposition (Nair et al 1974). Experiments (Nair and McEwen 1976) revealed that
although glucosinolates appeared to be the only oviposition inducing substances
present in crucifers, there was no correlation between the total glucosinolate content of
crucifer leaves and the oviposition response of the fly and that some glucosinolates
elicited more oviposition than others. It appeared that both the absence of glucosino-
lates and the presence of inhibitory chemicals could make a plant unacceptable for
oviposition. With more than 30 glucosinolates known from different plants, and with
several glucosinolates occurring together in a given species, Nair et al (1976) suggested
that a total 'glucosinolate pattern' may be more important in deciding the oviposition
response. In the light of Dethier's (1973) conclusions based on extensive elec-
trophysiological studies on the gustatory receptors of lepidopteran larvae, Nair et al
(1 976) also discussed how host selection may depend not on the presence or absence of a
single stimulant or deterrent, but upon the total sensory impression derived from an
integration of sensory responses to several plant constituents. Dethier (1973) has
discussed in detail how subtle differences in one or more plant constituents result in
different patterns of sensory input, and consequently, different magnitudes of response
of insects.
346 K S S Nair
In general, since a chain of sequential behavioural responses to food plant chemicals
is involved in food selection and since most of these responses can at least partially be
modified by other factors, the chances of utilizing any of the food plant chemicals to
control insects by radically modifying their behavioural response is meagre. Recent
studies have also shown (Ellis et al 1982) that the egg laying behaviour of the cabbage
root fly is also influenced by microbial activity in or around its host plants. To compete
with natural food plants, we need chemicals that will elicit superoptimal responses from
the insect, but we are beginning to understand that though the insect may act
"instinctively" rather than intelligently, optimal responses have been instinctively built
into their behavioural repertoire. For example, while low concentrations of AITC will
attract the cabbage root fly and promote egg laying, high concentrations of it will either
be ignored through sensory adaptation or may elicit the opposite reaction. For use in
controlling the insect, we should expect that a high concentration of attractive
chemicals will attract the insects more effectively than the optimal concentration
released by the natural hosts. Such responses have seldom been observed under natural
conditions, although the search must continue in the hope that it may turn out useful in
some cases.
4.2 Pheromones
There is vast literature on pheromones of insects and their potential uses (see Jacobson
1972); we shall limit our discussion to two examples of forest pests and examine the
usefulness of pheromones for their control.
The first example is the gypsy moth Lymantria dispar ( = Porthetria dispar)
(Lymantriidae). The caterpillar of this moth is a pest of oak, birches and maijy other
hardwoods in Europe, USSR and some parts of USA, particularly eastern New York and
New England. As early as 1925 it was suspected that the flightless virgin females of this
moth produced a sex attractant (pheromone) which lured the males. A series of
investigations over several years culminated in the identification of the active
component of the sex pheromone in 1970, designated as 'disparlure' (Bierl et al 1970). In
between, preparations called 'gyptol' and 'gyplure' were also isolated and/or syn-
thesized. Early attempts (in 1961) by the us Department of agriculture to control gypsy
moth populations by disruption of the mating communication systems (male confusion
method) with gyplure distributed by air craft over a 160 ha island located near New
Hampshire did not show any adverse effect on male mating activity, but this was
attributed to weak attractiveness of the commercial 'gyplure' used in the experiment
(Jacobson 1972). A subsequent field experiment in 1964 with preparations of 'gyplure'
previously shown to be effective in both laboratory and field tests, also did not yield
encouraging results. In later experiments using disparlure, traps baited with the
chemical were air dropped over large infested areas to attract and kill the males or strips
of paper impregnated with the chemical were dropped to confuse the males. Both
approaches appeared to be successful in areas of light infestation although not in areas
of heavy infestation (see Jacobson 1972).
While disparlure has not yet become a practical tool for controller se, it is widely used
as a surveillance tool, particularly to monitor the spread of this introduced pest within
the USA to undertake timely control measures. General conclusions on the usefulness of
these chemicals for control may be drawn after considering the second example, the
pine bark beetles.
Behavioural studies and forest pest management 347
Several species of the small scolytid beetles belonging to the genera Dendroctonus,
Ips, Scolytus, Orthotomicus, Xyleborus etc., attack pines and other trees in different
parts of the world. Most investigations have been made on the pine bark beetles,
Dendroctonus spp. in the USA and USSR and the literature is vast (see Wood 1970, 1980;
Stark 1973; etc). In general, infestation occurs in the following sequence. Depending on
the species, adult beetles of one of the sexes make the initial attack, usually on weakened
trees which apparently have a characteristic odour profile. Once established, the beetles
produce a population aggregating pheromorie, possibly using precursors ingested from
the tree, which is passed out through the frass and serves to attract other beetles to cause
mass attack. In severe infestations, healthy trees are also attacked and killed. Several
pheromones viz, frontalin, brevicomin, etc., have been isolated and synthesised from
different species of bark beetles. The results of field experiments for control using bark
beetle pheromones have been discussed by Wood (1970) and Roelofs (1975). In general,
pheromone mixtures have been found effective in trapping large numbers of bark
beetles, but unequivocal evidence of their effectiveness in reducing tree mortality under
field conditions is yet to come, although the results are reported to be promising.
We shall now consider the prospects of using pheromones for pest management in
forestry. The most obvious and profitable use of such chemicals is to detect the presence
of the insect, either to monitor its spread into an area or to time the application of
control measures. In the case of pests which appear all of a sudden in large numbers, like
the teak defoliator, pheromones will be of little value to detect the time of their
appearance. It is, however, an important research tool to elucidate many aspects of the
ecology of the insect; for example, to determine whether residual populations of the
insect occur in plantations or natural forests during periods when no visible defoliation
occurs.
Although high hopes have been raised on the possibility of using pheromones for
control of insects, either through mass trapping or the male confusion method, and
optimism still prevails, no significant practical achievement has so far been reported, in
spite of intensive efforts over the past 15 to 20 years -a sufficiently long period for
experimentation. An excellent and critical discussion, full of insight, of the problems
and prospects has been given by Green way et al (1977), which must be consulted by
anyone who contemplates use of pheromones for insect control. Some of the difficulties
in using pheromones for direct control are:
(1) Isolation, identification and synthesis of the pheromones have progressed very
fast, but our understanding of the responses they induce in individuals, and especially of
the responses they induce in populations has not progressed fast enough (Greenway et
al 1977). Biological response studies are more cumbersome and time consuming and tall
claims have often been made with preliminary data. As Greenway et al (1977) pointed
out, variability of responses in behavioural assays has not always been reported in
published studies.
(2) In many cases it has been found that the functional sex pheromone is not a single
compound but a mixture of compounds which constitute a 'pheromone system* (Minks
et al 1977). For example, four components are involved in the sex pheromone system of
the sugarcane stalk borer Chilo auricilius and effectiveness depends on an optimum
combination of these compounds (H. David personal communication). There are
several examples in which only specific combinations of certain chemicals are active
(Minks et al 1977). The greater problem, which has not been generally recognised, is the
348 K S S Nair
likelihood that different individuals in the given population may respond (reference
here is to optimum response) to different combinations of the chemicals.
(3) In general, many predictions based on laboratory results have not been realized
under field conditions due to various interfering factors. The behavioural reaction of a
living organism, more particularly of a population of organisms cannot be expected to
follow rigid principles, or perhaps, if they do, we are still ignorant of the principles
which govern them and unable to predict the outcome. In spite of such knowledge, in
control trials using pheromones we often expect that behavioural responses of the
insect in the field will be as predictable as the outcome of a chemical reaction.
(4) Because of the cost and effort involved, development and use of pheromones for
direct control may prove economically feasible only in high-value, intensively managed
crops, and not in extensive low-value plantings (Green way et al 1977) like forest
plantations, especially in developing countries.
(5) As noted in the few field trials on gypsy moths, artificially used pheromones were
effective in controlling the insect only when the pest population was low. When
enormous numbers of insects are involved, as in an epidemic of forest insects like the
teak defoliator or bark beetles, pheromone traps cannot cope up effectively with the
numbers. Theoretical calculations suggested (Roefols 1975) that an initial trap to
female ratio of at least 5 : 1 would be needed to obtain 95 % suppression of mating.
(6) The possibility of insects developing resistance to pheromones has generally been
ignored in discussions on the use of pheromones for insect control. Even Greenway et
al's (1977) otherwise elegant discussion is silent on this point. Green et al (1960),
however, discussed this problem. Development of pheromone resistance appears to be
as simple and imminent as development of insecticide resistance, if we resort to direct
control of insects with pheromones. One of the arguments in favour of use of sex
pheromones for control was the idea that an insect cannot develop resistance to a
chemical on which it depends for mating communication. However, resistance can
develop in the following manner. Most insects make use of a pheromone system
involving more than one chemical. As discussed above, different individuals of a given
population may show optimal response to different combinations or ratios of the
pheromones. Widespread use of a particular pheromone combination for mass
trapping or mating disruption will eliminate that part of the population which responds
to it, leaving the small unresponsive part of the population to mate and multiply. With
continued selection pressure, a new population will evolve which makes use of a new
slightly altered pheromone communication system. Such a population will no longer be
responsive to the standard pheromone system; in other words, resistance has
developed. If the pheromones are used only for survey purposes, it will not exert much
of selection pressure and therefore will not lead to development of resistance.
5. Conclusions
Behavioural observations are important in developing control measures against forest
pests. Simple observations on the behaviour of natural populations can often be used to
great advantage in developing strategies for management of pests. In the management
of forest pest populations behaviour modifying chemicals such as food lures, sex
pheromones and population aggregation pheromones are useful as research and survey
tools, but not for direct control
Behavioural studies and forest pest management 349
Most new ideas useful for management of pests are likely to come from behavioural
studies of populations, particularly natural populations, rathet than observations made
on individual insects in the laboratory. Populations exhibit properties that cannot be
understood by studying individual insects.
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Coaker T H and Finch S 1971 The cabbage root fly, Erioichia brassicae (Bouche'); Rep. Natl Veg. Res. Stn.
for 1970 (Wellesbourne England) pp. 23^2
Dethier V G 1 970a Chemical interactions between plants and insects; In Chemical ecology (eds) E Sondheimer
and J B Simeone (New York: Academic Press) pp. 83-102
Dethier V G 1970b Some general considerations of insects' responses to the chemicals in food plants; In
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and A K Minks (Wageningen: Centre for Agric. Publishing and Documentation) pp. 131-137
Finch S 1978 Volatile plant chemicals and their effect on host plant finding by the cabbage root fly (Delia
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Fraenkel G 1959 The raison d'etre of secondary plant substances; Science 129 1466-1470
Green N, Beroza M and Hall S A 1 960 Recent developments in chemical attractants for insects; In Advances in
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Green way A R, Lewis T, Mudd A, Scott G C and Wall C 1977 Some chemical and entomological problems in
the investigation and use of behaviour-controlling chemicals; In Crop protection agents — their biological
evaluation (ed.) N R McFarlane (London: Academic Press) pp. 167-185
Hawkes C and Coaker T H 1979 Factors affecting the behavioural responses of the adult cabbage root fly,
Delia brassicae to host plant odour; Entomol. Exp. Appl. 25 45-58
Jacobson M 1972 Insect sex pheromones (New York: Academic Press) 382 pp.
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© Printed in India.
Behavioural strategies of emergence, swarming, mating and
oviposition in mayflies
K G SIVARAMAKRISHNAN and K VENKATARAMAN
Department of Zoology, Madurai College, Madurai 62501 1, India
Abstract. Behavioural strategies of emergence, swarming, mating and oviposition in
mayflies are reviewed in the light of available literature.
Keywords. Behavioural strategies; emergence; swarming; mating; oviposition; mayflies.
L Introduction
Mayflies are unique among the insects in having two winged adult stages, the subimago
and the imago. The adults live from 1-2 hr to a few days and even up to 14 days in some
ovoviviparous species. The brevity of the adult stage is possible because the sole
function of the adult is to reproduce. In fact, the process of natural selection has
resulted in insects whose every adaptation is directed towards the process of
reproduction. Moreover, the abbreviated adult life of Ephemeroptera is an adaptation
to minimise exposure time to predators (Edmunds and Edmunds 1980). An attempt is
made to review the data on behavioural strategies of emergence, swarming, mating and
oviposition in mayflies.
2. Emergence
Emergence, the transition from the aquatic nymph to the terrestrial subimago, is a
critical period for mayflies. Shedding of the nymphal skin usually occurs at the water
surface on some object such as a stone or macrophyte stem or in mid-water (Brittain
1982). The latter is more typical of the burrowing species which inhabit deeper waters,
and of a number of river species. Genera such as Siphlonurus, Isonychia and Baetisca
crawl completely out of the water before they moult (Edmunds et al 1976). The
mechanism of emergence has been well documented in Baetisca rogersi (Pescador and
Peters 1974). The emergence process begins with a medial split of the thoracic notal
shield. The abdominal segments contract repeatedly in a peristalic fashion followed by
the outward bulging of the thorax until the entire medial line of the mesothoracic notal
shield opens. The split gradually progresses anteriorly and posteriorly. Anteriorly it
reaches the vertex of the head, usually between the compound eyes along the obscured
ecdysial line, but sometimes extend to the base of the frontal process of the head.
Posteriorly the split terminates at the posterior margin of the median carina. As the split
progresses, the subimago wriggles out from the old skin. The dorsum of the
subimaginal thorax emerges first, followed by the compound eyes and then the head. At
this point the emerging subimago assumes a slanted position with the head and anterior
351
352 KG Sivaramakrishnan and K Venkataraman
half of the thorax completely exposed, and the abdomen still encased in the old cuticle.
Quick jerky body movements and abdominal contractions complete the process with
the release of the abdominal segments and caudal filaments. Sometimes the subimago
spreads out its prothoracic legs immediately upon exposure and firmly anchors the
claws on the supporting objects. This probably helps the emerging subimago pull itself
from the nymphal skin. Normally the prothoracic legs and the mesothoracic legs
remain firmly drawn under the venter of the thorax until the metathoracic legs appear
and all three pairs spread out at the same time. At emergence the wings of the subimago
are moist and are often curled at the apex. A newly emerged subimago remains
motionless for a while, and then crawls up on the supporting object. This resting
behaviour probably allows the subimago time to regain its strength and dry its wings
(Pescador and Peters 1974).
Mayfly emergence patterns can be analysed under two categories:
2.1 Die! patterns
Edmunds and Edmunds (1980) makes an interesting comparison of emergence in
specialized short life ( < 2 hr) and longer adult life ( > 6 hr) unspecialized species of
Ephemeroptera in tropical and temperate regions (figure 1). Specialized genera have the
potential of being similar in the two regions. For example, the emergence of the short-
lived Caenidae invariably takes place either at dawn or dusk and appears to be
Specialized genera Specialized genera
temperate tropics
Unspecialized genera Unspecialized genera
temperate tropics
• Subimago > imago •
Figure 1. A generalized comparison of emergence and moulting to imago stages in
specialized and unspecialized species of Ephemeroptera in tropical and temperate regions
(after Edmunds and Edmunds 1980).
Behavioural strategies in Mayflies 353
controlled by light intensity (Muller-Liebenau 1960). On the other hand, whereas in the
unspecialized genera of the temperate region, emergence of subimagos is scattered, and
length of subimaginal and imaginal life is variable, unspecialized genera in the tropical
lowlands tend to be strongly restrained to a short daylight life, although exceptions are
known (Edmunds and Edmunds 1980). Furthermore, nearly 100% of the vulnerable
subimagos emerge in almost total darkness in tropical lowlands whereas emergence of
the subimago during full daylight occurs in many temperate species. The most common
time of emergence for most temperate species is from late afternoon through the first
hour of darkness. In warm temperate regions with suitable night-time temperatures
subimagos of various species may emerge during the night.
Edmunds and Edmunds (1980) point out that apparently many of the activity
patterns and adaptations of adult mayflies (subimagos and imagos) have formed in
response to selection pressure from predators. Mayfly subimagos are slow and clumsy
fliers and are highly vulnerable to predation. Flying imagos are much less vulnerable
but resting subimagos or imagos, being Paleoptera, are unable to fold the wings and
hide in leaf litter, crevices or other protected areas. The brevity of their winged lives is
itself an adaptation to reduce exposure to predation. Apparently the most significant
daytime predators in tropics are Odonata. Birds are secondary. Most mayflies are killed
also in spider webs. The only significant night time predators are bats. It is also clear
that predation on mayfly subimagos and imagos is several times as great in the day as it
is at night. Apparently the lowest predation rate of subimagos in the temperate regions
also would be during hours of darkness. However, the selection pressure which seems to
counteract selection for night-time emergence appears to be cool climate that slows
transformation from the subimago to the imago. In the lowland tropics, nights are
warm and most imagos which emerge in the first 1^ hours of darkness transform to the
imago stage before 0300 hr the next morning.
2.2 Seasonal patterns
Mayflies have distinct and finite emergence periods, especially in temperate and arctic
areas. In cold temperate and arctic areas, mayfly emergence is more or less restricted to
the summer months, owing to the physical barrier of ice cover and the low air
temperatures during the rest of the year (Boerger and Clifford 1975; Brittain 1982;
Ulfstrand 1969). Probably only a few species such as Baetis macani are able to emerge at
water temperatures below 7°C (Brittain 1975). As one approaches the tropics, and also
in more oceanic climates, there are fewer restrictions and emergence may occur
throughout much of the year, although most emergence still occurs during the warmer
months (Clifford 1981). In the tropics emergence is often non-seasonal, (Tjonneland
1960, 1970), although some species have clear emergence patterns. The lunar rhythm of
emergence of the African species, Povilla adusta, is well known from a number of lakes
(Hartland-Rowe 1958).
In habits with several mayfly species, peak emergence of the major species may be
separated in time, especially in congeneric species (Brittain 1982). Such temporal
separation over the emergence season may serve to reproductively separate species
(Friesen et al 1980). Recent authors who have studied emergence suggest the following
factors as possibly important in influencing or synchronizing emergence: temperature
of air and water, light, moon phase, flow, humidity, wind, rainfall, photoperiod,
354 KG Sivaramakrishnan and K Venkataraman
successive instars, hormones and endogenous rhythms (Corbet 1964; Humpesch 1971;
Thibault 1971; Fremling 1973 a, b; Langford 1975; Peters and Peters 1977). In species
with well defined emergence periods, males and females usually emerge synchronously,
which may ensure survival of adequate numbers of organisms for successful
reproduction (Friesen et al 1980). Emergence should be viewed as an integral part of the
species' overall life cycle strategy (Brittain 1980).
3. Swarming and mating
Swarming is a male activity, apart from the Caenidae and Tricorythidae where both
males and females may participate. The females fly into these swarms and mating
occurs almost immediately and usually in flight. The flight of mayflies is a sort of
wedding dance. Swarming may take place over the water itself, over the shore area, or
even remote from the water. For instance, the swarms of Baetis, Paraleptophlebia and
Rhithrogena have been observed up to several kilometers from the nymphal habitat
(Edmunds and Edmunds 1 980). Most swarms are oriented according to terrain markers
such as areas of vegetation, the shoreline, and trees (Savolainen 1978). The time of
swarming varies considerably, although dusk is the most common time of the day in
temperate areas. Light intensity and temperature are major factors in determining the
timing of swarming. The time of nuptial flight appears to be the result of selection to
reduce the time of daylight. Table 1 is the estimate of Edmunds and Edmunds (1980)
regarding the most to least frequent times of swarming in tropical and temperate
species.
The behavioural adaptations for mating as evident in the shape and location of eyes,
and foreleg modifications in males are really striking. The prominent turbinate eyes of
males, especially well-developed in the Baetidae and some Leptophlebiidae, provide
both high acuity and good sensitivity (Horridge 1976). This enables them to detect and
capture single females in a swarm at low light intensities. The forelegs of male are
unusually long for grasping and holding the female during mating. The other legs are
reduced. The backward bent of tarsus necessary for the suspension during mating is
made possible by a reversible joint at their bases.
Brinck (1956) observed mating in Parameletus chelifer. Males dominate during the
early swarming period. But soon numerous females mixed with the swarms. The male
pressed himself under the female abdomen and stretched the front legs forwards and
upwards along the sides of her body, until they reached the prothorax. Then the tarsus
Table 1. Nuptial flight time (after Edmunds and Edmunds
1980).
Tropics Temperate
fr
Morning
Dusk
1
First dark hours
Afternoon
I
Dawn
Afternoon/Morning
OJO
c
Midday
Morning
'1 1
r Dusk
Midday
£ Afternoon
Dawn
1
First dark hours
Behavioural strategies in Mayflies 35f
were bent so that each clasped round a wing-base. At the same time the abdomen was
curved and the forceps grasped the 8th or 9th abdominal segment of the female. The
female abdomen was usually held like an S so that the penis was easily pressed into the
female genital opening. The male cerci were stretched forward, fixing the female
abdomen at the same vertical plane as the male abdomen. The female cerci were
directed obliquely hindward (figures 2 A,B). The copulation lasted about 20 sec and
then the male took off, soon followed by the female.
The suspension of the male body in the anterior legs and the forceps is certainly very
safe. It is most probable that the above type of male suspension in anterior tarsus and
forceps is characteristic of this insect group. This is indicated by the presence of the
forceps in all males investigated and lengthened male front legs in all species known.
4. Oviposition
Specialized structure like ovipostor found in other groups of insects, is lacking in
mayflies. However some authors call the produced subgenital plate an ovipositor. We
prefer to call it 'egg channel' (Sivaramakrishnan 1984). The problem of downward
displacement of eggs and immature nymphs, can be compensated for by the adult
mayflies flying upstream before they lay eggs.
Oviposition behaviour of mayflies can be categorized under five basic types (Elliott
and Humpesch, Personal communication).
(i) Female goes underwater and eggs laid on substratum:
Baetis rhodani belongs to this category (Elliott 1972). The female lands on a partially
submerged stone in rapidly flowing water, folds her wings along the abdomen, then
walks under the water and searches for a suitable Oviposition site, usually on the
(B)
Figure 2. Parameletusfhelifer Bengts (after Brinck 1 957) A. Mating couple in flight. B. The
backward bent of the tarsus of male clasped round the wingbase of female.
356 K G Sivaramakrishnan and K Venkataraman
underside of the stone. This behaviour may permit assessment of water quality before
oviposition (SutclifFe and Carrick 1973). The female lays contiguous rows of eggs to
form a flat semi-circular plate. When oviposition ceases, the female may walk out of the
water and fly away, but she is usually swept away downstream.
(ii) Female rests on a stone above water and eggs laid on substratum under water:
Habroleptoides modesta (Pleskot 1953) belongs to this category in which the female
dips its abdomen in the water and lays eggs. The female is never totally submerged. The
tails are usually broken off before oviposition starts.
(iii) Female flies down to the water surface and eggs are released in a single mass:
In Ephemerella ignita (Elliott 1978) the egg mass forms a spherical greenish ball that is
carried at the genital aperture with the posterior abdominal segments curved
downwards and round the ball to hold it in position. The female flies upstream and
descends to the water surface, releasing the egg-ball on contact with the water.
(iv) Female flies down to the water surface and eggs are released in several batches:
In Rhithrogena semicolorata (Humpesch and Elliott 1980) the female flies upstream,
descends to the surface of the water and releases a few eggs by dipping the tip of her
abdomen at intervals. Most species belong to this category.
(v) Ovoviviparous species:
Cloeon dipterum is the only species in which the females rest for 10-14 days after
copulation and then lay their eggs on the surface of the water. As soon as the eggs come
into contact with the water, they hatch and the larvae swim away, (Degrange 1959).
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 4, August 1985, pp. 359-375.
© Printed in India.
Functional morphology of air-breathing fishes: A review
HIRAN M DUTTA and J S DATTA MUNSHI*
Department of Biological Sciences, Kent State University, Kent, Ohio 44242, USA
'Department of Zoology, Bhagalpur University, Bhagalpur 812007, India
MS received 10 December 1984; revised 9 April 1985
Abstract. This review of the air-breathing fishes deals with morphology, histochemical
analyses of respiratory membrane and muscles, morphometrics and development of re-
spiratory organs, hematology and other blood parameters, oxygen uptake by gills and skin,
physiological and behavioural aspects, ecology and effects of pollutants on the morphology
and behaviour. The scientific significance and application of the studies of functional
morphology and physiology in understanding the alteration caused by pollutants have also
been elucidated.
Keywords. Air-breathing fishes; morphology; hematology; histochemistry; morphometrics;
physiology; behaviour; pollutants.
1. Introduction
The air-breathing structures appeared in freshwater fish during the late Silurian or early
Devonian period and are thought to have evolved as adaptation to hypoxic water
conditions due to severe periodic droughts (Smith 1931; Johansen 1970). Recent studies
on air-breathing fish have demonstrated that various types of morphological and
physiological adaptations have made it possible for fish to utilize aquatic and aerial
respiration. The morphological and physiological adaptations in these fishes are
designed in a manner so that they derive maximum advantage of their surrounding
environment. There is an intimate relationship among the physico-chemical characters
of habitat, nature of biota, food chain and finally the morpho-physiological adaptation
of animals.
2. Morphology
The structures of the different types of respiratory organs have been studied for many
years by several scientists. Munshi (1961, 1962, 1962a, 1968), Hughes and Munshi
(1973), Dutta (1968), Jordan (1976), Peters (1978), and Burggren (1979) found that
certain air-breathing fishes such as Clarias batrachus, Heteropneustes fossilis, Blue
gourami, Trichogaster trichopterus, Anabas, Macropodus and Bella possess labyrin-
thine air-breathing organs. These organs are derived from the epibranchial regions of
the first and second branchial arches, and extend dorsally as plate-like organs to fill the
suprabranchial chamber and the air-sacs which are the extensions of the opercular
cavity or branchial chamber. The earlier hypothesis of Munshi (1962b) that the
"respiratory islets" of H.fossilis and C. batrachus are modified lamellar structures has
been confirmed later by electron microscopic studies of Hughes and Munshi (1973a).
359
360 Hiran M Dutta and J S Datta Munshi
According to them, the cells forming the vascular spaces in the dendritic organs and
respiratory islets are typical pillar cells of the gills.
Although the suprabranchial chambers in the Anabantidae group are extensions of
branchial chambers and the labyrinthine organs which have developed on the
epibranchials, the "respiratory islets" of the accessory respiratory organs have evolved
in different fashion. The hypothesis that the labyrinthine organs and the respiratory
islets of the suprabranchial chambers represent modified gill structure (Munshi 1968) is
now no longer tenable as the electron microscopic studies of Hughes and Munshi
(1973a) revealed that in Anabas testudineus the pillar cells of the air-breathing organs
do not have the same relationship as in gills, but are modified epithelial cells. Obviously
we are dealing with analogous rather than homologous structures that are used to serve
the same function, supporting the contiguous vascular units which make up these
respiratory organs. Such a supporting function is clearly of importance. During
evolution, different structural arrangements have been selected to provide surfaces with
minimum diffusion barrier between blood and the respiratory medium (Hughes and
Munshi 1968). Further, the blood capillaries of the respiratory islets of Anabas are
unique structures, having a series of most characteristic type of unicellular valves ever
discovered in any animal system so far (Hughes and Munshi 1973a). These valves
control the movement of blood through the respiratory islets. In Amphipnous cuchia
and Channa sp. the suprabranchial chambers are the extensions of the pharynx. The
vascular mucosa of the air-sacs have evolved independently (Hughes and Munshi
1973a) and not from the gill lamellae as it appears in the histological preparations under
light microscope (Munshi 1962a; Munshi and Singh 1968).
In Amphipnous (Monopterus) cuchia, the structure of the valve is very unique. It
projects freely into the papilla lumen and appears to be involved in the regulation of
blood flow through individual papilla (Hughes and Munshi 1973a). These structures
were discovered for the first time and are not only new contributions to knowledge, but
open up new lines of further research.
In Periophthalmus vulgaris and Boleophthalmus boddaereti, opercular chambers are
modified for air-breathing purposes. The accessory respiratory organs of P. vulgaris
provide an excellent example of adaptation by modification of opercular chambers
(Singh and Munshi 1969). In this fish, the opercular chambers have enlarged and become
vascularized for respiratory purposes. Intricate mechanisms for opening and closing of
the inhalent and exhalent apertures have evolved and the brarichiostegal apparatus has
developed a special type of safety valve which is workable by stripes of muscles.
Further, the studies on ultrastructure of gills of these air-breathing fishes have made
new contributions to our knowledge. The earlier hypothesis of Munshi and Singh
(1968a) that the pillar cells are modified smooth muscle ceils have been confirmed
(Munshi 1976).
Some other forms of accessory gas exchange organs have evolved in numerous
groups offish in order to obtain oxygen from air. These air-breathing organs may be in
the form of modified swimbladders, pharyngeal cavities, stomach and intestine
(Johansen 1966; Munshi 1976; Singh 1976). Kramer and McClure (1980) found that
similar to other callichthyids, the posterior end of the intestine of Corydoras aeneus
works as an accessory respiratory organ and its anterior end is provided with a
muscular bulb. Generally, these organs are utilized for oxygen uptake and gills are used
for the elimination of CO2, and these processes seem to be similar to aquatic respiration
(Randall et al 1978; Burggren 1979).
Functional morphology of air-breathing fishes 36 1
It has been observed by Jordan (1976) that some air-breathing fishes are facultative
air-breathers and can survive indefinitely on dissolved oxygen, while some such as
Protopterus, Lepidosiren and Electrophorus are obligate air-breathers that will drown
when access to air is denied. She also found that Clarias batrachus is a bimodally
breathing teleost. C. batrachus possesses an air-breathing organ with highly branched
dendritic organs or respiratory "trees" that develop as outgrowths of the second and
fourth gill arches, and are located in the suprabranchial chamber.
3. Histocfaemistry
3.1 Histochemical studies of the respiratory membrane
A complete knowledge of the cellular structure of the respiratory membrane of the air-
breathing organ is essential to understand its physiology. The histochemical study of
the respiratory membrane reveals five kinds of specialized cells: (a) mucous cells,
(b) acidophil granular cells, (c) basophil mast cells, (d) large bi- or trinucleate
glandular cells, and (e) mitochondria rich chloride cells in the gills and accessory
respiratory organs of many air-breathing fishes (Munshi 1960).
The typical goblet type of mucous glands are present in large numbers in freshwater
fishes such as, Catla catla, Labeo rohita, Channa punctatus, Mastacembellus armatus,
Clarias batrachus, Heteropneustesfossilis. In C. catla these cells respond to the chloride
test (Munshi 1964). This means that besides mucous secretion, they also play an
important role in chloride regulation, In air-breathing fishes viz C. punctatus,
C. batrachus and H. fossilis only few mucous cells give positive reaction with
AgNO3/HNO3 test for chloride. The medullary hormones of adrenal have profound
effect on the mucous cells of air-sacs and the gills of H. fossilis and M. aculeatum
respectively (Guha et al 1967). The sulphated acid mucopolysaccharide component of
the mucous keeps the air-sac moist and lubricated during gaseous exchange.
The acidophil granular cells of the gill epithelia are diastase resistant, pAS-positive.
Those belonging to the connective tissue system are pAS-negative, however, almost all
the granular cells are PAS-positive after extraction of lipids. The granules of these cells
are composed of tyrosine-rich protein. The cells also appear to contain a large amount
of RNA. The eosinophilic granular cells appear to contain carbohydrate, protein and
lipid firmly bound with each other. Some of the cells give positive reactions for alkaline
phosphatase. These cells do not respond to Ag NO3/HNO3 test for chloride (Singh and
Munshi 1968).
The basophilic mast cells are present in large numbers in the sub-epithelial
connective tissues of the gill lamellae of Hilsa ilisha. They are closely associated with
blood capillaries (Munshi 1960). It is quite meaningful that mast cells (which are
reservoirs of heparin and histamine) are found in the gills of fishes.
The bi- and trinucleate glandular cells are found in the gills of siluroid fishes. They
have been derived from the glands of skin of these fishes (Munshi 1960). The
cytomorphosis of these glands has been studied for the first time and throw light on
their origin (Mittal and Munshi 1970).
Chloride cells are found in good numbers in many of the air-breathing fishes viz
Anabas and Clarias which live in brackish waters also. The endoplasmic reticulum is
very well developed and a large number of mitochondria is found in these cells (Hughes
and Munshi 1973a).
362 Hiran M Dutta and J S Datta Munshi
Large amounts of reserve fats have been discovered in true air-breathing organs of
amphibious fishes (Singh et al 1973). The stored lipids lie in the well-developed fat cells
of the connective tissue layer between the respiratory islets and the muscles of the air-
sacs. A direct correlation exists between the vascularity of the organs and the
concentration of the fat globules, and they contain both acidic and neutral fats. The
pharmacological action of adrenalin and atropine was effective in bringing about
complete mobilization of the fat deposits of the air-sacs of Saccobranchusfossilis in vivo
condition.
3.2 Enzyme histochemistry of the respiratory muscles
The gill ventilation is under the influence of buccal pressure and opercular suction
pumps. These pumps are operated by means of a series of respiratory muscles. The
enzyme histochemistry of these respiratory muscles has opened up a new field of
investigation and it also reveals that the muscles operating these respiratory pumps are
composite in nature. Red, white and intermediate muscle fibers have been distinguished
in the respiratory muscles depending on their intensity of reaction for succinic
dehydrcgenase (Munshi et al 1975). It has been noted that the muscles innervated by
the facialis nerve are dominated by red fibers, whereas those innervated by trigeminal
are dominated by white muscle fibers (Ojha and Munshi 1975). The cytochernical
differentiation of the muscle fibers will reflect their metabolic activities during gill
ventilation. The combined study of ultrastructure and enzyme activities of muscle fibers
provides an indepth understanding of the muscle physiology which can be correlated
with behavioural patterns of air-breathing fishes.
Both electron microscopy and enzyme determination techniques were used by
Hochachka et al (1978) and Johnston (1979) to determine the ultras tructures and
enzyme activities of white and red muscle fibers of Aruana and Arapaima, both obligate
air breathers. Their study reveals that the white muscle fibers in both species possess a
rather similar ultrastructure, characterized by large diameter, very few mitochondria,
and few capillaries. They also found that white muscle fibers of Aruana displayed higher
levels of enzyme activity, while enzymes in aerobic metabolism occurred at about one-
half the levels in Arapaima. No red muscle was found in Aruana, but it was present in
Arapaima and was fueled by glycogen and lipid droplets. Their studies led to a revealing
conclusion that the surface skimmer sustained a higher oxidative capacity in its
myotomal muscles than that of the facultative air-breather.
4. Morphometrics and development of respiratory organs
An earlier study (Das 1927) detailed only the morphometric aspect of the air-breathing
organ of Channa striatus and C. punctatus during their ontogenic development.
Whereas the more recent morphometric studies (Hughes et al 1973; Hakim et al 1978)
explain the role of the gas exchange machinery of the amphibious fishes during their
development and growth. Recently, Dube and Munshi (1974) have observed that
Anabas testudineus of the lower weight group survived for a longer period than that of
higher weight group, when prevented from surfacing. They reasoned that as the fish
grow, the. rate of increase in gill surface becomes less than that in the surface of the
accessory respiratory (labyrinthine) organ.
Functional morphology of air-breathing fishes 363
Dube and Munshi (1 974) also found out that the O2 gas-diffusing capacity of the gill
of Anabas testudineus decreases at faster rate with increasing body weight than that of
Clarias and Heteropneustes. These findings explain why Anabas of higher weight group
dies when not allowed to breathe atmospheric air whereas the gills and skin of Clarias
and Heteropneustes are efficient enough to take care of the total metabolic demands of
the fishes as they grow in size. The estimation of the diffusion capacity of the accessory
respiratory organs of the air-sacs ofAmphipnous are less suited for oxygen uptake than
that of Anabas.
The morphometry of respiratory surface area can be used for gas transfer by using
the following equation (Hughes and Morgan 1973)
F02 =K(A P02)/T.
where FO2 = O2 uptake in ml O2/min; A is the area in cm2 for gaseous exchange; PO2 is
the mean difference between the oxygen tensions of water and blood; K is the
permeation coefficients (rnl O2/m/cm2 mm Hg/min).
On the basis of morphometric deduction of the surface area, its physiological aspects
can be inferred. These studies further have practical importance in rearing and
transporting of these air-breathing fishes (Munshi and Ojha 1974; Munshi and Dube
1974; Munshi et al 1974). Other researchers (Lenfant and Johansen 1968; Farber and
Rahn 1970; Hughes and Singh 1970a, b, 1971; Singh and Hughes 1971; Magid and
Babiker 1 975; Stevens and Holeton 1 978; Magnuson et al 1 982; Ischimatsu and Itazawa
1983a, b) have also shown that there are considerable variations among air-breathing
fishes in the degree of dependence on aquatic or aerial respiration depending on degree
of development and efficiency of the respiratory and related structures, environmental
limitations and metabolic needs of the fish.
To correlate form with function, the morphometric study of these respiratory (both
aerial and aquatic) structures during development is extremely important. A correlation
exists between the surfacing behaviour and different stages of ontogenic development
of Channa striatus. The surfacing frequency is most erratic in the fingerling and
becomes regular in the fish of 750 mg. The individuals weighing more than 30 g also can
survive on aquatic breathing for more than 16hr, when prevented from surfacing. It
seems that this fish size can accumulate sufficient energy-rich substances that they are
able to switch over to anaerobic respiration (Vivekanandan 1977). Babiker (1979) has
also found out that the dependence on aerial respiration appeared to develop gradually
with age in Clarias but occurred over limited age range (200-300 g) in Protopterus.
It has also been observed by Natarajan (1977) that labyrinthine organs of certain air-
breathing fishes such as Anabas scandens have the capacity of regeneration. It was
observed that when the first branchial arch was amputated at the level of the origin it
never regenerated, whereas regeneration was detected in those fishes which have a
remnant of labyrinthine tissue at their base. This suggests that the base of the
labyrinthine organ acts as a root for regeneration. Therefore, a knowledge of the
process of regeneration will definitely promote the understanding of the developmental
mystery of the respiratory organs.
5. Ecology, pollutants and air-breathing fishes
The extensive use of insecticides is continuously polluting fresh water. There are
manifold effects of insecticides on living organisms including economically important
364 Hiran M Dutta and J S Datta Munshi
fishes. They are also responsible for a number of physiological and biochemical
disturbances. Metasystox is known to be the most widely used insecticide against paddy
sucking aphids, spiders, mites, saw flies etc. It has been proven by Natarajan (1981) that
in the pesticide contaminated water, the air-breathing organs ofChanna striatus play a
very important role by extracting O2 from air during their stay in such contaminated
water. He also observed the existence of DDT and Dieldrin-induced anemia as shown by
the low erythrocyte count, low Hb content, light MCH and colour index in Channa
punctatus. The progressive decrease in erythrocyte count, Hb concentration and total
leucocyte count were found in C. punctatus exposed to malathion and methyl
parathion. It has also been observed by Natarajan (1978) that when a climbing perch,
Anabas scandens is exposed to a lethal dose of sumithion, it used its air-breathing
organs extensively to overcome the pollution stress for survival.
The effect of pollutants on air-breathing fishes has also been investigated by Munshi
and Singh (1971). Different aspects of the biology of air-breathing fishes (reproduction,
feeding habits, and body composition) have been correlated with the ecology of ponds
and swamps (Bilgrami 1977).
The presence of bacteria in high concentration on the surface of supra-branchial
chambers of Anabas has been recorded in many collections made by Munshi from
several localities of W. Bengal and Bihar, This is an interesting association and
practically nothing is known about the role of these bacteria. The possibility of their
playing some decisive role connected with respiration or other physiological processes
cannot be ruled out. It is also quite likely that some of these bacteria produce biotic
substances that can protect these fishes against possible injury under stressed
physiological environment. The bacteria in turn get their oxygen supply readily
available in the suprabranchial chambers.
6. Hematology
6.1 Hematology and other blood parameters
A series of hematological studies on Amphipnous, Anabas, Channa and Heteropneustes
indicate that the oxygen-carrying capacity of blood is related to body size of these
amphibious fishes (Dube and Munshi 1973; Mishra et al 1911 \ Pandey et al 1977).
Recently, a comparative study of the bloods of 45 species of Amazonian fishes (Powers
et al 1979) pointed out that there is a significant difference between water and air-
breathers CO2 tension in the blood. Air-breathing fishes have much higher CO2 tension
in the blood and the arterial CO2 tension of water breathers is generally below 5 torr
(Rahn 1966). Whereas, in the air breathers CO2 tension ranges from 1 5 to 43 torr (Rahn
and Garey 1973).
Thus, it is apparent that with the evolution of air-breathing mechanism, adjustments
occur at the molecular level of CO2 which is the causative factor for efficient
hemoglobin function to counteract the increased CO2 load. Therefore, some of the
differences in the hemoglobin of air and water breathers are related to the effect of
carbon dioxide on the hemoglobins of the air-breathers (Farmer 1979). Further, while
studying the effects of CO2 on hemoglobin function of air-breathing fish, Farmer
investigated three major points. First, to what extent are the oxygen binding properties
of non-mammalian hemoglobins get influenced by CO2 and whether these properties
Functional morphology of air-breathing fishes 365
are independent of pH or not. Second, whether or not the hemoglobins of water
breathers differ from the hemoglobins of air-breathers with respect to the magnitude of
the effect of carbamino CO2 affinity. Third, whether or not there is a correlation
between Bohr effect and effect of carbamino CO2 on the O2 affinity of hemoglobin. He
also found that the blood CO2 content of air-breathing fish and amphibians is much
higher than that of water breathers, but hemoglobin showed no adaptation to an
increased CO2 load. But the drop in oxygen affinity of hemoglobin caused by CO2 is
increased by increasing pH for each hemoglobin examined.
Blood oxygen, erythrocyte nucleoside triphosphate (NTP) concentration and several
other hematological parameters in facultative air-breathing fish from the Amazon
acclimated in normoxic and hypoxic water were measured by Weber et al (1979). They
found that the armored catfish Hypostomus sp and Pterygoplichthys sp in hypoxic
water undertake surfacing intermittently and result in lower NTP level.
Benesch and Benesch (1967) found in eel, Anguilla that the decrease of NTP under
hypoxic condition increases blood O2 affinity, by reducing the direct allosteric
interaction and also affecting the Donnan distribution of proteins across the red ceil
membranes. These changes help to increase the intraerythrocytic pH of eels in hypoxic
water and thus increases the O2 affinity of the hemoglobin via the Bohr effect (Wood
and Johansen 1973).
Similar studies on the catfish indicated changes in acid-base balance and blood-
buffering associated with hypoxic acclimation (Wood et al 1979).
6.2 Determination of blood proteins
Although some work has been done on serum and plasma proteins of freshwater fishes,
little is known about the air-breathing fishes specially those exposed to the pollutants.
Comparative electrophoretic studies of serum and plasma proteins of different fish
have been made (Lepkovsky 1929; Deutsch and Goodloe 1945; Drilhan 1954;
CTRourke 1960; Nyman 1965; Badawi 1971 and Hattingh, 1972). Most of them deal
with blood protein patterns of bass, carp, trout, catfish, perch, pike and whitefish.
Badawi (1971) investigated blood protein patterns of four Tilapia species. Hattingh
(1972) studied the plasma and serum samples by SDS- PAGE of five South African species.
Hattingh's results indicated that plasma and serum of these fishes differ in elec-
trophoretic pattern, and also in the number of fractions and the protein concentration
of fractions. It appears that each fish has its own characteristic pattern of elec-
trophoresis, which is fairly constant in mature and healthy fishes within a species
(Hattingh 1972). Badawi (1971) and Lysak and Wojcik (1960) postulated that the
variable amounts of protein found in blood samples might be related to diet and/or
season.
The serum proteins of bluegills, Lepomis macrochirus, exposed to methyl mercuric
chloride were electrophoretically investigated by Dutta et al (1983). They found
significant changes in the qualitative and quantitative profiles at 24 and 48 hr. A variety
of peptides, showing differential loci, staining intensity, mobility and molecular weight
were seen at 48 hr compared to 24 hr. However, 72 hr samples showed a reduction in
polypeptide bands approximating a profile similar to controls. A few other investi-
gators have found changes in serum protein of fishes exposed to MeHg (Lagler et al
1977; Hilmy et al 1980).
366 Hiran M Dutta and J S Dana Munshi
7. Oxygen uptake by gills and skin in relation to body size of amphibious fishes
The exponent b values related to oxygen consumption and the body weight show a wide
range of variation among the teleostean species. Winberg (1956) reported an average
value of 0-81 for a number of species, whereas Paloheimo and Dickie (1965) suggested
0-80 to be the characteristic value of most teleosts. These exponent values are based on
data available on water breathing forms.
The rate of oxygen consumption (VO2) through gills and skin (Munshi et al 1982)
was measured in air-breathing fishes of different body weights under two experimental
conditions, viz (i) when access to air was allowed and (ii) when it was prevented.
Characteristics of the regression lines relating the logarithm of oxygen consumption to
log body weight were calculated by the method of least squares. These physiological
studies revealed that in Anabas testudineus, Channa punctatus and Clarias batrachus the
oxygen consumption through gills and gill and skin (Clarias) increases by a power of
0-67, 0-79 and 0-869 respectively when these fishes have free access to air. Further,
lowering of the exponent values was indicated in A. testudineus (b — 0-53), C. punctatus
(b = 0-62) and C. batrachus (b = 0*841) when they were not allowed to breathe
atmospheric air. The lowering of the exponent value was perhaps related to the stress
condition of the air-breathing fishes when they were not allowed to breathe
atmospheric air. The fall of "b" value due to stress condition in air-breathing fishes is a
phenomenon newly discovered in the physiology of these fishes.
8. Other physiological and behavioural aspects of air-breathing fishes
During the last decade or so, a large number of physiologists have become interested in
the study of various adaptive features of the amphibious vertebrates (Hughes and
Munshi 1979; Johansen 1966, 1970; Rahn etall97l; Lenfant and Johansen 1968; Liem
1980). Gas exchange and respiratory patterns of Indian air-breathing fishes such as
Anabas testudineus, Heteropneustesfossilis and Clarias batrachus have been studied in
detail by Hughes in his Research Unit for Comparative Animal Respiration, University
of Bristol, U.K. These studies clearly indicate that there are large variations in the
metabolic rates among the species of these fishes inhabiting more or less similar waters.
They also indicate differences in the relative dependance on air and water for the
oxygen and CO2 exchange. The morphometric studies have been interpreted in relation
to the physiology of respiratory system (Hakim et al 1978).
9. Acclimation of air-breathing fishes
The bimodal (aerial and aquatic) gas exchange capacity of hypoxia-acclimated
Ancistrus chagresi, the armored catfish has been investigated by Graham (1983). He
found that hypoxia-acclimated Ancistrus have higher blood O2 affinity, more
hemoglobin (Hb) and can maintain a higher aquatic oxygen consumption rate (VO2) in
hypoxic water. These fishes also have a 25 % larger stomach which they use as an air-
breathing organ. Johnston and Bernard (1983) have inquired in detail the aquatic and
aerial respiration rates, muscle capillary supply and mitochondrial volume density in
Functional morphology of air-breathing fishes 367
air-breathing catfish (Clarias mossambicus) acclimated to either aerated or hypoxic
water. They determined that hypoxia acclimation did not cause changes in either
muscle mitochondrial density or capillary density. They ascertained that increased
ventilation of the suprabranchial chambers and greater oxygen extraction across the
gills inhibit the need for modifications in the above mentioned structures.
Pettit and Beitinger (1980) have searched into the respiration of the reedfish,
Calamoichthys calabaricus which encounters hypoxic conditions over much of its
range. Their experiments have confirmed that Calamoichthys can exist exclusively on
aerial respiration when exposed to hypoxic aquatic environment. Burggren and
Randall (1978) demonstrated that the sturgeon, Acipenser transmontanus can reduce its
aerobic metabolism drastically in response to hypoxic water. Though Pettit and
Beitinger (1980) have also found a remarkable reduction in the respiration rates with
time in seven experiments (in hypoxic water), they attributed this reduction to the stress
caused by handling.
Babiker (1979) discovered that when the fish was allowed to partition oxygen uptake
in hypoxic water, the rate of pulmonary ventilation in Protopterus and oxygen
consumption in Clarias also increased with enhanced hypoxia. However, this increase
was very noticeable in Clarias and insignificant in Protopterus. This finding shows that
the independence of pulmonary ventilation from the level of oxygen in the water in the
case of dipnoans and dependence of Clarias on accessory respiratory organ.
Enhancement of the respiratory frequency in hypoxic water has been observed in
many other air breathing teleosts (Gans 1970; Hughes and Singh 1970a, b, 1971; Singh
and Hughes 1971; Stevens and Holeton 1978, 1978a; Gee and Graham 1978). Effects of
hypoxia acclimation on the respiratory system of air-breathing fishes have also been
studied by Weber et al (1979), Gee (1980), and Graham and Baird (1982).
It has been observed that all air-breathing fish utilize aquatic gas exchange to some
extent, because air-breathing organs have a low gas exchange ratio. Since carbon
dioxide is highly soluble in water, it is naturally eliminated mainly through gill or skin
(Singh and Hughes 1971, 1973).
The oxygen uptake of air-breathing fishes varies in various environmental con-
ditions. Jordan (1976) and Munshi et al (1982) have measured the rate of oxygen uptake
(VO2) of Clarias batrachus and Heteropneustes fossilis under various experimental
situations, such as, in normoxic water, forcible submergence, and air exposure.
Most studies on oxygen uptake of fishes have been carried out under experimental
conditions and controlled environment. In the future investigation it is proposed to
measure the rate of O2 uptake under natural environment with different types of
waters.
Pandey and Chanchal (1977) demonstrated that if access to atmospheric air was not
allowed C punctatus and A. testudineus could not survive or asphyxiate if the oxygen
content of the water fell below 2-79 and 2-93 ml/L respectively. Immediate death may
occur due to brain anoxia.
There are some air-breathing fishes such as Piabacinafistae, a central American fish,
which breathes air frequently even in air-saturated water, yet, it is not an obligate air-
breather. Without an access to air this fish can maintain VO2 by aquatic respiration
down to a PO2 of about 70 torr. Its vascularized respiratory compartments or cells
located in the second chamber of the physostomus gas bladder function for aerial
respiration (Graham et al 1977).
368 Hiran M Dutta and J S Datta Munshi
10. Behaviour
Behaviour such as way of aerial respiration and time required for the same can be
accurately observed by cinematography. Some of the earlier investigators have
recorded the time required for gas release and gas intake. Gradwell (1 971) has recorded
the time needed for entire cycle from breaking the surface to gas release (which requires
about 0-2 sec) and inspiration (which requires less than 0-07 sec) for Plecostomus
(Hypostomus) punctatus, the Loricariid catfish. Whereas, Lepidosiren needs about
3-3 sec for entire cycle (Bishop and Foxon 1968) and Protopterus 1-1 sec (McMahon
1969). Garfish Lepisosteus requires 0-9 sec. (Rahn et al 1971) and Hopterythrinus (a
teleost) needs 0-86 sec (Kramer 1978). The shorter breath duration of Corydoras has
been related to the continuous rather than tidal ventilation (Kramer and McClure
1980). Longer breath duration may be related to the size of the above mentioned
species. Kramer and McClure (1980) have searched into the surfacing behaviour of the
catfish Corydoras aeneus (Callichthyidae) by using cinematography, which made it
possible to determine the sequences and timing of the ventilation movement of this fish.
They also studied the effect of dissolved oxygen on surfacing and depth on surfacing
rate and activity. They concluded that brief and infrequent surface visits are essential
for survival when PO2 falls below 1 5 torr and surfacing is not obligatory at high oxygen
level. They confirmed Gee and Graham's (1978) findings which revealed that air-
breathing was not obligatory in the Callichthyids. Compared to impressive research
made in ultrastructure, physiology, and biochemistry of the respiratory organs of air-
breathing fishes, knowledge of air ventilation mechanism has been largely neglected.
Very few researchers have paid attention to neuromuscular and biomechanical aspects
of intake and expulsion of air into the accessory respiratory organs. The aerial
respiration and the movements of the related bony elements ofAnabas testudineus were
recorded cinematographically by Dutta (1968), establishing a network of functional
correlation between the specific bones, muscles and the air-breathing mechanism of the
fish. It has been noted that Anabas uses only upper and lower jaws, opercular bones,
branchial chamber and the hyoid apparatus during normal respiration. The sus-
pensorium is not involved and a small expansion of the buccal cavity seems to be
conducted only by the ventral movement of the hyoid apparatus. The aerial respiration
(gulping) is realized by the above mentioned elements of the head, but in this case a
concurrent lateral movement of the suspensorium is also recorded. There is a
remarkable similarity between the aerial respiration (gulping) and feeding mechanisms
of Anabas. The same functional elements of the head are involved in carrying out both
functions. To create a suction-force in the oral cavity, expansion of this cavity is
essential which is performed by the lateral movement of the suspensorium, caused by
the contraction of the musculus levator arcus palatini. Similar type of expansion of the
oral cavity has been observed when Anabas engages itself to engulf fresh air.
In Anabas, the branchial chamber is expected to be much reduced, because of the
large antero-dorsally situated labyrinthine organs. On the contrary, the branchial
chamber is not small. The moderate sized branchial chamber seems to be maintained by
the caudal displacement of the pectoral girdle, which is reflected by the long
supracleithrum, supratemporal, and post-temporal bones. The caudal displacement of
the pectoral girdle can be considered as a classic example of an interelemental
dependence. It seems that in order to accommodate the labyrinthine organs, the size,
Functional morphology of air-breathing fishes 369
shape and even entire architecture of the head had to make certain compromise.
Therefore, air-breathing organs of any fish should be analyzed in a functional-
morphological perspective which emphasizes the mechanical and spatial arrangements
of the structural features in accordance with their use or function. Dutta (1975) has also
correlated the functional and structural aspects of the suspensorium of another air-
breather, Ctenopoma acutirostre with those of Anabas testudineus. It has been
cinematographically recorded that Ctenopoma's suspensorium is involved in aquatic
and aerial respirations as well as in feeding, whereas, in Anabas the same bone functions
only during air-breathing and feeding. Further, the suspensorium has been analyzed as
a part of the architectonic structure of the entire head by using a diagrammatic model
based on mutual influence, integration and couplings including other functional
elements.
Dutta (1979) has traced the structural configurations and functional mechanics of
jaws of Macropodus opercularis, Ctenopoma acutirostre and Anabas testudineus (air-
breathing fishes) in feeding. The mechanics of air-breathing in all the three fishes are
similar during feeding. In these fishes the protrusion of the upper jaw has been
discerned during feeding and air-breathing. The ventral groove of the premaxillae and
caudo-lateral articular process in Macropodus and Ctenopoma, the median condyle and
the antero-lateral articular process on premaxillae in Anabas are vital structures for the
protrusion of the upper-jaw. In Ctenopoma the dorsally inclined mouth seems to be
correlated to surface feeding as well as air-breathing. Easy access to the surface may be a
functional advantage obtained by this fish by having the afore-mentioned structural
modification. In between the air-breathing and feeding behaviours, Ctenopoma rests on
the bottom of the aquarium. The condylar articular process on the articular bones of
the lower jaw in Ctenopoma is a structural modification, which is adapted to work as
balancing wheel when fish is in resting stage.
The movement of the jaws is a vital mechanics which is involved in life-sustaining
functions like respiration and feeding. The patterns of articulation between premaxilla
and maxilla, maxilla and vomer, palatine and ethmoid, maxilla and palatine, the
structural configuration of rostral cartilage and the surrounding ligaments have an
integrally vital role in realization of upper-jaw protrusion (Dutta and Chen 1983). Any
change in the structural and functional patterns of these elements will have a definite
unfavourable effect on the function of the fish, whether the function is an aerial
respiration or feeding, even though the fish might have a well-developed labyrinthine
organ, swim-bladder or any other kind of air-breathing organs.
Jaw protrusion of a fish is not only influenced by its closely associated functional
elements but a distantly located element, hyomandibula, also, has a greater impact on
the protractibility of both lower and upper jaws. Dutta (1980) discussed the
comparative structural and functional aspects of the hyomandibula in three air-
breathing fishes, Macropodus opercularis, Ctenopoma acutirostre, and Anabas tes-
tudineus and concluded that the structural analysis of the hyomandibula and its
relationship with the function like aquatic and aerial-respirations depict the inter-
relation between form and function. Features such as the vertical ridge and articu-
lations like the hyomandibulo-neurocranial, the hyomandibulo-opercular, and the
hyomandibulo-interhyal have been specially analyzed in relation to the respiratory
function in the above three air-breathers. Peters (1978) not only indicates that
hyomandibula is one of the apparatus which form the lateral wall of the suprabranchial
370 Hiran M Dutta and J S Dana Munshl
chamber in Anabantoids, but uthe air ventilation is regarded as being caused by the
activity of a double pumping mechanism consisting of the buccal and opercular
apparatus." Therefore, the hyomandibula being one of the functional elements of the
buccal apparatus plays equally an important role in air ventilation in Anabantoids
(Dutta 1980). Peters (1978) and Kramer (1978) have used cineradiography to analyze
the mechanics of air ventilation of the air chamber and respiratory gas bladder in some
teleosts and have achieved remarkable results.
The physiological origin of the mechanisms underlying air ventilation in air
breathing fish of different phylogenetic lineages has also been researched by using high
speed cinematography, cineradiography and electrornyography (Liem 1980). With the
help of these experiments Liem was able to disprove the hypotheses of Bader (1937) and
Mishra and Munshi (1958). Munshi (1968) attributed a key functional role to the
constrictor suprabranchialis during ventilation. With the electromyographic profiles
and more intense kinematic analyses Liem (1980) revealed that the "monophasic"
pattern of air breathing as indicated by Peters (1978) to be explicitly "triphasic" and the
"diphasic" is actually "quadriphasic." Therefore, with the help of modern experimental
tools and kinematic analyses Liem has accurately traced out the "monophasic",
"diphasic", "triphasic" and "quadriphasic" air ventilations in Anabantoidei.
Based on kinematic and electromyographic profiles ofHelostoma and Anabas, Liem
(1980) interpreted the air ventilation mechanism as follows: as the fish rises to the
surface it compresses its buccal cavity by the contraction of adductor arcus palatine,
geniohyoideus and adductor mandibulae to minimize the volume of the buc-
copharyngeal cavity in order to expel as much water as possible from the buccal cavity
as preparatory phase for air intake. Then the air is sucked in by the sudden expansion of
the buccal cavity conducted by the contraction of the levator arcus palatini which then
move the suspensorium laterally. Sternohyoideus muscle engages itself to lower the
floor of the buccal cavity.
Therefore, cinematography provides the precise movements of the bony elements
and the synchronous electromyography reveals the related muscles' potential which are
involved in carrying out the air-ventilation in air-breathing fishes.
Not only the mechanisms, but the behavioural patterns of air-breathers can be
studied elusively by high speed cinematography. Behavioural aspects will unfold the yet
unknown nature of these fish at the time of their aerial respiration. A synchronous
aerial respiration by the walking catfish in Florida has been studied by Loftus (1979).
According to him, in turbid water the synchronous air-breathing amongst several
catfishes is done by detecting the vibrations of respiring fish with their lateral line
system. Whereas, Kramer and Graham (1976) stated that in low turbid water air-
breathing fishes seem to maintain synchrony by visual means. They have also observed
catfish Hoplosternum to breathe synchronously in very murky water, probably without
any visual aid.
11. Scientific significance and application of functional morphological work
A fundamental knowledge of the morphology and physiology of the air-breathing
organs of these fishes is indispensable in understanding the alterations caused by
pollutants.
Development of normal fishes will reveal the sequential changes which incur in the
Functional morphology of air-breathing fishes 37 1
progressive stages of ontogeny of their air-breathing organs. The developmental
studies will indeed unravel the interesting adaptations and many convergences of these
fishes to the ancestral vertebrates. Development study will also give us the indepth
understanding of the structural adaptations and will also reveal how they have helped
the vertebrates to achieve their transition from water to land. For example such studies
might include the morphometrics of the developing gills and the accessory respiratory
organs by employing the modern stereological methods (Weibel 1973) and structural
patterns of the bimodal gas exchange system.
The developmental studies of the pollutant-exposed fishes will give us the knowledge
of the varied alterations in the structures and the functions in their different stages and
pinpoint the stages which are most susceptible to the pollutants. The developmental
study includes the fertilized eggs, larvae, juvenile and adult specimens.
Further, an indepth knowledge of their physiology, micro and macroecology, the
development of the respiratory organs and general growth would be especially
important in relation to any work envisaged in the culture of these fishes. Many of these
species form an important food source in India. A knowledge of their mode of life and
environmental conditions can improve methods for their successful culture and help to
protect them from dangers of environmental pollution.
12. Conclusions
Being a warm water tropical fish the air-breathers are not only widespread in India, but
the Indian biologists have made significant contributions to the understanding of their
functional morphology. Gross morphology and the behavioural aspects of the air-
breathing fishes have been researched more intensively than the applied aspects of
physiology and effects of pollutants on morphology. With the use of more chemicals in
our food and growing environmental pollution, the air-breathing fishes are likely to
undergo varied changes in their functional morphology. Studies in this aspect,
therefore, will have new openings of far reaching significance.
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Singh B N and Hughes G M 1971 Respiration in an air-breathing catfish, Clarias batrachus (Linn.); J. Exp.
Biol. 55 421-434
Singh B N and Hughes G M 1973 Cardiac and respiratory responses in the climbing perch Anabas
testudineus; J. Comp. Physioi 84 205-226
Singh B N and Munshi J S D 1968 Cytology and cytochemistry of the granular cells found in the gill of certain
freshwater fishes; Mikroskopie 23 1 1-25
Singh B N and Munshi J S D 1969 On the respiratory organs of Periopt halmus vulgaris (Eggert); Zool. Anz.
18392-110
Singh B R, Guha G and Munshi J S D 1973 Fat depot in the air-breathing organs of some teleosts; Z.
Mikroskopie. Anal. Forsch. 87 242-248
Smith H W 1931 Observation on the African lungfish Protopterus aetheopius and evolution from water to
land environment; Ecology 12 164-181
Stevens E D and Holeton G F 1978a The partitioning of oxygen uptake from air and from water by the large
obligate air-breathing teleost pirarucu (Arapaima gigas); Can. J. Zool 56 974-976
Stevens E D and Holeton G F 1978 The partitioning of oxygen uptake from air and from water by
erythrinids; Can. J. Zool. 56 965-969
Vivekanandan E 1977 Ontogenetic development of surfacing behavior in the obligatory air-breathing fish
Channa ( — Ophiocephalus) striatus; Physioi. Behavior 18 559-562
Weber R E, Wood S C, Davis B J 1979 Acclimation to hypoxic water in facultative air-breathing fish and
Blood oxygen affinity and allosteric effectors; Comp. Biochem. Physioi A62 125-130
Weibel E R 1973 Stereological methods In Principles and techniques of electron microscopy, (ed.) M A Hayat
(New York, Van Nostrand Reinhold) Vol. 3
Winberg G 1956 Rate of metabolism and food requirements of fishes. Belorussian University Minsk (Fish,
Res. Bd. Canada Trans. Series No. 194)
Wood S C and Johansen K 1973 Blood oxygen transport and acid-base balance in eels during hypoxia; Am. J.
Physioi 225 849-851
Wood S C, Weber R E and Davis B J 1979 Effects of air-breathing on acid-base balance in the catfish,
Hypostomus sp; Comp. Biochem. Physioi. A62 185-188
Proc. Indian Acad. Sci. (Anim. Sci,)» Vol. 94, No. 4, August 1985, pp. 377-381.
© Printed in India.
Bionomics of hill-stream cyprinids. L Food, parasites and length-
weight relationship of iMbeo dyocheilus (McClell)*
SANDEEP K MALHOTRA
Parasitology Laboratory, Department of Zoology, University of Allahabad, Allahabad
211002, India.
MS received 15 February 1985; revised 29 May 1985
Abstract. Food habits, parasites and length-weight relationship in the hill-stream fish Labeo
dyocheilus were examined. The frequency distribution of the pseudophyllidean cestode,
Bothriocephalus teleostei in this fish has been reported to be log-normal.
Keywords. Parasites; riverine ecosystems; cyprinids; parasitization index; Labeo dyocheilus.
1. Introduction
Studies on the biological and statistical significance of length-weight relationship
among ichthyofauna of Himalayan riverine ecosystems are available on only three fish
species: Barilius bola (Ham) (Chauhan and Malhotra 1981). Labeo dero (Ham)
(Malhotra and Chauhan 1984) and Tor tor (Ham) (Malhotra 1982). The present
investigation is aimed at analysing the biology and length-weight relationship in
L. dyocheilus (McClell) in high altitude ecosystems.
2. Materials and methods
Methods of collection of fish samples (N = 152) and their analyses were published
earlier (Malhotra 1982). L. dyocheilus (9-5-49 cm length range) collected from River
Nayar (650 meters ASL) were used in the present investigation. The length- weight
relationship was estimated by the formula, W = aLn where PFis the weight, L the body
length, and a and n are constants. Logarithmic transformation of the equation may be
written as log W— log a + n log L where, log Wis the dependent variable (y\ log L the
independent variable (x), n the regression coefficient or slope (b), and log a the
y-intercept. Analysis of co variance and student's t-test were applied and the coefficient
of determination (Croxton 1953), the values of least squares regression slopes and the
proportion of correlated variance (Zeller and Carmines 1978) were computed.
3. Results
3.1 Food
Qualitative and quantitative (percentage by weight) analysis of gut contents including
food and parasites showed 0496 % worms, and 99*504 % Cladophora sp., Spirogyra sp.,
Sphaerocystis sp., Volvox colonies and plant debris.
377
378 Sandeep K Malhotra
3.2 Parasites
The frequency of parasites (mean worm burden, 2-3(2) in the alimentary canal of the
examined fishes was 049 % cestodes (female, 0-248 %; male, 0-248 %). Bothriocephalus
teleostei Malhotra, 1984a was the only cestode recorded. Interestingly, no trematode,
nematode or acanthocephalan infection was found in L. dyocheilus. The parasitization
index and prominence values of the cestode were 0-004-0-009 and 1-277-1-592
respectively. The optimum water temperature and humidity during infection period
were 13-75 ±0-25/0-18°C (13-5-14°C) and 85 ±5/3-54% (80-90%) respectively.
3.3 Length-weight relationship
Various body measurements in the ratio of total and standard length offish including
body weight are given in table 1.
3.4 Estimated regressions
An initial assessment of the fishes of the length range 9-5-49 cm suggested that the same
equation would not fit the data for the entire length range and that a break occurred
around <17cm and > 17. 1cm groups. Separate parabolic equations and linear
regressions were therefore computed for both the sexes and length classes as mentioned
in table 2. The significance of the differences between the regression coefficients (b) was
Table 1 . Mean values of body weight and ratios of total vis-a-vis standard length of Labeo
dyocheilus.
Mean ± S. E.
Category
Sample
size
Total length
(cm)
Standard length
(cm)
TL/SL ratio
Body weight
(g)
Female
Male
<17cm
> 17-1 cm
Pooled
86
66
76
7o
152
22-61 16 ±0-9789
205955 + 0-9744
14-956 ±0-2062
32-391 ±0-6251
21-7362 ±07016
19-6209 ± 0-7323
17-2773 ± 0-7458
14-4605 ±0-27 18
22-7461 ± 0-7849
18-6033 ±0-5342
1-1524 + 0-0123
1-1921 ±0-0039
1-0343 + 0-0110
1-4240 ±0-0379
1-1684 + 0-0631
178-9419 + 45-5443
132-2879 ±29-0144
49-84 + 3-6949
268-2368 ± 54-5471
159-0395 ±28-7343
Table 2. Regression equations describing length-weight relationship in Labeo
dyocheilus.
Logarithmic regression equations
Category (logW + JogL)
Parabolic equations
Female
= 1-946 +2-2103
0-001 1324L2-2103
Male
= 15998 + 2-1396
0-00251304L2-1396
Pooled
= 18199 + 2-0871
0-00151391L2-0871
<17cm
= 1-7670+1-7559
0-00171002L1'7559
> 17-1 cm
= 3-0889 + 2-2850
0-000814892L2-2850
Bionomics of cyprinids
379
tested by the method of analysis of co variance. The relevant data has been presented in
table 3. The test for heterogeneity of regressions revealed that the differences between
the regression coefficients were significant at 1 % level (sum of squares 0-0239149, mean
square 0-00797163, d/3, F = 8-337, F1% = 3-91). The test of heterogeneity was again
performed for the sexes (within and with each of the two length classes) and length
classes (within). It was observed that the differences between the regression coefficients
between male and female (F = 0-002, F5% = 3-91 d/1; 151) and between < 17cm and
> 17-1 cm (F = 0-139, F5«/ = 3-91, d/= 1; 151) were not significant while those
between sexes and < 17 cm (F = 141-521, Ft o, = 4-71, d/2; 226) and between sexes and
> 17.1 cm (F = 10-693, Fl% = 4-71, d/2; 226) were significant at 1 % level.
The application of Mest revealed that the departures of regression coefficients from
the isometric growth value of 3 were significant at 1 % level in < 17 cm (b — 3, — 1-1609;
t, - 19-720; d/, 74; t{ % = 2-66), > 17-1 cm (b - 3, - 0-8842; t, - 7-392; d/, 74; t{ % = 2-66)
and sexes (ft -3, -0-9129; t, -31-325; d/, 150; t1% = 2-58).
A comparison of the regression lines of the length-weight relationship of
L. dyocheilus has been presented in table 4. According to the standardized least squares
linear regression line, for each standard unit of length, the fish gained, 0-8703-0-8722;
0-7888-0-7918; 0-8192-0-8291; 0-5509-0-5584; and 088-0-9074 of a standard unit of
weight for females, males and pooled, and < 17cm and > 17.1cm classes of fish,
respectively. In both the sexes and weight classes r is significant.
Table 3. Analysis of covariance between the regression coefficients (b) for Labeo dyocheilus.
N
Female
86
Male
66
Pooled
152
< 17cm
76
> 17.1cm
76
Z(X-X)2
3-5984
3-3848
3-8244
2-6300
3-5445
L(T~F)2
7-1849
6-5631
7-2805
4-8910
7-2417
2,(X-X)(Y-Y)
5-3318
4-8717
5-4684
3-5046
5-3443
bE(X-X) (Y-Y)
11-7849
10-4235
7-9819
6-1537
12-2117
P2
0-7590
0-6245
0-6792
0-3077
0-7986
r2
0-7572
0-6233
0-6874
0-3035
0-7979
N = numer of observations; p2 = proportion of correlated variance; r2 :
determination.
= coefficient of
Table 4. Comparison of the regression lines of the length-weight relationship of Labeo
dyocheilus.
Variance
Standardized least squares
regression slope predicting
Category
aampie -
size
Length
Weight
— v-u v ail lauwc -
X from 7
Y from X
*
Female
86
1-6639
5-2504
3-3973
0-8703
0-8722
P<0-001
Male
66
1-5652
4-7435
3-0522
0-7888
0-7918
P < 0-001
Pooled
152
1-6426
5-0987
3-2866
0-8192
0-8291
P< 0-001
< 17cm
76
0-7492
3-0102
1-6238
0-5509
0-5584
P < 0-001
> 17-1 cm
76
1-6637
5-3609
3-4634
0-9074
0-8800
P< 0-001
380 Sandeep K Malhotra
4. Discussion
4.1 Food and parasites
The analysis of food reveals that L. dyocheilus maintains a predominantly herbivorous
feeding habit. The curves for frequency distribution of B. teleostei during infection
period, fish age, size and weight classes have been reported to be a simple logarithmic
function and revealed the familiar gaussian hump when plotted in octaves (Malhotra
1984b). The implications of this tendency have already been discussed by the author in
the earlier study (Malhotra 1984b).
4.2 Length-weight relationship
To conform to the results based on analysis of samples of L. dero collected from natural
habitats in the same locality (Malhotra 1984b) no major difference was noted in the
ratio values of total vis-a-vis standard length of L. dyocheilus. The agreement between
fish length and weight was good (P < 0-001) in female, male and pooled fishes and by
< 17cm and > 17-1 cm classes offish (table 4). Based on coefficient of determination
more than 75 % of the variation in weight in females, 62 % in males, 68 % in pooled,
30 % in < 17 cm length class and 79 % in > 17-1 cm length class was attributable to the
variation in length of L. dyocheilus. Similarly the proportion of correlated variance (p2)
suggests that 75-9071% variance in length in female fishes, 62-4532% in males,
67-9212 % in pooled, 30-7659 % in fishes of < 17 cm length class, and 79-861 1 % in fishes
of > 17*1 cm length class was associated with weight.
The length-weight relationship for female, male, pooled, < 17cm and > 17- 1cm
length classes of L. dyocheilus is defined and illustrated in this investigation. Similar to
the earlier observations in T. tor (Malhotra 1982) and L. dero (Malhotra 1984b) in the
Himalayan riverine ecosystem the larger fishes (> 17- 1cm) showed higher value of
regression coefficient (b — 2-2850) than the smaller ones i.e. < 17 cm (b = 1-7559). This
conclusion illustrates a relatively rapid change in body outline of the fishes of > 1 7- 1 cm
length class as they increase in length compared to those of the fishes of smaller length
class (< 17cm).
As a depends upon the obesity of the fish (LeCren 1951), by comparing log a values it
becomes obvious that there is no significant difference (F1;151 = 0-002) in the general
fatness of the two sexes in the present study similar to the findings of Malhotra (1982).
The regression for the pooled lot of fishes was calculated. The value of n (2-0871)
indicated that the growth rate is lesser than the cube length. Similar deviations have
been discussed earlier by Malhotra (1984b) on another species of the genus i.e. L. dero
from the same locality. In the present case too the departures of regression coefficients
from 3 were found highly significant (1 % level) for sexes and both length classes. As
emphasized earlier (Malhotra 1984b) the suitability of the exponential formula
W= aLn used in the present analysis to the cubic formula W~ CL3 (C, constant) has
been justified by several authors (Sekharan 1968) in similar studies. Beverton and Holt
(1957) discussed the merit of both allometric and cubic formula and reported that the
former works much better as a and n of the allometric formula vary within a wide range
for very similar data and are very sensitive to even quite unimportant variations in n.
Hence the high value of coefficient of correlation indicates that the allometric
relationship of length and weight is suitable for the fish.
Bionomics of cyprinids 38 1
References
Beverton R J and Holt S J 1957 On the dynamics of exploited fish populations; Fish. Invest. 19 533 pp
Chauhan R S and Malhotra S K 1981 Bionomics of hill-stream cyprinids. II. Barilius bola (Ham.); Biology 3
34-36
Chauhan B S and Ramakrishna J 1953 Fauna of Balangir district (formerly Patna state), Orissa. I; Fish. Rec.
Ind. Mus. 51 404 pp
Croxton F E 1953 Elementary statistics with applications in medicine and the biological sciences (New York:
Dovar) pp. 376
LeCren ED 1951 The length- weight relationship and seasonal cycle in gonad weight and condition in the
perch (Perca fluviatilis)', J. Anim. Ecol. 21 201-219
Malhotra S K1982 Bionomics of hill-stream cyprinids. III. Food, parasites and length-weight relationship of
Garhwal mahaseer, Tor tor (Ham.); Proc. Indian Acad. Sci. (Anim. Sci.) 91 479-485
Malhotra S K 1984a Cestode fauna of hill-stream fishes in Garhwal Himalayas, India. II. Bothriocephalus
teleostei n. sp. from Barilius bola and Schizothorax richardsonii; Bol. Chile. Parasit. 39 6-9
Malhotra S K 1984b Log-normal distributions in parasitology; Proc. Indian Acad. Sci. (Anim. Sci.) 93
591-598
Malhotra S K and Chauhan R S 1984 Bionomics of hill-stream cyprinids. IV. Length- weight relationship of
Labeo dero (Ham.) from India; Proc. Indian Acad. Sci. (Anim. Sci.) 93 411-417
Sekharan K V 1968 Length- weight relationship in Sardinella albella (Val) and S. gibbosa (Bleek); Indian J.
Fish. 15 166-174
Srivastava G J 1968 Fishes of Eastern Uttar Pradesh (Varanasi: Vishwavidyalaya Prakashan) 150pp
Sultan Salim and Shamsi M J K 1981 Morphometric study ofPuntius sarana (Ham) of the river Kali; Geobios.
8 17-21
Zeller R A and Carmines E G 1978 Statistical analysis of social data (Chicago: Rand McNally College Publ.
Co.) 398 pp
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 4, August 1985, pp. 383-387.
© Printed in India.
pH and dissolution of crystalline style in some bivalve molluscs of Porto
Novo coastal waters
P SHAHUL HAMEED
Centre of Advanced Study in Marine Biology, Parangipettai 608 502, India
Present address: Post-Graduate Department of Zoology, Jamal Mohamed College,
Tiruchirapalli 620020, India.
MS received 20 December 1984; revised 26 April 1985
Abstract. A comparative study of- the pH of crystalline style, digestive diverticula and
stomach fluid in six bivalve species, Anadara rhombea, Crassostrea madrasensis, Meretrix
meretrix, M. casta, Katelysia opima and Donax cuneatus revealed that the digestive diverticula
are more acidic while crystalline style is either slightly acidic or nearly neutral. The
significance of this finding is discussed. In vitro dissolution of crystalline style of all the six
species in buffer solutions of varying pH from 3-6 to 9-0 was observed and the study indicated
that the optimum pH for style dissolution is around 8-0 in ail species excepting Donax cuneatus
where some dissolution was recorded only at pH 9-0. Based on the physical changes observed
in the style, a mechanism of style dissolution has been put forward.
Keywords. Bivalve molluscs; crystalline style; style dissolution; Anadara; Crassostrea',
Meretrix', Katelysia', Donax.
1. Introduction
The crystalline style is a long, transparent rod of glycoprotein, lodged in the midgut of
most bivalves and some gastropods aiding in the processes of feeding and digestion
through its dissolution. Dissolution of the crystalline style had been shown to be pH
dependent by Yonge (1925, 1926), Venugopalan (1955), Mathers (1974, 1976) Mathers
et al (1979) and Alyakrinskaya (1977, 1979). Yonge (1925, 1926) proposed a theory that
the style was the most acidic part of the gut in bivalves and that the dissolution of the
style in the stomach was effected by the higher pH of the stomach content. On the other
hand, Purchon (1971) and Morton (1973) found that the style was not the most acidic
part of the gut and that the style pH was near neutral or slightly acidic. Further, the pH
in different regions of the gut was reported to be variable (Mathers 1974; Owen 1974).
Therefore, the present study was undertaken to clarify the actual situation in the
bivalve species of Indian coastal waters.
2. Materials and metliods
The bivalve species, Anadara rhombea (Bom), Crassostrea madrasensis (Preston),
Meretrix meretrix (Linnaeus), M. casta (Chemitz) and Katelysia opima (Gmelin) were
collected from the mud flats of Vellar estuary joining Porto Novo sea shore (1 1°29' N
lat. and 79°46' E long.) and D. cuneatus from the marine intertidal zone. The pH of
aqueous solutions of the crystalline style, the digestive diverticula and the stomach
383
384 P Shahul Hameed
content was measured in a digital pH meter (Toshniwal Cat. No. CL 46) to the nearest
0-01 pH following the method of Mathers (1974). In vitro dissolution of the style was
observed in buffer solutions with the pH range from 3-6 to 9*0 and in distilled water, sea
water and estuarine water at room temperature (34° C). The time required for the
complete dissolution was recorded following the method of Mathers (1974). Changes
occurring in the style during dissolution was observed under a microscope. Three
estimations were made at each pH and the mean dissolution time (t) and the rate of
dissolution (I//) were calculated.
3. Results
Table 1 presents the mean pH of the crystalline style and the digestive diverticula in six
species and that of stomach fluid on four species. The mean pH was either slightly
acidic or neutral. It is interesting to note that pH of any of these three regions of the gut
was not stable but variable within a limited range (table 1) and the digestive diverticula
was the most acidic part in all the species tested. The relationship between the pH of the
medium and the rate of style dissolution (1/r) was studied on all the six species (figure
1). In general, at low pH (3-6 to 4-6) the styles were stable for a prolonged period with
low rate of dissolution. The rate of style dissolution was rather slow at pH range of 5 to
7. Faster dissolution was observed at pH 8*0 in all species tested excepting D. cuneatus.
At this optimum pH, a high rate of dissolution was recorded in C. madrasensis (3 min)
and in A. rhombea (9 min). The pattern of style dissolution in D. cuneatus was quite
distinct from that of other species tested in that the style of this species was highly
resistant to dissolution in the pH range of 3-6 to 8-0. At pH 9-0 the style dissolved at a
slow rate (175 min) which was close to the dissolution time in sea water (215 min).
The data on the style dissolution in distilled water (pH 7-0), estuarine water (pH 8-2)
and sea water (pH 8-5) indicated that the style dissolved faster in natural media than in
distilled water (table 2).
Observation on the mode of style of dissolution revealed two distinct patterns. In
D. cuneatus the style underwent a process of lengthening before dissolution. In all other
species dissolution was followed by the shortening of the style. In both cases the style
had a central granular matrix around which concentric crystalline layers were found.
During dissolution, the granular matrix was observed to be continuously flowing out
largely through the anterior end and to a small extent through the posterior end.
4. Discussion
It is evident from the present study that the pH of the crystalline style of bivalve is either
slightly acidic or neutral. Morton (1969, 1970, 1971) recorded that the pH of styles of
Dreissena (pH 7-0) and Ostrea fpH 6-8) is approximately neutral whereas that of
Cardium (pH 6-4) is slightly acidic. Similar results were also reported by Langton and
Gabbott (1974) in Ostrea, Langton (1977) in Mytilus and Mathers (1974, 1976) in
Ostrea, Crassostrea and Pecten. In a given individual, the digestive diverticula were
always more acidic than its crystalline style. The pH of the style, digestive diverticula
and stomach content were not stable but fluctuated within a range. Fluctuating pH in
these three regions of the gut was also recorded by Mathers (1974, 1976) and Morton
pH and style dissolution in bivalve molluscs
Table 1. pH of style, digestive diverticula (DD) and stomach content of bivalves
385
Species
Mean0
Style pH
Range
Mean0
DDpH
Range
Stomach
Mean*
content pH
Range
A. rhombea
6-39
5-99-6-98
5-83
5-63-6-06
6-2
5-75-6-65
C. madrasensis
7-17
6-88-7-52
6-43
6-3 1-6-79
6-4
6-41-7-10
M. meretrix
7-11
6-11-7-46
6-42
6-58H5-66
6-5
6-50-7-00
M. casta
7-37
7-08-7-57
6-56
6-76-7-32
—
—
K. opima
6-41
6-05-6-59
5-80
5-70-5-86
6-0
5-90-6-90
D. cuneatus
6-79
6-45-7-03
6-31
6-09-6-59
—
—
^Average of 10 estimates; ^Average of 5 estimates; — not determined.
Table 2. Dissolution of crystalline style of bivalve species in natural media
and distilled water
Mean dissolution time (Minutes)
Estuarine Distilled
water Sea water water
Species (pH8-2) (pH 8-5) (pH 7-0)
Anadara rhombea
53 —
174
Crassostrea madrasensis
12 —
16
Meretrix meretrix
65 —
180
Meretrix casta
23 —
203
Katelysia opima
44 __
174
Donax cuneatus
— 215
2580
(1969, 1970). The present work confirms the current concept that in bivalves, the most
acidic part of the gut is the digestive diverticula and not the crystalline style; the pH of
the crystalline style, digestive diverticula and stomach content fluctuates considerably
and that the stomach pH is not stable and not buffered by style dissolution (Purchon
1971; Morton 1973; Owen 1974).
Bivalve crystalline style had long been known to dissolve at a faster rate in alkaline
medium and stable in acidic medium (Yonge 1925, 1926; Venugopalan 1955; Mathers
1974, 1976; Mathers et al 1979; Kristensen 1972; Alyakrinskaya 1977, 1979). On
the basis of the rate of dissolution, the crystalline styles of the bivalve species examined
in the present work could be classed into two types: (i) rapidly dissolving styles and
(ii) slowly dissolving styles. The rapidly dissolving styles were found to be housed in the
style sac conjoined with midgut as observed in A. rhombea, C. madrasensis, K. opima,
M. meretrix and M. casta. These estuarine bivalves had a high rate of dissolution at
pH 8-0 (figure 1) and the complete dissolution time ranged between 3 minutes in C.
madrasensis and 25 minutes in M . meretrix. On the other hand, D. cuneatus possessed a
slowly dissolving style and its style sac is entirely separated from the midgut. Even at
the pH of 9-0 its dissolution rate is very slow (22 hr). Probably the rate of dissolution
depends on other factors as well such as the organic content of the style (Alyakrinskaya
1979).
386
P Shahul Hameed
o.;o-
0.20
0.10
o
(D
0.06
LU
CO
0.04
LU
»—
o: 0.02
0.0100
0.0050
' Crassest rea madrosinsn
' Anadarq rhomb^a
3.0
6.0 7.0
pH
Figure 1. Relationship between the rate of style dissolution and pH.
The optimum pH for dissolution of the style coincided with the pH of the medium
(table 2). It would seem that during feeding the entry of water into the stomach would
raise the pH facilitating an increase in the rate of style dissolution and the consequent
release of digestive enzymes. The inference is in conformity with the observations of
Mathers (1974, 1976) in Ostrea and Pecten.
According to Yonge (1925, 1926, 1949) the dissolution of the style was thought to.be
restricted to its anterior end projecting into stomach lumen. But in vitro observations
on the mode of style dissolution did not support this view. Dissolution of bivalve style
is a physical phenomenon involving the entire style body. Kristensen (1972) reported
that the outer crystalline layers of the style are relatively hard and more resistant to
dissolution while the central core is hygroscopic. It is suggested that the dissolution of
the style is brought about by the liquefaction of the crystalline layers into granular core
through uptake of water. The actual transport of the dissolved materials was observed
in the present study as an anteriorly directed streaming movement of the central
granular matrix.
pH and style dissolution in bivalve molluscs 387
Acknowledgement
The author sincerely thanks Dr A L Paulpandian for his guidance, Dr R Natarajan
Director, C.A.S. in Marine Biology, Parangipettai, for encouragement and to UGC
New Delhi for the award of a fellowship.
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some groups of animals (Moscow: Nauka) 142-150
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Ostrea edulis L: Mar. Bioi 24 181-187
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Mathers N F 1974 Digestion and pH variation in two species of oysters; Proc. MalacoL Soc. London 41
37-46
Mathers N F 1976 The effects of tidal currents on the rhythm of feeding and digestion on Pecten maximus L;
J. Exp. Mar. Bioi. Ecol 24 271-283
Mathers N F, Smith T and Coline N 1979 Monophasic and diphasic digestive cycles in Venerupis decusata
and Chlayms varis; J. Moll. Stud. 45 68-81
Morton B S 1 969 Studies on the biology of Dreissena polymorpha Pall. II. Correlation of rhythms oi
adductor activity, feeding digestive and excretion; Proc. MalacoL Soc. London 38 401-414
Morton B S 1970 The tidal rhythm and rhythm of feeding and digestion in Cardium edulis: J. Mar. Bioi.
Assoc. U. K. 50499-512
Morton B S 1971 The daily rhythm and tidal rhythm of feeding and digestion in Ostrea edulis'. Bioi. J. Linn.
Soc. London 3 329-342
Morton B S 1973 A new theory of feeding and digestion in filter feeding lamellibranchia; Malacologia 14
63-69
Owen G 1974 Feeding and digestion in Bivalvia; Adv. Comp. Physiol. Biochem. 5 1-35
Purchon R D 1971 Digestion in filter feeding bivalves, a new concept; Proc. MalacoL Soc. London 39 253-
262
Venugopalan V K 1955 Studies in the anatomy and physiology of Sanguinolaria diphos (Gmelin); M.Sc.
Thesis, Annamalai University
Yonge CM 1925 The hydrogen ion concentration in the gut of certain lamellibranchs and gastropods; J.
Mar. Bioi. Assoc. U.K. 13 938-957
Yonge C M 1926 Structure and physiology of organs of feeding and digestion in Ostrea edulis; J. Mar. BioL
Assoc. U.K. 14 295-386
Yonge CM 1949 On the structure and adaptations of the Tellinacea, deposit feeding Eulemellibranchia:
Philos. Trans. R. Soc. (London) B234 29-76
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 4, August 1985, pp. 389-398.
© Printed in India.
Studies on the induced spawning and larval rearing of a freshwater
catfish, Mystus punctatus ( Jerdon)
N RAMANATHAN, P NATARAJAN and N SUKUMARAN
Fisheries College, Tamil Nadu Agricultural University, Tuticorin 628 008, India
MS received 21 March 1984; revised 30 March 1985
Abstract. The freshwater catfish Mystus punctatus was successfully bred in the laboratory by
injecting the pituitary extract of the marine catfish Tachysurus maculatus. The number of eggs
released by a single female fish was 9050 ± 700, the relative fecundity was 43-1 and the
fertilization success was 85 ± 2-8 per cent. The average diameter of the unfertilized eggs ranged
from 1-27 to 1-35 mm and the fertilized eggs ranged from 1-45 to 1-50 mm. The eggs hatched
within 18 to 24 hr at a water temperature of 28-5 ± 1-8°C, and the percentage of hatching was
78 %. The larvae metamorphosed into juveniles within fifteen days of their hatching. A
description of the egg and larval development to metamorphosis is given.
Keywords. Mystus punctatus; induced breeding; pituitary extract; hatching; metamorphosis;
larval development.
1. Introduction
The freshwater catfish Mystus punctatus is one of the good table fishes in the inland ,
regions of India and hence has good culture potential.
The effect of pituitary gland extract from the marine catfish Tachysurus maculatus to
induce the breeding of freshwater catfish Clarias batrachus was studied by Devaraj et al
(1972). In the present study the freshwater catfish Mystus punctatus was successfully
induced to spawn in the laboratory by injecting marine catfish pituitary gland extract as
adopted by Devaraj et al (1972). The larvae that hatched out from the fertilized eggs
were reared in the laboratory and their development was studied.
2. Material and methods
The matured adult fish of both the sexes were collected from natural waters of
Kalladaippu tank near Tuticorin of Tirunelveli district of Tamil Nadu (South India)
during January to February 1981. The fish thus collected were maintained in fibreglass
tanks of size 200 x 50 x 50 cm, and were fed with artificial pelleted feeds (Tilapia flesh,
groundnut oil cake and rice bran at a ratio of 2 : 1 : 1) at a rate of 5 % body weight offish.
The water temperature maintained was 28-5 + 1-8°C. The pituitary glands were
collected from the marine catfish Tachysurus maculatus. The breeding experiments were
conducted in three fibreglass tanks of 200 x 50 x 50 cm size, in which a water depth of
25 cm was maintained.
The females were injected with the extract containing 6 mg of dry pituitary /kg of
body weight in two split doses with an interval of 3 hr while the males were given the
extract of 3 mg/kg of body weight in single dose at the time of second injection to the
389
390 N Ramanathan, P Natarajan and N Sukumaran
female. Six males (68 to 73 g) and 3 females (197 to 223 g) were used for the experiment.
Very clear water was maintained in the tank. The water temperature was 28-5 ± 1-8°C.
The hatched larvae were reared in nursery tanks of size 5 x 5 m with a water depth of
75cm. In these nursery tanks the water temperature was 28-5 ± 1-8°C. The larval
development was studied and discussed (Mansueti and Hardy 1967).
3. Results and discussion
After 15 minutes of the second doze of the pituitary extract injection, the experimental
fish started the courtship behaviour. The males started chasing the females and twisted
around the body of the females with the tail. The females released the eggs eight hours
after administering the pituitary injection.
The eggs were siphoned from the tank after removing the spent fish and placed in
clear water in another tank of the same size, and the temperature of the water was 28-5
± 1-8°C. The fertilization of the eggs was estimated as 85 %. The average number of
eggs released was 9050 and the diameter of the fertilized egg was 1-45 to 1-50 mm.
The eggs hatched out within 18 to 24 hr after fertilization. The percentage of
hatching was 78 %. The standard deviation (x ± so) for number offish, number of eggs
collected, relative fecundity, percentage of fertilization, egg diameter and the number of
degrees-days for spawning and incubation are given in table 1. The hatchiings had a
large yolk sac and the larvae were found attached to the sides and bottom of the tank.
The yolk was completely absorbed on the third day of hatching and subsequently the
larvae were fed with plankton obtained from the ponds. The larvae started feeding from
the fourth day onwards. They were then transferred to a manured cement nursery tank
of size 5 x 5 m with a water depth of 75 cm. The postlarvae metamorphosed into
juveniles within 15 days from hatching.
A description of the eggs to the fully metamorphosed juvenile is given below in order
to facilitate its identification at any stage of its larval development as it was not
described earlier.
3.1 Unfertilized ova
The ova (figure 1) are light yellowish in colour with a denser area in the centre. Their diam-
eter ranges from 1-27 to 1-35 mm. The outer surface is smooth and concretions are visible.
Table 1. Standard deviation (X ± SD), of the fecundity description
Experiment Control
Number of fish ^_3;5_6 o-_2;C_i
Fishsize o 69±4g; $ 210± 13g 6*69g, 71g; $210g
No. of eggs released 9057 ±700 —
Relative fecundity 43-1 —
Percentage of fertilization 85 ± 2-8. —
Egg diameter i Unfertilized eggs ) 1-27-1 -35 mm —
[Fertilized eggs ) 1-45 -1-50 mm — -
Number of degrees for spawning )
and incubation j ~ ^ —
Temperature 28-5 ± 1 -8°C 28-5 ± 1 -8°C
Induced spawning and larval rearing of catfish
3.2 Fertilized egg
391
The fertilized eggs (figure 2) are also light yellowish in colour and the diameter ranges
from 145 to 1-5 mm. The outer surface of the egg is somewhat thickened when
compared to that of the unfertilized ova. The developing embryo is clearly visible under
a microscope.
1 mm 1
1 mm
1 mm 3
1 mm
392 AT Ramanathan> P Natarajan and N Sukumaran
1 mm
1 mm
10
1 mm
Figures 8-11.
11
Induced spawning and larval rearing of catfish
393
Figures 12-15.
394
N Ramanathan, P Natarajan and N Sukumaran
2 mm
17
Figures 16-17.
3.3 Just hatched out larva
The just hatched out larva (figure 3) is laden with yolk sac. The larvae measure 3-1 to
3-3 mm in total length and the yolk sac 0-8 mm on an average. The mouth has not yet
formed, while the optical sac has already developed.
The larvae settle at the bottom and sides of the tank and their movements are
restricted on account of the large yolk sac. The body is devoid of any chromatophores.
3.4 One-day larva
The one-day old larvae (figure 4) measure 3-4 to 3-6 mm in total length and the yolk still
persists measuring 1-6 mm on an average. As in the just hatched out larva, the mouth
has not yet developed. The anal opening has developed by the constriction of the
ventral fin fold in the middle. The dorsal and ventral margins of the body have a row of
simple chromatophores and the larvae do not swim except when disturbed.
3.5 Two-day old larva
The larvae (figure 5) measure 4-3 to 4-6 mm in total length. The yolk is almost absorbed.
Three pairs of barbels have developed (one pair maxillary and two pairs mandibular).
Induced spawning and larval rearing of catfish
395
396 N Ramanathan, P Natarajan and N Sukimaran
The mouth has just formed. The chromatophores are distributed over the head and
along the dorsal and ventral margins of the body. Chromatophores are also seen
scattered over the opercular region. The larvae are able to swim freely.
3.6 Three-day old larva
They (figure 6) measure 4-6 to 5-0 mm in total length. The nostril is formed and the
mouth developed. The operculum is seen as a slit. The chromatophores are found
scattered over the head, below the opercular slit and along the dorsal and ventral
margins of the body. The caudal rays have begun to develop. The larvae are active and
freely swimming at this stage.
3.7 Four-day old larva
They (figure 7) measure 4-7 to 5-1 mm in total length. The eyes and nostrils are well
formed. The size of maxillary barbels at this stage is more than that of the mandibular
barbels. The chromatophores are scattered over the head, dorsal and ventral margins of
the body, except along the posterior half of the ventral margin. The caudal fin is slightly
constricted from the dorsal and ventral fin folds.
3.8 Five-day old larva
They (figure 8) measure 5-2 to 5-4 mm in total length. The pectoral fins are developed.
The dorsal and anal fins are separated from the fin folds. The chromatophores are
scattered over the head, on the dorsal margin of body and above the pectoral fin. The
body is dark compared to the four day old larva.
3.9 Six-day old larva
They (figure 9) measure 64 to 6-7 mm in total length. The rays of the pectoral fins and
the lower lobe of the caudal fin are calcified. The dorsal fin rays have developed and so
also the ventral and caudal fin lobes. The chromatophores are scattered over the head,
around the orbit, along the dorsal margin of body and over the belly region.
3.10 Seven-day old larva
They (figure 10) measure 9-8 to 10-1 mm in total length. Nostrils have formed. The rays
of dorsal and upper lobe of caudal fin are calcified. The chromatophores are scattered
throughout the body. A spine is developing in the dorsal fin.
3.1 1 Eight-day old larvae
They (figurell)m^urelO-3to 10-6 mm intotal length. The first dorsal fin spine is well
calcified and the chromatophores are star-shaped over the head.
Induced spawning and larval rearing of catfish 397
3.12 Nine-day old larva
They (figure 12) measure 10-4 to 10-6 mm in total length. The anal fin spines and rays are
calcified. The caudal fin is clearly forked. The chromatophores are scattered throughout
the body, but are more concentrated over the dorsal region of the body.
3.13 Ten-day old larva
They (figure 13) measure 10-6 to 10-9 mm in total length. The ventral fin rays are
calcified and the caudal fin is clearly forked as in the juveniles. The adipose second
dorsal fin is still attached to the upper lobe of the caudal fin. The chromatophores are
more concentrated on the dorsal surface of the body.
3.14 Eleven-day old larva
They (figure 14) measure 12-3 to 12-7 mm in total length. The adipose second dorsal fin
has just started detaching itself from the caudal fin. The spines in the anal fin are fully
calcified. The chromatophores are found only along the dorsal surface of the body.
3.15 Twelve, Thirteen- and fourteen-day old larvae
These larvae (figures 15-17) show -only minute differences in their morphology. In the
twelve-day old larva, the chromatophores are very few and started disappearing. The
thirteen- and fourteen-day old larvae are devoid of any chromatophores. The adipose
second dorsal fin has completely separated from the caudal lobe in the fourteen day old
larva and appears almost similar to that of a juvenile catfish.
3.16 Juvenile catfish
The juveniles (figure 18) just metamorphosed from the post-larvae show all the
morphological characters of the adult. The metamorphosis from postlarvae to juvenile
stage takes place mostly in about fifteen days after hatching.
Acknowledgements
We record our sincere thanks to Dr S J de Groot, Netherlands Institute for Fishery
Investigation, The Netherlands for critically going through the manuscripts and giving
suggestions. Thanks are also due to Dr R Natarajan, Director and Professor, C. A. S.
in Marine Biology, Annamalai University, Parangipettai for providing facilities to
complete the figures.
398 N Ramanathan, P Natarajan and N Sukumaran
References
Alikunhi K H, Vijayalakshmanan M A and Ibrahim K H 1960 Preliminary observations on the spawning of
Indian major carps, induced by injection of pituitary harmones; Indian J. Fish 71-19
Chaudhuri H 1 960 Experiments on induced spawning of Indian major carps with pituitary injections; Indian
J. Fish 1 2CM8
Devaraj K V, Varghese T J and Sathyanarayana Rao G P 1972 Induced breeding of the freshwater cat fish
Glorias batrachus (Linn.) by using pituitary glands from marine cat fish; Curr. Sci. 41 468-870
Jhingran V G 1975 Fish and fisheries of India (New Delhi: Hindustan Publishing Corporation) 508-510
Mansueti A J and Hardy J D Jr 1967 Development of fishes of the Chesapeake Bay region — An Atlas of Egg.
Larval and Juvenile Stages, Part I. (ed.) E E Daubler Jr Natural Resources Institute, (Baltimore:
University of Maryland Press) 202 pp.
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 4, August 1985, pp. 399-406.
© Printed in India.
Wing raicrosctilptiiring in the Brazilian termite family Serritermitidae
(Serritermes serrifer, Isoptera), and its bearing on phylogeny
M L ROONWAL and N S RATHORE
Desert Regional Station, Zoological Survey of India, Jodhpur 342006, India
MS received 28 January 1985; revised 24 April 1985
Abstract. The results of our studies on the Brazilian family Serritermitidae are presented
here. Microsculpturing is simple and consists of a few rows of small, tongue-shaped papillae on
the anterior and posterior wing margins and a few rows of angular arrowheads in the anterior
one-third of the wings. Hairs are almost absent, a few small ones being scattered on some of the
veins. The bearing of wing microsculpturing on the phylogeny of the Serritermitidae is
discussed. It is concluded that the family arose as a lone sideline from the common ancient
rhinotermitid stock. The other line from this stock gave rise to the Styiotermitidae on the one
hand and the Rhinotermitidae on the other.
Keywords. Wing microsculpturing; Isoptera; Serritermitidae; Serritermes serrifer, phylogeny
1. Introduction
In a long series of papers, Roonwal and co-workers (1967-1983) have published the
results of studies on wing microsculpturing in all the major families and subfamilies of
termites except two tiny single-genus families, the Serritermitidae (neotropical, Brazil)
and the Indotermitidae (oriental). The occurrence of a thick carpet of eight different
types of microsculpturing elements on both wing surfaces has been demonstrated, with
densities as high as over 12500/mm2.
Imagoes of Serritermes serrifer (Bates in Hagen), the sole representative of this rare
and unique neotropical (Brazilian) family, have, after years of effort, recently become
available, and the results of its study are presented here.
2. Materials and methods
Imagoes collected from Coxipo de Ponte (about 5 km SE of Cuyaba, Mato Grosso
Province, Brazil; lat. 15-35 S, long. 56-00 W) were studied. Glycerine mounts of wings
gave excellent delineation of the microsculpturing elements.
3. Results
3.1 Family SERRITERMITIDAE Holmgren
Various authors have placed the subfamily Serritermitinae in either the family
Rhinotermitidae or the Termitidae. Emerson (1965, p. 17) raised it to family rank
399
400
M L Roonwal and N S Rathore
(characters later elaborated by Emerson and Krishna 1975), and we accept that status
(also vide Krishna 1970; Weidner 1970).
Genus Serritermes Wasmann
Serritermes serrifer (Bates in Hagen)
Wings (figures 1-3)
Wings small (size without scale : forewing 5- lxl-2 mm; hindwing 4-Oxl -3 mm),
transparent, colourless, veins somewhat dark yellowish; almost hairless, a few small
hairs (length 28-40 jon) scattered on some of the veins; margins smooth and totally
hairless; at the distal end the margin is wavy and rugose (figure 3C). Venation variable.
In the two pairs of wings examined by us the media is absent (according to Emerson and
Krishna 1975, it is present in some wings and absent in others).
Microsculpturing (figures 2 and 3)
Consisting of papillae and arrowheads on both the dorsal and ventral wing surfaces. No
micrasters or any other type present.
Papillae: Consisting of 2 or 3 rows of distally directed, tongue-shaped papillae on
anterior margin of first vein (costal-subcosta), and 1 or 2 rows of similar but smaller
papillae on the posterior margin. Size of anterior papillae 6-1 1 /an x 4-5 jum. Density
(in the concerned region) ca 6400/mm2.
Arrowheads: Consisting of 3 or 4 rows of distally directed, angular, pointed,
arrowhead-like bodies on the second vein (radius) and 2 or 3 rows of similar but smaller
C+Sc.
R. Cu.
B
I mm
Figure 1. Serritermes serrifer. Right wings. A. Forewing. B. Hindwing. C-fSc, Costa-
subcosta (costal margin of Emerson); R, radius (radial sector of Emerson); Cu, cubitus.
Wing microsculpturing in Brazilian termite
401
h.
lOOjum
Figure 2. Serritermes serrifer. Portions of right wings, magnified to show microsculpturing.
A. Middle of anterior margin of forewing, dorsal surface. Papillae (on anterior edge of first
vein) and arrowheads (on first and second veins), B. Same, posterior margin (papillae), C.
Middle of anterior margin of hindwing, ventral surface, a., arrowheads; h., hairs; p., papillae.
structures on the first vein below the papillae. Size (on the second vein) 8-1 1 /zm
x 6-9 ^m. Density (on second vein) ca 3060/mm2, Arrowheads mostly V-shaped with a
sharp, narrow to broad angle, and with a thick base and pointed tips; those on the first
vein tend to be smaller and somewhat subcresentic.
4. Discussion
4.1 General
No information was hitherto available on wing microsculpturing in Serritermes serrifer.
Emerson and Krishna (1975) gave a preliminary and incomplete account as follows:
402
M L Roonwal and N S Rathore
B
50pm
Figure 3. Serritermes serrifer. Portions of right forewing, dorsal surface, greatly magnified to
show microsculpturing. A. On first vein (below anterior edge), B. On second vein. C Distal
margin to show rugosity of outer edge, a., arrowheads; h., hairs.
(i) Hairs are "absent on the costal margins, inner margins, membranes, and strong
veins beyond the humeral suture ..." (p. 12). The absence of hairs was regarded as a
derivative (i.e. secondary) condition, (ii) Wing membranes "lack punctations" (p. 16).
The precise meaning of punctation was, however, not made clear, and further down the
same page they mentioned the presence of "rounded punctations or micrasters" in
several genera of other families, e.g. Coptotermes, Psammotermes, Heterotermes,
Termitogeton, Stylotermes and Parastylotermes. Some, but, not all, of these genera
possess true, multi-armed, asteroid micrasters, and it is clear that these authors neither
distinguished punctations from micrasters, nor gave their precise meaning.
The presence of weak papillae and simple arrowheads (and no other form of
microsculpturing) provide valuable clues to the phylogenetic affinities of the
Serritermitidae, as discussed below.
Wing microsculpturing in Brazilian termite
403
4.2 Phylogeny
Serritermes serrifer is a rare and peculiar species whose phylogenetic position has been
the subject of much diversity of opinion. Hagen (1858) placed it with the
Kalotermitidae (his Calotermes serrifer Bates). Wasmann (1897) erected for it a new
subgenus Serritermes (of genus Calotermes), while Silvestri (1901, 1903) raised it to full
generic rank. Holmgren (1911) placed it in a new subfamily, Serritermitinae (of his
family Mesotermitidae = Rhinotermitidae). Subsequent authors placed the
Serritermitinae either with the Rhinotermitidae (Grasse 1949) or the Termitidae
(Snyder 1949). Emerson (1965) raised it to family rank. Emerson and Krishna (1975)
regarded it as close to the rhinotermitid subfamily Psammotermitinae (genera
Psammotermes and Glossotermes). They concluded (p. 28) that "the Serritermitidae can
be traced backward to an origin from the base of the Psammotermitinae, and in
sequence back to the primitive rhinotermid stem, to the hodotermitid-rhinotermitid
stem, or to the primitive isopteran stem that arose from primitive blattoids possibly as
early as Permian times."
We may now discuss briefly the significance of the more important characters,
including wing microsculpturing, which have a bearing in determining the phylogenetic
positions of the Serritermitidae.
The very small size of this termite, its characteristics imago-worker mandibles with a
single marginal tooth (versus two or three in others), the peculiar, long, sword-like,
serrated soldier mandibles (figure 4A) and the soldier pronotum (figure 4D) with a very
deep, median indentation in both the anterior and posterior margins are characters
which distinguish Serritermes from all other genera.
Figure 4. Serritermes serrifer. Soldier. A. Mandibles.
D. Pronotum.
B. Labrum. C. Postmentum.
404
M L Roonwal and N S Rathore
Several characters suggest a rhinotermitid or a stylotermitid affinity. The simple,
elongate, tongue-shaped soldier labrum (figure 4B) recalls the similar labrum of the
primitive rhinotermitids (Psammotermes, Coptotermes), but lacks the pair of long apical
bristles. The differences from the latter genera are also emphasised by the small
hexagonal postmentum in Serriterrnes (figure 4C) and a long one in the other two.
Regarding wing microsculpturing, unlike the Psammotermitinae (Roonwal et al
1979b) where only pimpules are present in addition to the papillae, there are no
pimpules in Serriterrnes but only arrowheads in addition to the universally occurring
papillae. Arrowheads first appeared in the family Stylotermitidae (Stylotermes,
Roonwal et al 1979a; Roonwai 1981, 1983b) and are present in considerable abundance
in addition to pimpules and papillae. They are only occasionally present in a few
Rhinotermitidae and Termitidae (Roonwal 1983b), but their main concentration seems
to be in the Stylotermitidae. Their presence, in fair abundance, in the Serritermitidae
would thus suggest its affinity with the Stylotermitidae, but this is offset by the
important fact that the tarsi are 3-segmented in all the three legs of the Stylotermitidae
(Roonwal 1975) and 4-segmented in all the legs of the Serritermitidae (figures 5 A-D).
Emerson and Krishna (1975) have suggested that the origins of the Serritermitidae
can be traced back not only to the primitive rhinotermitid stem at the base of the
Psammotermitinae, but even earlier, near the primitive blattoid stock. The two most
primitive families which also arose from primitive blattoid stocks are the
Mastotermitidae (Mastotermes) and the Termopsidae (Archotermopsis). But the
Serritermitidae can be separated from these primitive genera by the fact that the apical
tibial spurs are naked in Serriterrnes (Figure 5 E) and clothed with scaly papillae in the
other two.
Figure 5. Serriterrnes serrifer. Soldier, legs of left side. A. Foreleg. B. Middle leg. C. Hindleg.
D. Lower part of hindleg, more magnified. E. An apical tibial spur.
Wing microsculpturing in Brazilian termite
405
On the whole, it may be concluded that the Serritermitidae, while being a primitive
and isolated family, differs from the most primitive ones and has close affinities with the
Rhinotermitidae, especially the Psammotermitinae. It seems (also vide Roonwal 1983b,
p. 367, chart) that the ancestral rhinotermitid stock (with 4-segmented tarsi and
potentiality for varied sorts of wing microsculpturing) evolved into two main lines
(figure 6), viz (i) a lone serritermitid line (with 4-segmented tarsi and simple wing
microsculpturing consisting of only papillae and arrowheads); and (ii) a rhinotermitid-
stylotermitid line with (initially) 4-segmented tarsi and varied types of microsculptur-
ings. This latter line further evolved into two branches: (a) A small Stylotermitidae
branch (with a simple wing microsculpturing consisting of only 3 kinds, e.g. papillae,
pimpules and arrowheads, and with the number of tarsal segments reduced to 3); and
(b) a large Rhinotermitidae branch with 4-segmented tarsi and wing microsculpturing
consisting of 5 types of structures, e.g. papillae, arrowheads, pimpules, tubercles and
micrasters, though all the five do not occur together in the same species.
Ui
<
o
ANCESTRAL
RHINOTERMTID
STOCK
Figure 6. Diagram to illustrate the probable phylogeny of the Serritermitidae from the
ancient rhinotermitid stock. Numerals indicate the number of tarsal segments (for details see
text.)
Acknowledgements
We are indebted to the following for assistance: to Dr Eliana M Cancello (Zoology
Museum, Sao Paolo University, Sao Paolo, Brazil) for kindly sending examples of
Serritermes serrifer; to Dr R C Sharma (Zoological Survey of India, Jodhpur) for
assistance with some of the drawings.
406 M L Roonwal and N S Rathore
References
Emerson A E 1965 A review of the Mastotermitidae (Isoptera), including a new fossil genus from Brazil; Am.
Mus. Novitat. No. 2236, 1-46
Emerson A E and Krishna K 1 975 The termite family Serritermitidae (Isoptera); Am. Mus. Novitat. No. 2570,
1-31
Grasse P P 1949 Ordre de Isoptere ou termite (Isoptere Brulle 1832). In Traite de Zooi (Anat., Syst., Biol.) IX.
Insecta, (Paris; Masson et Cie) pp. 408-567
Hagen H A 1858 Monographic der Termiten. Part 2; Linn. Ent. 12 1-342
Holmgren N 1911 Termitenstudien 2. Systematik der Termiten. Die Familien Mastotermitidae,
Protermitidae und Mesotermitidae; Kungi Svensk. Vet. Akad. Handl. 46 1-86, 6 pis
Krishna K 1970 Taxonomy, phylogeny and distribution of termites. In Biology of termites (eds) K Krishna
and F M Weesner, (New York: Academic Press) Vol. 2, pp. 127-152
Roonwal M L 1975 Phylogeny and status of termite families Stylotermitidae and Indotermitidae with three-
segmented tarsi, and the evolution of tarsal segmentation in the Isoptera; Biol Zentralbl. 94 27-43
Roonwal M L 1 98 1 Evolution and systematic significance of wing microsculpturing in termites (Isoptera). XL
Some hitherto unstudied genera and species in five families; Proc. Indian Natl. Sci. Acad. B47 467-473
Roonwal M L 1983a Universal occurrence of external cuticular, microscopic papillae and allied structures as
an essential character of termites (Isoptera, Insecta), and redefinition of the order; Zooi Anz. 211 137-144
Roonwal M L 1983b Evolution and systematic significance of wing microsculpturing in termites. XIII. Order
Isoptera; Proc. Indian Natl. Sci. Acad. B49 359-391
Roonwal M L and Chhotani O B 1967 Wing microsculpturing in termite genera Odontotermes, Hypotermes
and Microtermes (Termitidae: Macrotermitinae); Zooi Anz. 178 236-262
Roonwal M L, Chhotani O B and Verma S C 1981 Evolution and systematic significance of wing
microsculpturing in termites (Isoptera). X. Subfamily Nasutitermitinae of family Termitidae; Proc. Indian
Natl. Sci. Acad. B47 341-369
Roonwal M L, Verma S C and Thakur M L 1979a Evolution and systematic significance of wing
microsculpturing in termites (Isoptera). V. Families Mastotermitidae, Termopsidae, Hodotermitidae and
Stylotermitidae; Proc. Indian Natl. Sci. Acad. B45 115-128
Roonwal M L, Verma S C and Thakur M L 1979b Evolution and systematic significance of wing
microsculpturing in termites (Isoptera). VI. Family Rhinotermitidae; Proc.. Indian Natl. Sci. Acad. B45
332-353
Silvestri F 1901 Note preliminare sui termitidi sudamericani; Bol. Mus. Zooi. Anat. compar. Torino 16 1 -8
Silvestri F 1903 Contribuzione alia conoscenza dei termitidi e termitofili dell* America meridionale; Redia 1
1-234
Snyder T E 1949 Catalog of termites (Isoptera) of the world; Smithson. Misc. Colls. 112 1-490
Wasmann E 1897 Termiten von Madagaskar und Ostafrica; Abhandl. Senkenbergischen nat. Gesell. 21
137-182
Weidner H 1 970 Isoptera (Termiten). In Handbuch der Zooi. Vol. 4 (Anhropoda), Sec. 2 (Insecta), Pt. 2
(Special), Lief. 13, No. 14, pp. 1-147 (Berlin: W. de Gruyter)
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 4, August 1985, pp. 407-41 1.
© Printed in India.
Electrical stimulation — Effects on the protein in the ventral nerve cord
of cockroach, Periplaneta amencana
S L MAHESWARI, K BALASUNDARAM and V R SELVARAJAN
Division of Neurobiology, Department of Zoology, University of Madras, Madras 600 005,
India
MS received 15 June 1984; revised 8 September 1984
Abstract. Changes in protein concentration due to electrical stimulation have been
investigated at different time intervals in cockroach ventral nerve cord. Significant increase in
total protein concentration was observed at 15 min interval of stimulation. Increase in protein
concentration was also observed in ventral nerve cords incubated with exogenous glucose.
Microdisc polyacrylamide gel electrophoresis at 15 min interval has revealed an increase in low
molecular weight protein fractions in nerve cords from stimulated group. The data clearly
depict the changes in protein due to electrical stimulation stress.
Keywords. Cockroach; electrical stimulation; ventral nerve cord; protein; microdisc poly-
acrylamide gel electrophoresis.
L Introduction
Studies on the correlation of the functional activity of the brain with metabolic
parameters due to chemical, electrical and light stimuli are available on vertebrates
(Chitre and Talwar 1963; Chitre et al 1964; Jones 1972; Jones and Macllwain 1971;
Kol's et al 1974; Luxuro 1960; Selvanayagam and Habibulla 1979; Talwar et al 1961,
1966). Review of literature shows absence of knowledge on the effects of electrical
stimulation on the metabolic parameters of nervous system (NS) of invertebrates.
Hence the present study was undertaken to gain information of electrical stimulation
on the protein concentration and pattern in an invertebrate central nervous system.
2. Material and methods
In order to eliminate the effect of sex on protein concentration, only male animals were
chosen for study. Male cockroaches, Periplaneta amencana were taken in two groups.
One served as control and the other as the experimental group. From each group,
atleast ten animals were used for removal of ventral nerve cord. The dissection was
carried out in insect ringer solution (Orchard and Finlayson 1977). One set of the nerve
cords from the control group was incubated in ringer with glucose and another set in
ringer free of glucose for 15 min (Edstrom and Mattison 1972). The nerve cords from
the experimental group were incubated in two sets similar to control before
stimulation. After the incubation period, all the sets of ventral nerve cords were hooked
separately to a pair of silver-silver chloride electrodes, supported by a wax bath in a
petridish. The preparation was prevented from drying by periodically adding insect
ringer. The nerve cords of the experimental sets alone were given a repetitive stimuli of
407
408 S L Maheswari et al
0-1 m sec duration with a current strength of 3 V at a frequency of I/sec using an
electronic stimulator (Palmer, England Electronic square-wave stimulator) for 5, 10, 15,
30 and 60 min.
2.1 Total protein estimation
Both control and stimulated nerve cords were removed, washed with insect ringer and
weighed separately after carefully blotting out the moisture. They were homogenised
separately in prechilled glass-glass homogeniser with 80 % ethanol and centrifuged at
3500 rpm for 5 min. The residue was dissolved in IN NaOH solution and the proteins
were estimated following the method of Lowry et al (1951).
The results were tested for significance using student's t test.
2.2 Microdisc electrophoresis
Protein pattern was analysed by homogenising the ventral nerve cord from both the
sets of control and experimental groups in 0-01 M Tris-HCl buffer (pH 7-4) and
subjecting the supernatent to microdisc polyacrylamide gel electrophoresis (Ganesan
et al 1979). The gels were stained in 0-25% coomassie brilliant blue, destained and
stored in 7 % acetic acid (Smith 1968). Gels were photographed over an x-ray viewer as
suggested by Oliver and Chalkley (1971) and scanned using Shimadzu-Dual length
TLC scanner CS-910 with C-RIA chromotrophe recorder at 640 and 430 nm.
3. Results
3.1 Total proteins
The electrical stimulation of the cockroach ventral nerve cord (incubated in exogenous
glucose) at 15 min interval has revealed highly significant (p < 0-001) increase in total
protein concentration (40-81 ±5-56 mg/g). Though the protein concentration in-
creases from 5 min up to 15 min, a decrease is observed after that period (table 1).
Table 1. Effect of electrical stimulation on the total protein concentration (mg/g)
from the cockroach ventral nerve cord with exogenous glucose.
Total
protein
Duration of
A Vicnlnfp
stimulation
Control
Experimental
difference
P value
5
24-30 ±2- 14
29-54 + 2-54
+ 5-24
< 0-005
10
25-91 ±3-26
35-86 ±1-89
+ 9-95
< 0-005
15
26-02 ±4-21
40-81 ±5-56
+ 14-79
< 0-001
30
24-90 ±5-04
20-79 ±4- 15
- 4-11
NS
60
30-00 ±6-01
19-10 ±5-76
-10-90
< 0-010
Mean + S.D. of 6 observations; +: indicates increase; -: indicates decrease;
NS: not significant.
Electrical stimulation and protein in cockroach nerve cord 409
Table 2. Effect of electrical stimulation on the total protein concentration (mg/g)
from the cockroach ventral nerve cord without exogenous glucose.
Total protein
Duration of
A Kc/\1iitj»
stimulation
Control
Experimental
difference
P value
5
21-05 ±1-85
20-11 ±1-02
- 094
NS
10
22-23 ±2-12
20-23 + 2-03
- 2-00
NS
15
30-72 ±2-92
21-98 ±1-65
- 8-74
<O001
30
20-56 ±1-99
9-89 ±010
-10-67
< 0-001
60
21-81 ±2-942
7-12 ±0-59
-14-69
< 0-001
Mean ± S.D. of 6 observations; - : indicates decrease; NS: not significant.
In nerve cords incubated free of exogenous glucose, stimulation showed a decreasing
trend in the total protein concentration significantly (p < 0-001) (table 2). The decrease
was consistently found at all intervals of stimulation.
3.2 Protein pattern
Densitometric scanning of the gels from unstimulated and stimulated ventral nerve
cords are shown in figure 1. The number of fractions resolved from the unstimulated
control group was eleven and from the stimulated group was nineteen. The control
group thus depicted only six high molecular weight protein fractions. But the
experimental group showed an increase by two fractions over the control group.
Similarly the low molecular weight protein fractions were only five in control groups
but the experimental group revealed an increase by six fractions. Thus the rise in total
protein concentration in the stimulated nerve cord may be due to the increase in both
the low and high molecular weight protein fractions.
4. Discussion
Electrophoretic and immunological studies have shown that brain contains several
proteins which presumably involved in specific neural functions (Bock 1978). Hence,
factors regulating protein synthesis are known to play an important role in the
functioning of the NS. The changes observed in the total protein concentration with and
without exogenous glucose in the present study suggests that glucose exhibits
considerable role as an energy source to meet the altered condition. Selvanayagam and
Habibulla (1979) report glucose as an energy source in the absence of adequate energy
supply in frog sciatic nerve.
The significant increase in total protein concentration in electrically stimulated
nerves with glucose suggests the increased rate of protein biosynthesis. The observed
increase in total protein concentration up to 1 5 min is in accordance with the findings of
Jones (1972) who has stated that the increase could be due to the electrical activity
causing localized increases in concentration of leucine or neutral amino acids which are
known to enhance the incorporation of amino acids into proteins. The decrease
410 S L Maheswari et al
observed after 15 min may be due to the induced hyperactivity for longer duration
resulting in protein catabolism leading to fatigue and exhaustion as suggested by
Selvanayagam and Habibulla (1979).
The polyacrylamide gel electrophoresis depicts a greater increase in the total number
of low molecular weight protein fractions. Such a change suggests the dissociation of
complex proteins into simple low molecular weight proteins to meet the stimulation
stress. Luxuro (1960) has put forth the view that a small fraction of nerve proteins may
split probably to the level of amino acids during the nerve activity. Maheswari (1983)
has also reported similar observation of dissociation of proteins in the nervous system
of Scylla serrata due to pesticide stress. The present investigation has clearly shown
alteration in proteins and is probably a geared mechanism to functional demand.
However, further investigation on protein change to stress condition is needed.
Acknowledgement
The authors thank Prof. Dr K Ramalingam for encouragement. Valuable assistance
was provided by Prof. Dr P Govindarajulu in densitometric scanning of gels. SLM
acknowledges the financial support of CSIR, New Delhi.
References
Bock E 1978 Nervous system specific proteins; J, Neurochem. 30 7-14
Chitre V S and Talwar G P 1963 Correlation of electrical activity of brain with metabolic parameters. Part III.
Pentose nucleic acid content of isolated cerebral cortex during various phases of electrical activity
following topical application of metrazol; Indian J. med. Res. 51 80-91
Chitre V S, Chopra S P and Talwar G P 1964 Changes in the ribonucleic acid content of the brain during
experimentally induced convulsions; J. Neurochem. 11 439-448
Edstrom A and Mattisson H 1972 Fast axonal transport in vitro in the sciatic system of the frog;
J. Neurochem. 19 205-221
Ganesan A, Murthy V A and Thiruneelakantan K 1979 Electrophoresis on polyacrylamide microgradient
gels in Proc. Natl Symp. on food proteins. Protein research unit Loyola College Madras 58-62 p.
Jones D A 1972 The relationship between amino acid incorporation into protein in isolated neocortex slices
and the tissue content of free amino acid; J. Neurochem. 19 779 — 790
Jones D A and Mcllwain H 1971 Amino acid distribution and incorporation into proteins in isolated
electrically stimulated cerebral tissues; J. Neurochem. 18 41-58
Kol's O R, Fedorov G E, Maksimov G V and Burlakova E V 1974 Change of ATP ase activity of the crab nerve
in relation to the stimulation regime; Dokl Akad. Nauk SSSR Ser. Biol. 216 943-945
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with the folin phenol
reagent; J. biol Chem. 193 265-275
Luxuro M 1960 Incorporation of amino acids labelled with carbon-14 in nerve proteins during activity and
recovery; Nature (London) 188 1119-1120
Maheswari S L 1983 Neurological manifestations ofphosalonet an organophosphate pesticide in the edible crab
Scylla serrata (Forskal) (Crustacea: Decapoda) Ph.D. Thesis University of Madras, India
Oliver D and Chalkley R 1971 An improved photographic system for polyacrylamide gels; Anal. Biochem. 44
540-542
Orchard I and Finlayson L H 1977 Electrical properties of identified neurosecretory cells in the stick insect;
Comp. Biochem. Physiol. A58 87-91
Selvanayagam P F L and Habibulla H 1979 Electrical stimulation effects on the levels of free amino acids and
proteins in peripheral nervous system of frog; Indian J. Exp. Biol. 17 300-302
Smith I 1968 Chromatographic and electrophoretic techniques (London: William Heineman Medical Book
Ltd) Vol II pp. 365-418.
Electrical stimulation and protein in cockroach nerve cord 41 1
Talwar G P, Sadasivudu B and Chitre V S 1961 Changes in the pentose-nucleic acid content of subcellular
fraction of the brain of the rat during 'MetrazoF convulsions; Nature (London) 191 1007-1008
Talwar G P, Chopra S P, Goel B K and Monte B D 1966 Correlation of the functional activity of the brain
with metabolic parameters-Ill. Protein metabolism of the occipital cortex in relation to light stimulus;
J. Neurochem. 13 109-116
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 4, August 1985, pp. 413-419.
© Printed in India.
Studies on the silk gland of Bombyx mori: A comparative analysis
during fifth instar development
S K SARANGI
R & D Programme in Sericulture, Bangalore University, Bangalore 560 009, India.
MS received 11 June 1984; revised 1 January 1985
Abstract. Middle and posterior parts of the silk gland of fifth instar bivoltine and
multivoltine races of Bombyx mori and their hybrid were analysed for the concentration of
fibroin, DNA, RNAand total protein. Fibroin content of the silk gland increased rapidly from the
beginning of fifth instar upto the spinning stage. Concentration of DNA in the middle silk gland
was maximum at 24 hr and decreased thereafter. In the posterior silk gland, the concentration
of DNA increased upto 72 hr and then decreased. RNA concentration was maximum at 72 hr and
120 hr, in the middle and posterior silk gland respectively. The total protein content increased
gradually upto the spinning stage in the middle silk gland whereas it increased upto 1 20 hr and
decreased sharply thereafter in the posterior silk gland. The difference in the concentration of
these constituents in the silk gland was correlated with the differential silk output in both the
pure races and their hybrid.
Keywords. Bombyx mori; bivoltine; multivoltine; hybrid; silk gland; fibroin; DNA; RNA; total
protein.
1. Introduction
Structural and functional aspects of the silk gland of silkworm Bombyx mori have
thoroughly been investigated (Dhavalikar 1962; Lucas 1966; Machida 1970; Tashiro
and Otsuki 1 970; Sasaki and Noda 1 973 a, b; Prudhomme et al 1 973; Tashiro et al 1 976).
Variations in the concentration of fibroin, DNA and RNA in the silk gland of several
strains of bivoltine race and their hybrids have also been reported (Shigematsu and
Takeshita 1968; Tashiro et al 1968; Shigematsu and Moriyama 1970; Moriuchi et al
1972; Shigematsu et al 1974). However, no information is available on the quantitative
variations in the silk gland of multivoltine race. Bivoltines are shown to produce more
quantity of silk compared to multivoltines (Tanaka 1964). It is therefore interesting to
undertake a comparative study on the quantitative analysis of the silk gland of both
bivoltine and multivoltine races of Bombyx mori and their hybrid to highlight the
influence of these variations on the differential silk output by the silkworm varieties.
The silk gland grows enormously during the fifth instar development of silkworm
(Sakaguchi 1978). The middle and posterior parts of the silk gland are known to
synthesize sericin and fibroin respectively (Machida 1927; Oba 1957; Shibukawa 1959).
Further it has been shown that fibroin is synthesized very rapidly during the fifth instar
development (Shimura et al 1955; Noguchi et al 1974) being associated with an increase
in DNA, RNA and total protein content of the posterior silk gland (Tashiro et al 1968).
The present communication deals with a comparative account of the middle and
posterior silk gland of the fifth instar bivoltine and multivoltine races of Bombyx mori
and their hybrid. A few important commercial characters are analysed in different
silkworm varieties to correlate with the variations in the silk gland.
413
414 S K Sarangi
2. Materials and methods
2.1 Silkworms
Bivoltine (NB18), multivoltine (Pure Mysore) races of Bombyx mori and their hybrid
(NB18 c? X PM ?) were maintained under standard laboratory conditions at a
temperature of 25-28°C and relative humidity of 75-90 % with good quality mulberry
leaves (M5 variety). Fifth instar larvae of average body weight were used at different
time intervals.
2.2 Analyses of the silk gland
The silk gland was dissected and washed with 0-9 % NaCl. The intraglandular fibroin
was extracted separately from the middle and posterior parts of the silk gland according
to the procedure of Tashiro et al (1968). After complete extraction of fibroin, the
extracts from both parts of the silk gland were pooled and weighed. The nucleic acid
was extracted from the middle and posterior silk gland separately and estimated
according to Schneider (1957). The concentration of DNA was determined by
diphenylamine method using calf thymus DNA as standard and the concentration of RNA
was determined by orcinol method using rat liver RNA as standard. The total protein
from the middle and posterior silk gland was extracted by the reduction of disulphide
bonds, according to the procedure of Gamo et al (1977) and estimated according to
Lowry et al (1951). Usually 4-5 larvae were used for each determination and average
value of four independent determinations was calculated.
2.3 Analyses of cocoon characters
Four to five lots of 10 cocoons each, were taken after harvesting from bivoltine,
multivoltine and the hybrid silkworm varieties for analysis of cocoon characters.
Important characters like cocoon weight, shell weight percentage, floss weight
percentage and filament length were determined. The shell weight percentage was
calculated as the ratio between the cocoon shell and the whole cocoon, while the floss
weight percentage was the ratio between the total floss and the whole cocoon. Mean
values of the lots with standard deviation are presented in table 1.
3. Results
As shown in figure 1, total fibroin content increased significantly (P = <0-01) from 24
hr to reach the maximum level prior to spinning in both the pure races. In hybrid male, a
significant increase (P = < 0-01) in fibroin content was observed between 72 brand 120
hr which remained more or less at the same level upto the spinning stage. On the other
hand, the fibroin content increased significantly upto 120 hr followed by a slight
increase thereafter in hybrid female. However, the concentration of fibroin at the final
stages of fifth instar was high in bivoltine and low in multivoltine, while hybrid showed
a sex difference in fibroin content being high in male and low in female.
Silk gland of B. mori
415
Table 1. Important cocoon characters of bivoltine, multivoltine and the hybrid silkworm
varieties
Silkworm
variety
Av. Cocoon
weight (g)
Mean SD
Shell weight
(%)
Mean SD
Floss weight
(%)
Mean SD
Av. filament
length (m)
Mean SD
NB18
(bivoltine)
Pure Mysore
(multivoltine)
Hybrid male
Hybrid female
1-28 ±0*06
0-88 ±0*04
1-08 ±0-06
0-94 ±0-06
14-35 ±0-80
9-59 ±1-02
12-02 ±1-05
11 -00 ±0-90
0-79 ±0-08
2-25 ±0-1 8
1-38 + O09
1-40±0-11
780 ±55
350 ±25
620 ±35
575 ±50
20
« N B 18
o Pure Mysore
m Hybrid mole
a Hybrid female
24
72 120
TIME (hours)
168
Figure L Increase in fibroin content of the silk gland (middle plus posterior) during fifth
instar development.
The concentration of DNA in the middle silk gland was maximum at 24 hr which
decreased gradually upto 120 hr and remained at a more or less constant level (figure
2A). The DNA concentration was high in bivoltine and both sexes of the hybrid while in
multivoltine it was significantly low (P = < 0-005). As shown in figure 2B, the
concentration of DNA in the posterior silk gland increased to reach the maximum level at
72 hr and decreased thereafter. However, DNA concentration was different in different
races being high in bivoltine and low in multivoltine. Further, at the final stages of fifth
instar the DNA content remained constant between 0*4 and 0-5 mg/g wet wt in both the
pure races and the hybrid.
RNA concentration increased upto 72 hr and then decreased to a more or less constant
level by 120 hr in the middle silk gland of bivoltine and hybrid male, whereas it was
maximum at the beginning of fifth instar (24 hr) and decreased thereafter in
multivoltine and hybrid female (figure 3A). The level of RNA in the middle silk gland
of bivoltine and both sexes of the hybrid was high compared to a significantly low
(P = < 0-001) level in multivoltine. The concentration of RNA in the posterior silk
gland increased gradually to reach the maximum at 120 hr and decreased significantly
416 S K Sarangi
06 -
I ! I I I I 1
72 120
TIME "( hours)
168
Figure 2. Change in DNA content of the middle (A) and posterior (B) silk gland during the
fifth instar.
(P = < 0-001) thereafter in bivoltine and hybrid male (figure 3B). But in multivoltine
and hybrid female, the RNA content was maximum at 72 hr and then decreased
gradually. The concentration of RNA in the posterior silk gland was significantly high
(P = <0-01) in bivoltine and hybrid male as compared to multivoltine and hybrid
female.
There was a significant increase (P = < 0*001) in the amount of total protein in the
middle silk gland from the beginning to the end of fifth instar (figure 4A). The final
concentration was about five times that at the beginning of the fifth instar. Further, a
significant difference in the concentration of total protein was observed in different
races, being high in bivoltine (160 mg/g wet wt) and low in multivoltine (112 mg/g wet
wt) prior to spinning. As shown in figure 4B, the concentration of total protein in the
posterior silk gland increased upto 120 hr, reached the maximum level and then
decreased significantly (P = < 0-005), unlike in the middle silk gland. Further, the
concentration of total protein in the posterior silk gland at 120 hr was significantly
different in the silkworm varieties studied being high in bivoltine and low in hybrid
female.
Table 1 shows the difference in a few important commercial characters of the cocoon
from different silkworm varieties. The average cocoon weight, shell weight percentage
and the average filament length were significantly high in case of bivoltine as compared
to multivoltine. But the hybrid showed an improvement of all these characters over the
Silk gland of B. man*
417
(A)
12
I
J_
24
72 120
TIME (hours)
168
Figure 3. Change in RNA content of the middle (A) and posterior (B) silk gland during the
fifth instar.
multivoltine parent. Further, a difference, though not significant, was observed between
both sexes of the hybrid.
4. Discussion
The amount of fibroin in the silk gland increases significantly from the beginning to the
end of fifth instar. This can be correlated with the high rate of fibroin synthesis in the
silk gland during fifth instar development (Shigematsu and Takeshita 1968; Tashiro et
al 1968; Noguchi et al 1974). A higher concentration of fibroin in the silk gland of
bivoltine might account for more quantity of silk produced as compared to
multivoltine. The concentration of fibroin, in the silk gland of hybrid male is more
concomitant with larger quantity of silk produced as compared to the female. However,
no significant sex difference in the concentration of fibroin is observed in pure races.
High level of DNA at the beginning of fifth instar shows that the synthetic activity
starts early in the middle silk gland. In contrast, there is a time-lag for the increase in
DNA content of the posterior silk gland suggesting that the fibroin synthesis starts a little
late during the fifth instar. Incorporation studies have also shown that the synthesis of
fibroin is significant only from 96 hr of the fifth instar development (Fukuda and
Florkin 1 959). This result, however, contradicts the earlier reports of Tashiro et al (1 968)
who reported no time-lag for the increase in DNA content of the posterior silk gland. The
difference in the DNA content of both middle and posterior silk gland accounts for the
418 S K Sarangi
150
100
50
0
60
30.
(A)
- (B)
24
72
TIME ( hours)
120
168
Figure 4. Change in total protein content of the middle (A) and posterior (B) silk gland
during the fifth instar.
difference in the degree of their synthetic activity. Further, the DNA content of the silk
gland of both the races is different showing that it is probably specific to the race.
The concentration of RNA in the middle silk gland of multivoltine is low concomitant
with a low DNA concentration. This reflects the lower rate of synthetic activity in the
middle silk gland of multivoltine compared to bivoltine and both sexes of the hybrid.
Maximum level of RNA at 72 hr and 1 20 hr in the posterior silk gland of multivoltine and
bivoltine respectively, suggests that the synthetic activity in the posterior silk gland
starts later than in the middle silk gland.
The initial increase in the total protein content of the middle silk gland might be due
to the synthesis and accumulation of sericin as suggested by an early increase in its DNA
and RNA contents. The rapid increase in the amount of protein after 72 hr, supports the
view that there is an inflow of fibroin to the middle silk gland for storage (Fukuda and
Florkin 1959; Game et al 1911 \ Sakaguchi 1978). The significant decrease in total
protein content of the posterior silk gland after 120 hr is in accordance with earlier
reports (Tashiro et al 1968). This suggests that the outflow of fibroin from posterior silk
gland is more rapid during the later part of the fifth instar.
Thus it is evident that the concentration of fibroin, DNA, RNA and total protein in the
middle and posterior silk gland is different in both the pure races and that the hybrid
shows an increase in the concentration of these constituents over the multivoltine
parent. The final silk output, however, is directly correlated with the concentration of
these constituents in the silk gland of the silkworm varieties studied.
Silk gland of B. mori 419
Acknowledgement
Grateful acknowledgements are due to the World Bank for financial assistance and to
the Co-ordinator, R & D Programme in Sericulture, Bangalore University for facilities.
The author thanks Mr P M Chandrasekhar for technical help.
References
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Ind. Res. C21 303-304
Fukuda T and Florkin M 1959 Contributions to silkworm biochemistry, (VII) ordered progression of
fibrinogen in the reservoir of the silk gland during the fifth instar. Arch. Int. Physiol. Biochem. 67 214-222
Gamo T, Inokuchi T and Laufer H 1977 Polypeptides of fibroin and sericin secreted from the different
sections of the silk gland in Bombyx mori; Insect. Biochem. 1 285-295
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with folin phenol reagent;
J. Biol Chem. 193 265-275
Lucas F 1966 Cystine content of silk fibroin (Bombyx mori) Nature (London). 210 952-953
Machida J 1927 On the secretion of the silk substance in the silkworm (Bombyx mori L.); J. Coll. Agric. Imp.
Univ. Tokyo 9 119-138
Machida Y 1970 Studies on the silk glands of silkworms Bombyx mori L; Fukuoka Women's junior coll.
Studies. 3 1-21 (not referred to in original)
Moriuchi A, Koga K, Yamada T and Akune S 1972 DNA synthesis and activities of pyrimidine synthesizing
enzymes in the silk gland of Bombyx mori; J. Insect Physiol. 18 1463-1476
Noguchi A, Takeshita H and Shigematsu H 1 974 Interrelationship between the silk gland and other tissues in
protein metabolism in the latest larval stage of the silkworm, Bombyx mori; J. Insect Physiol. 20 783-794
Oba H 1957 Studies on the secretion and the current of liquid silk during spinning period in the silkworm
Bombyx mori L; Bull. Nagano Sericult. Exp. Station 10 9-25
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fibroin of Bombyx mori L; Arch. Biochem. Biophys. 159 97-104
Sakaguchi B 1978 In: The Silkworm: An important laboratory tool, (ed.) Y Tazima (Tokyo: Kodansha Ltd) pp.
31-51
Sasaki T and Noda H 1973a Studies on silk fibroin of Bombyx mori directly extracted from the silk gland;
Biochim. Biophys. Acta 310 76-90
Sasaki T and Noda H 1973b Studies on silk fibroin of Bombyx mori directly extracted from the silk gland;
Biochim. Biophys. Acta 310 91-103
Schneider W C 1957 Determination of nucleic acids in tissues by pentose analysis; Methods Enzymol. 3
680-4584
Shibukawa A 1959 Studies on the silk substance within the silk gland of the silkworm, Bombyx mori L; Bull
Sericult. Exp. Sta. Tokyo. 15 383-401
Shigematsu H and Moriyama H 1970 Effect of ecdysterone on fibroin synthesis- in the posterior division of
the silk gland of the silkworm, Bombyx mori; J. Insect Physiol 16 2015-2022
Shigematsu H and Takeshita H 1968 Formation of silk proteins by the silkworm, Bombyx mori, after
gamma ray irradiation in the embryonic stage; J. Insect Physiol. 14 1013-1024
Shigematsu H, Moriyama H and Arai N 1 974 Growth and silk formation of silkworm larvae influenced by
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Shimura K, Koide F, Itabashi H and Fukai H 1955 Protein synthesis in excised silk gland of silkworm; J.
Biochem. 42 285-294
Tanaka Y 1964 In: Sericology. V. Silkworm races (Bombay: Central Silk Board) pp. 99-104
Tashiro Y and Otsuki E 1970 Studies on the posterior silk gland of the silkworm Bombyx mori; J. Cell. Biol.
46 1-16
Tashiro Y, Morimoto T, Matsumura S and Nagata S 1968 Studies on the posterior silk gland of the silkworm,
Bombyx mori;J. Cell. Biol. 38 574-587
Tashiro Y, Matsumura S and Hata K 1,976 Ultrastructural observations on the organogenesis of the
posterior silk gland of the silkworm, Bombyx mori; J. Insect Physiol. 22 273-283
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 4, August 1985, pp. 421-426.
© Printed in India.
Studies on mating, spawning and development of egg in
Macrobrachium nobilii (Henderson and Matfaai)
C BALASUNDARAM and A K KUMARAGURU*
School of Biological Sciences, * School of Energy, Environment and Natural Resources,
Madurai Kamaraj University, Madurai 625021, India.
MS received 14 September 1984; revised 2 April 1985
Abstract. The chronomorphological events during the development of the egg of the
freshwater caridean prawn, Macrobrachium nobilii, were followed from spawning to hatching.
The female is receptive only for a 30-minute period after the premating moult. After mating,
spawning ensued within 9 ± 3 hours and was completed within 15-20 seconds. Unmated
females also spawned but the eggs did not survive. The rate of egg development increased 2-5*
for a temperature rise of 10°C. Hatching was synchronised under in vitro conditions indicating
uniform development of a clutch.
Keywords. Macrobrachium nobilii', mating; spawning; egg development.
I. Introduction
Macrobrachium nobilii, a caridean freshwater prawn moults and breeds once in 1 9 days.
In carideans, there is a premating moult before spawning. A freshly moulted female
remains receptive to a male for only a limited period (Ling 1964). If a male is not
available during the receptive period, then fertilization of a brood will be missed and
such a brood will be lost. Hence, it is important for culture purpose to know the
receptive period. Therefore, studies were carried out on the sexual receptiveness and
mating of M . nobilii.
Like most other decapods, M. nobilii incubates the developing eggs in the ovigerous
setae of the pleopods till hatching. However, incubation by the mother is less
productive in terms of larvae for culture needs (Balasundaram 1980). To circumvent the
disadvantages associated with incubation, the eggs can be hatched under artificial
conditions, simulating the ventilation technique of the mother (Balasundaram and
Pandian 1981). An average female (39 ± 5 mm : total length from tip of rostrum to
telson) incubates about 2,200 eggs/clutch. For mass incubation, eggs from more than
one female are needed. Incubation of egg masses of identical age will enable
simultaneous hatching. The percentage of hatching is influenced by the age of egg mass
at the time of relieving (Balasundaram and Pandian 1981).
Hence, it is advantageous to procure egg masses of identical age for mass incubation
under in vitro conditions. Therefore, an attempt has been made in this study to know
the age of eggs based on simple chronomorphological features of the developing egg
from 0 hour to hatching.
In general, temperature is known to influence egg development of decapods (Wear
1974). This indicates that the time required for development can be accelerated by
421
422 C Balasundaram and A K Kumaraguru
controlling the temperature. Hence, the effect of temperature on development of egg
and hatching also was studied.
2. Material and methods
Healthy individuals of M. nobilii were collected from the river Cauvery at Tiruchi
(10°5'N; 78°43'E). They were maintained in 90 litre/laboratory tanks at a density of
1 male to 7 females. The water temperature was 28 ± 2°C (mean ± SD). The pH and
dissolved oxygen content were 7-5 ± 1 and 6 ± 1 ml/1 respectively. The photo-period
was 14L: 10D. The prawns were fed once daily, ad libitum, with a mixture of beef, goat
liver and boiled Bengal gram (Cicer arietinum). Water was changed once a day. The
female about to moult and spawn was identified by its fully developed dark green
ovaries beneath the transparent carapace extending upto the third rostral spine
anteriorly and into the first abdominal somite posteriorly (Balasundaram 1980). Such
females were transferred into circular troughs at a density of 1 male to 1 female and
observed for moulting and spawning.
Sexual receptivity of the female was studied by introducing several freshly moulted
individuals in separate troughs. Males were released individually into each one of these
troughs at different time intervals (5, 10, 15, ... minutes) after moult, till sexual
attractiveness ceased to exist when the prawns ignored each other.
Two different techniques were tried to obtain eggs from the mother, for in vitro
incubation. In the first technique, 6 of the freshly moulted and mated females were
selected. Each one was gently stretched and enveloped with a flexible aluminium wire
mesh (0-5 cm mesh) around the body. This prevented the animal from bending its
abdomen to form the brood chamber. However, the enclosure was loose enough to
allow appendage movements. This arrangement enabled the eggs, on release, to pass
through the wire mesh to be collected at the bottom of the trough. The collected eggs
were washed with sterile water and transferred to incubating chambers. The incubating
chambers were 100 ml conical flasks with 50 ml water disinfected by boiling. Each
incubating chamber contained 10 eggs. The flasks were then covered loosely with
cotton plugs to avoid atmospheric contamination.
The second technique involved the removal after 3 hours of berrying of small egg
masses (25 ± 5 eggs/mass) from a clutch. These small masses were then carefully teased
manually with the help of needles into individual eggs and incubated as in the previous
method.
A batch of 6 berried females were allowed to incubate their eggs normally. Eggs were
removed from these females and examined under a light microscope once an hour. The
morphological stages attained by these eggs were compared with those of similar age
incubated in vitro.
To study the effect of temperature on egg development and hatching, samples of egg
mass from freshly berried females (3 hour old), reared at room temperature of 28
± 2°C, were teased individually and transferred into disinfected water in incubation
chambers as described above. The chambers were kept at 19, 22, 25, 29, 32 and 34
± 0-5°C at normal photoperiod (14L: 10D). Six replicates were maintained at each
temperature till the eggs hatched. The water in the chambers was carefully decanted and
fresh disinfected water at the same temperature was added everyday.
Studies on M. nobilii 423
3. Results and discussion
3.1 Mating and spawning
When a male was introduced into a trough containing a freshly moulted female with
ripe ovaries, the male quickly rushed to the female, turned it over and mated. In less
than 5 minutes the female, lying between the maxillipeds of the male, started moving
freely and kept away from the male darting backwards even on chance encounters.
Spawning occurred within 9 ± 3 hr after mating and lasted for a period of 15-20
seconds. The eggs on release were fertilized externally and passed into the brood
chamber. The pleopods secreted a glue-like substance into which the eggs were
enveloped and finally kept attached to the ovigerous setae of the pleopods as observed
by Ling (1969) in M. rosenbergii.
The female after undertaking the premating moult, remained receptive only for 30
minutes. After this period when a male was introduced, they ignored each other. In M.
rosenbergii such attractiveness persisted for 3 hours (Ling 1964) and Palaemonetes
was receptive only for 20 minutes (Burkenroad 1947).
Females which were not mated also spawned and got berried as usual but these
unfertilized eggs turned golden yellow owing to; cytolysis and were lost subsequently.
Such females with golden egg mass have also been collected from the field (3 out of 456
females over a period of one year). These eggs when examined under a light microscope
were similar to the unfertilized eggs obtained from unmated females in the laboratory.
This clearly reveals the importance of the male availability at the right time for mating
to ensure fertilisation of the eggs.
Table 1 . Macrobrachium nobilii'. Chronomorphological features of the developing egg from
0 hour to hatching
Time:
Hour Identification features
0 Egg-oval in shape and measures 570 /i long and 437 /i wide. Uniformly granulated.
7 Cleavage commences as furrows at 4 equidistant points — 8-celled stage.
14 Many hexagonal cells are seen — Egg pale green in colour.
25 Egg appears olive green in the centre due to the presence of large yolk cells and pale green
ectodermal cells in the periphery (see also Anderson 1973)
35 White opaque blastoderm appears
60 Blastoderm extends anteriorly
80 Blastoderm occupies 8-3 % of the egg's surface area
120 Blastoderm occupies 16-7% of the egg's surface area
160 Blastoderm occupies 36-7% of the egg's surface area
170 Heart beat begins— Eyes appear as crescentic streaks
205 Crescentic eye becomes oval in shape — Carapace appears
220 Length of egg increases from 570 to 608^ — Breadth from 437 to 491 fj.
240 Cornea formed — Mouth parts twitch occasionally
250 Setae of exopodite extend beyond carapace posteriorly
270 The outer and inner flagellae of antennule, their aesthetes and setae are seen distinctly — larva
twitches often and tries to stretch out abdomenr— Hatching imminent.
424
C Balasundaram and A K Kumaraguru
3.2 Egg development
The eggs on release were fertilized externally and passed into the brood chamber. The
pleopods secreted a glue-like substance into which the eggs were enveloped and kept
attached to the ovigerous setae. Ling (1969) made similar observations in M.
rosenbergii. Eggs removed from the pleopods within the first 3 hours of berrying
developed abnormally. This timing appears to vary in different species. For example in
Homarus it takes at least 29 hours before the eggs can be removed for normal
development to proceed (Templeman 1940). Patel and Crisp (1960) too observed that
initial development stages are highly sensitive and develop abnormally when incubated
in vitro.
The chronomorphological events occurring in the developing egg of M. nobilii
incubated at 29 -f 0-5° C is summarised in table 1. The segmentation of the egg was
completed on the first day and the blastoderm appeared on the second day. On the
seventh day the eyes appeared and heart beat began (figure Ib). The size of the egg
TEISQN
»HQRA:IC AWE NDAC* s
COMPOUND
Figure 1. Macrobrachium nobilii: Chronomorphological features of the developing egg.
Studies on M. nobilii
425
increased l-6x on the tenth day (figure Id). Such volumetric expansion of eggs due to
imbibition of water has been reported by Pandian (1984) for several species of decapods
and range from 1*2* in Palirurus gammarus (Berry 1971) to 5-4x in- Petrolisthes
elongatus (Greenwood 1965). On the 12th day the egg hatched as zoea (figure If).
3.3 Effect of temperature
The developmental stages attained by the eggs of M. nobilii at 22, 25, 29 and 32°C were
observed. The eggs required 333, 300, 270 and 173 hours respectively to complete
development to hatch (table 2). At any chosen temperature once the egg development is
completed, hatching of simultaneously incubated eggs lasts for a period of 6 hours
irrespective of the time of the day. The regression lines fitted by the method of least
squares bear a linear relationship between the stages attained and time taken for
development (figure 2). However, the percentage hatchability did not vary significantly
and ranged from 73-78 % (P < 0-05) (table 2). At 19 and 34°C the eggs failed to develop
and suffered total mortality due to cytolysis within 9 ± 3 hours of incubation.
16
CO
13
10
220
100 200
TIME (HOURS)
300
Figure 2. Macrobrachium nobilii: Effect of temperature on egg development.
Table 2. Effect of temperature (±0-5°C) on incub-
ation period and hatchability of eggs of
Macrobrachium nobilii.
Temperature
Time
Hatchability
(°Q
Hour
(%)
22
333 ±6-6
73±9-l
25
300±7-1
77 ±5-2
29
270 ±6-9
78 ±6-4
32
173 ±6-6
75 ±8-3
Each value (mean ± SD) is based on 6 observations
426 C Balasundaram and A K Kumar aguru
The development of egg at the lowest temperature (22°C) was the least. The rate
increased 2-5x times for a 10°C rise in temperature. This is comparable to the period
required for the development of several species of barnacles (Patel and Crisp 1960).
Under in vitro conditions all the eggs of a single batch incubated at any particular
temperature hatched simultaneously. This indicates the synchronous nature of the
development of all the eggs. The rate of egg development when incubated by the mother
(29 ± 1°C) was also uniform as in. amphipods (Fish 1975), Cirrepeds (Patel and Crisp
1960) and decapods (Bensam and Kartha 1967).
The present study indicates that whether incubated by the mother in situ or under in
vitro conditions, all the eggs of a brood commence development at the same time. They
are at the same stage of development at any given time during incubation. However,
when incubated by the mother the eggs are hatched in intermittent batches
(Balasundaram and Pandian 1981).
Information on the chronomorphological events during the development will help to
procure egg masses of identical age so that hatching can be synchronised. At room
temperature (28 ± 2°C) the eggs take 12 days to hatch whereas at 32°C they need only 8
days. This indicates that hatching can be timed to suit the need for larvae.
References
Anderson D T 1973 Embryology and phylogeny in annelids and arthropods; Int. Ser. Monogr. in Pure and
Appi Biol. 50 263-364
Balasundaram C 1980 Ecophysiological studies in prawn culture (Macrobrachium nobilii), Ph.D. thesis,
Madurai Kamaraj University, Madurai, 123 pp.
Balasundaram C and Pandian T J 1981 In vitro culture of Macrobrachium eggs; Hydrobiology 77 203-207
Bensam P and Kartha K N 1967 Notes on the eggs and early larval stages of H ippolysmata ensirostris Kemp;
Proc. Symp. Crustacea. Mar. Biol Ass. India, Part II, 736-743
Berry P F 1971 The biology of spiny lobster Panulirus homarus (Lin.) off the coast of Southern Africa; Investl.
Rep. Oceanogr. Res. Inst., Durban. 28 1-75
Burkenroad M D 1947 Reproductive activities of decapod Crustacea; Am. Nat. 81 392-398
Fish J D 1975 Development, hatching and brood size in Bathyporeia pilosa and E. pelagica (Crustacea:
Amphipoda); J. Mar. Biol. Ass. U.K., 55 357-368
Greenwood J G 1965 The larval development of Petrolisthes elongatus (H. Milne-Edwards) and
P. novazelandiae Filhol (Anomura: Porcellanidae) with notes on breeding; Crustaceana 8 285-307
Ling S W 1964 A general account on the biology of the giant prawn Macrobrachium rosenbergii and methods
for its rearing and culturing. Paper presented at IPFC llth session, Contrib. paper no. 40
Ling S W 1969 The general biology and development of Macrobrachium rosenbergii (de Man); FAO Fisheries
Report, 57 589-606
Pandian T T 1970 Ecophysiological studies on developing eggs and embryos of the European lobster
Homarus americanus; Mar. Biol 5 153-157
Pandian T J 1984 Propagation and reproductive strategy of crustaceans. In Reproductive biology of
invertebrates, (eds) K G Adiyodi and R G Adiyodi Vol. 6 (in press), (Chichester: John Wiley)
Patel B and Crisp D J 1960 Rates of development of embryos of several species of barnacles; Physiol Zool 33
104-119
Templeman W 1940 Embryonic developmental rates and egg laying of Canadian lobsters; J. Fish Res. Bd.
Can. 5 71-83
Wear R G 1974 Incubation in British decapod Crustacea and the effects of temperature on the rate and success
of embryonic development; /. Mar. Biol Ass. U.K. 54 745-762
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No, 4, August 1985, pp. 427-433.
© Printed in India.
Effect of castration and androgen treatment on the androgen dependent
parameters in the accessory glands of the slender loris, loris
Tardigradus lydekkerianus (Cabra).
A MANJULA* and K M KADAM
Department of Zoology, Bangalore University, Bangalore 560 056, India.
* Karnataka State Sericulture Development Institute, Thalaghattapura, Bangalore 560 062,
India.
MS received 29 November 1984; revised 26 March 1985
Abstract. The accessory glands of reproduction in slender loris exhibit reduction in secretory
activity following castration for 15 days and 30 days. Administration of androgens
(testosterone — propionate and 5a dihydrotestosterone) to castrated animals had a differen-
tial effect in maintaining the biochemical parameters like citric acid and fructose.
Keywords. Loris Tardigradus lydekkerianus; slender loris; castration; androgen treatment.
1. Introduction
Of the several secretions of the male sex accessory glands of mammals, fructose and
citric acid are important and are found in large quantities in the semen. Their synthesis
and secretion are entirely regulated by the androgens secreted by the testis (Price and
William Ashman 1961; Prasad et al 1973a,b). Amongst the non-human primates,
besides the monkey (Dinakar et al 1974a,b) not much information about the prosimian
primates is available. The present investigation deals with the secretions and their
androgen control in the accessory sex glands of the male slender loris, Loris tardigradus
lydekkerianus (cabra).
2. Materials and Methods
Lorises, also known by the native names as ceylon sloths, sherminds, unhappen luma
and thevangu, occur in some of the forested areas of Southern India and Ceylon from
sea level to an elevation of about 1800 inches. Lorises are found mostly in casurina,
tamarind and pongamia trees, quite close to rural habitation. They were collected from
the forests around Bangalore, brought to the laboratory, maintained in cages and
provided with food and water.
Adult male lorises used for the experimental groups were divided into nine groups of
3/5 animals in each (table 1). They were castrated by opening the inguinal passage under
asceptic conditions using sodium pentobarbitone (Nembutol, Abbot laboratories) as
anesthetic. Through a small incision in the inguinal passage, the testis were exposed,
carefully freed from the epididymis after ligating the efferent ductules and testicular
blood vessels without damaging the vascular supply to the epididymis and ductus
deferens. The different doses of testosterone propionate (TP) used were 125 /zg, 250 ^g
427
428 A Manjula and K M Kadam
Table 1. Changes in the weights (mg) of the accessory glands in the castrated,
testosterone propionate (TP), and 5a-dihydro testosterone (5a-DHT) treated
looses (Mean±S.E.)
Body
weight
Treatment (g) Seminal vesicles Prostate gland Cowper's glands
Intact control
279(5)
293-4 ± 6-3
75-4 ± 14-9
104-0 ± 3-2
$ for 15 days
252(3)
1954 ±9-Of.
55-2 ± 7-Oc
85-4 ± 12-4C
$ for 30 days
260(3)
192-1 ±4-8*
53-3 ± 3-6*
83-9 ±11-1*
$ +500^gof IP/day
241(5)
315-0±38-46
137-0 ± 9-0*
122-7 ± 9-2b
$ +250 ^g of IP/day
283(5)
393-0 ± 48-Oc
71-7 ± 3-5"
174-1 ± 3-1*
$ + 125/igofTP/day
259(5)
292-2 ± 89-7a
70-7 ± 19-3fl
114-5 ±13-7"
$ + 250/xgof DHT/day
285(5)
299-1 ±25-6*
71-3 ±10-5"
117-0 ±18-0*
$ + 50 n% of DHT/day
284(5)
286-0 ± 20-4a
56-5 ± 6-4
116-0 ±21-5fl
$ + 5 ^g of DHT/day
272(5)
216-6 ± 10-4
58-9 ± 3-8
74-3 ± 7-9
Figures in parentheses represent the number of animals used in each group. Levels of
significance compared with intact control animals. °P< 0-05; bP < 0-01; CP< 0-05. oT= Castrated.
and 500 ^g/day and of 5a- dihydrotestosterone (5a-DHT) were 5 ^g, 50 //g and
250/ig/day. The androgens were administered subcutaneously in 0-2 ml of olive oil
daily for 15 days from the next day of castration. Intact and castrated looses of
comparable body weights received the vehicle only and they served as controls.
The lorises were autopsied 24 hr after the last injection; one castrated group without
androgen treatment was autopsied 30 days after castration. The accessory glands were
removed, cleaned of fat and connective tissue and weighed to the nearest 0-1 mg in a
torsion balance; fructose was estimated in the accessory glands by the method of Roe
(1934) as modified by Under and Mann (1960) and citric acid by the method of Ettinger
et al (1952). The results were analysed statistically using student's t test.
3. Results
Response of the accessory glands of reproduction in the lorises to the administration of
different dosages of xp/5a-DHTin terms of change in weight is shown in table 2. Changes
in- the content and concentration of fructose in the accessory glands resulting from
castration and androgen replacement are shown in table 3. In loris, fructose is secreted
by the prostate gland in maximum quantities. The seminal vesicles and Cowper's glands
also secrete some quantities of the fructose. Castration for 15 days resulted in
significant decrease of fructose concentration in the accessory glands and a further
decrease was observed in 30-day castrated animals. More than 500 /*g of xpor 5 /zg of
5a-DHT/day maintained the fructose concentration in the seminal vesicles but there was
a three-fold increase in the fructose concentration in the animals treated with 50 /ig of
5a-DHT. Cowper's gland required 250 /xg of Tpor more than 50 /*g of 5a-DHT for the
maintenance of normal fructose concentration and in the prostate gland, it required
250 fig of TP/5a-DHT/day.
Changes in the levels of citric acid in the accessory glands of loris resulting from
castration and androgen replacement is shown in table 3. Citric acid is mainly secreted
Castration and androgen treatment in slender loris
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Castration and androgen treatment in slender ton's 43 1
by the seminal vesicles in maximum quantities. Like fructose, citric acid in the accessory
glands also depends on the androgens. Prostate and Cowper's glands also contribute
citric acid to the seminal plasma in the slender loris. In the 1 5-day castrated animals the
concentration of citric acid had decreased significantly and it was completely absent
from the glands of 30-day castrated animals. Requirement of androgens for the
maintenance of citric acid concentration by the different glands varied. 500 ^g of Tpor
250 /^g of 5a-DHT maintained the citric acid concentration in the seminal vesicles while
more than 500 jug of TP or more than 250 jug of 5a-DHT was required for the
maintenance of the citric acid concentration in the prostate and Cowper's glands.
4. Discussion
A decrease in the weights and secretory activity of the male reproductive system of
slender loris has been observed following castration (Manjula and Kadam 1980) for
15 days. These tissues showed a further significant decrease in weight after 30 days of
castration. A similar decrease has been observed in laboratory rodents (Price and
William Ashman 1961; Mann 1964; Gupta et al 1974), hamster (Ortiz 1953; Karkun et
al 1974), and rhesus monkey (Dinakar et al 1974a,b).
Fructose has been found in the semen of man, monkey, ram, guinea pig, rat and other
mammals (Mann 1964). Androgen is necessary for the production of fructose (Mann
1964; Gassner and Hopwood 1952) in the male reproductive system. Fructose is
secreted mainly by the prostate gland in the slender loris, but the seminal vesicles and
Cowper's gland also contribute. In the castrated guinea pig and rats (Harold and Clara
1955a,b) a lower dosage of testosterone (0-5/xg) was sufficient to restore the fructose
content. In the accessory glands of mouse, fructose level had decreased significantly
following castration for 3 days (Mawhinney et al 1970). In the castrated loris, a higher
dose of androgens, 500 /^g of TP and 250 jug of SCX-DHT, was required by the prostate and
Cowper's glands to restore the secretory activity in 15-day castrated animals. The
seminal vesicles required 250 jug of TP and a lower dose of 5a-DHT (5 ^g). Thus 5a-DHT
appears to be more potent than Tpin restoring and maintaining the secretory activity of
the accessory glands. Similarly in the rhesus monkey, the seminal vesicles required four
implants of 5a-DHT and eight implants of testosterone (Dinakar et al 1974a,b). The
content of fructose in dorsolateral prostate was maintained at control levels with
500 /^g of TP or 250 /ig of 5a-DHT in rats (Gupta et al 1 974). In loris, as in monkey and rat,
5a-DHT is more potent, whereas in hamster (Karkun et al 1974) testosterone appears to
be more potent. This shows that testosterone and SOC-DHT were not equipotent in
stimulating the production of fructose in the slender loris.
Citric acid is also an androgen-dependent parameter in the accessory glands. Most of
the higher mammals (Barron and Huggins 1946a,b; Humphrey and Mann 1948, 1949;
Schersten 1929, 1936), have a high concentration of citric acid in semen. A direct
relationship exists between the plasma testosterone and citric acid in seminal plasma in
man and it is a good index of androgen secreted by the gonads (Dondero et al 1972).
Decrease in the levels of citric acid in the seminal vesicles of rabbit following
castration has been observed (Humphrey and Mann 1948) and it reappears following
administration of testosterone. In the accessory glands of loris, castration for 15 days
resulted in a significant decrease in the levels of citric acid and it was completely absent
432 A Manjula and K M Kadam
in the seminal vesicles of 30-day castrated animals, prostate and Cowper's glands also
showed significant reduction in the citric acid content. In the castrated loris, seminal
vesicles required a low dosage of 5a-DHT (50 /zg) and a high dosage of TP (500 /zg) for the
maintenance of citric acid content. The prostate and Cowper's glands required more
than 500 /*g of TP or 250 jug of SOC-DHT, of both the androgens, 5a-DHT is more potent in
low doses than TP in maintaining citric acid content in the seminal vesicles thus
indicating greater sensitiveness to low doses of 5a-DHT, than to testosterone itself.
Androstendione and 5/J-androsten 3-01-1 7-one have been found in the seminal
vesicles after administration of testosterone (Harding and Samules 1962; Kinson 1962;
Evalkar et al 1964) and 5a-androstane 17-01-3-one is found bound to the macromole-
cular fraction of the homogenate of the seminal vesicles in the ventral prostate of the rat
(Unjem and Tveter 1969). In the slender loris the results indicate that 5a-DHT is more
potent than TP and hence it is possible that similar DHT binding sites to macromolecules
are present in the accessory glands of loris also.
Acknowledgement
One of the authors (AM) is grateful to the UGC, New Delhi, for the award of a
fellowship.
References
Barren E S G and Huggins C 1 946a The citric acid and acqnitase content of the prostate; Proc. Soc. Exp. Biol.
Med. 63 85-87
Barron E S G and Huggins C 1946b The Metabolism of the prostate transamination and citric acid; J. Urol
55 385-390
Dinakar N, Renu A and Prasad M R N 1 974a Effects of microquantities of testosterone on the epididymis
and accessory glands of castrated rhesus monkey, Macaca Mulatta; J; Endocrinoi 60 399-408
Dinakar N, Arora R and Prasad M R N 1 974b Effects of micro quantities of 5a-dihydrotestosterone on the
epididymis and accessory glands of the castrated rhesus monkey, Macaca mulatta', Int. J. Fen. 19
133-139
Dondero F, Sciarra F and Isidori A 1972 Evaluation of relationships between plasma testosterone and
human seminal citric acid; Fen. Ster. 23 168-171
Ettinger R H, Goldbaum L H and Smith L H 1952 A simplified photometric method for the estimation of
citric acid in biological fluids; J. Biol. Chem. 199 531-536
Evalker K O, Mosebach O and Dirscherl W 1964 Acta Endocr. Kopen. 45 437
Gassner F X and Hopwood M C 1952 Seminal amino acid and carbohydrate pattern of bulls with normal
and abnormal testis function; Proc. Soc. Exp. Biol. Med. 81 37-43
Gupta G, Rajalakshmi M and Prasad M R N 1974 Regional differences in androgen threshold of the
epididymis of the castrated rat; Steroids 24 575-586
Harold A L and Clara M S 1955a The effect of androgens on fructose production by the sex accessories of
male guinea pig and rat; Endocrinology 59 404-412
Harold A L and Clara M S 1955b Effects of castration and androgen administration on metabolic
characteristics of the guinea pig seminal vesicles; Am. J. Physiol. 183 371-376
Harding B W and Samules L T 1962 The uptake and sub cellular distribution of C1 ^labelled steroid in rat
ventral prostrate following in vivo administration of C1 ^testosterone; Endocrinology 70 109-118
Humphrey G F and Mann T 1948 Citric acid in Semen. Nature (London) 161 352-353
Humphrey G F and Mann T 1949 Studies in the metabolism of semen: Citric acid in semen; Biochem. J. 44
97-105
Karkun T B, Rajalakshmi M and Prasad M R N 1974 Maintenance of the epididymis in the castrated golden
hamster by testosterone and dihydrotestosterone; Contraception 9 417
Castration and androgen treatment in slender loris 433
Kinson G 1962 Fed. Proc. 21 24
Under H R and Mann T 1960 A relationship between the content of androgenic steroids in the testis and
secretory activity of the seminal vesicles of bull; J. Endocrinol. 21 341-360
Manjula A and Kadam K M 1980 Effect of testosterone propionate and 5a-dihydrotestosterone on
epididymis and accessory glands of the castrated slender loris. Loris tardigradus lydekkerianus (cabra);
Indian J. Exp. Biol. 18 466-^70
Mann T 1964 Biochemistry of semen and of the male reproductive trat (London: Methuen)
Mawhinney M G, Knytck J R E T and Thomas J A 1970 Relationship of fructose and fructose phosphate
esters in the accessory sex organs of the mouse; J. Endocrinol. 46 545-546
Ortiz F 1953 The effects of castration on the reproductive system of the golden hamster; Anat. Rec. Ill
65-93
Prasad M R N, Rajalakshmi M, Gupta G and Karkun T 1973a Control of epididymal function; J. Reprod.
Fert.Suppl. 18 215-222
Prasad M R N, Rajalakshmi M, Gupta G, Dinakar N, Arora A and Karkun T 1973b Epididymal
environment and maturation of spermatozoa. Presented at the Int. Conf. for the study of fertility and
sterility in the male, Milan, Italy
Price D and William Ashman H G 1961 The accessory reproductive glands of mammals. In: Sex and internal
secretions eds. (Baltimore: W C Young Williams and Wilkins Co.) Vol. 1, pp. 366, III ed.
Roe J H 1934 A colorimetric method for the determination of fructose in blood and urine; J. Biol. Chem. 107
15-22
Schertsen B Skandinav 1929 Arch. Physiol. 58 90
Schertsen B Skandinav 1936 Arch. Physiol. 74 90
Unjem O and Tveter K U 1969 Preliminary characterisation of an androgen macromolecular complex from
the rat ventral prostate; Acta Endocrinol. Copenh. 62 153-154
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 4, August 1985, pp. 435-441.
© Printed in India.
Community and succession of the round-head borers (Coieoptera:
Cerambycidae) infesting the felled logs of White Dhup, Canarium
euphyllum Kurz
T N KHAN
Zoological Survey of India, Andaman & Nicobar Regional Station, Port Blair 744 102, India
* Present address: Department of Zoology, Darjeeling Government College, Darjeeling
734 101, India
MS received 28 September 1984; revised 20 April 1985
Abstract. Succession and assemblage of the round-head borers infesting Canarium eu-
phyllum Kurz have been described. The borer pests have been categorized into two major
groups. The first group includes the borers of standing trees and freshly felled logs, while the
second constitutes the species infesting the dead logs after some seasoning. Even among the
borers belonging to each of these groups, there is a clear sequence of succession of species
infesting the logs depending upon the period lapse after felling and subsequent conditions of
the host.
Keywords. Canarium euphyllum Kurz; Cerambycidae; community and ecological succession;
overlapping of species
1. Introduction
It is well known that various stages of a gradually disintegrating log or tree harbour
particular groups of insects, some in the living or dying parts, some in the recently dead
material, others in the moderately dry wood and still others in the wood which has
seasoned for several years (Shelford 1913; Adams 1915). Round-head borers are
generally recognized as the 'second group of invaders', preceded by the bark- and
ambrosia-beetles (Scolytidae and Platypodidae), infesting the host-material when it is
still green (Howden and Vogt 1951; Sen-Sarma 1983; Maiti et al 1983).
However, the round-head borers themselves exhibit a clear succession of species in
infesting the wood, depending upon its progressive drying and decaying due to many
physico-chemical and biological factors (Khan 1984), The present communication
summarizes the succession and assemblage of the round-head borers infesting the logs
of different period lapse after felling of an economically important timber yielding
plant, Canarium euphyllum Kurz.
2. Materials and methods
The work was carried out in several localities of Andaman and Nicobar islands (latitude
6°40' to 13°41' N and longitude 92°11' to 94°10/E) from 1978 to 1981. Observations
were made in different forest areas, timber extraction and logging centres, timber
depots, wood-based industries, etc. The frequency of infestation of different round-
head borers attacking the standing trees and felled logs of C. euphyllum was regularly
435
436 T N Khan
recorded. The period lapse after the felling of the logs was also recorded, as could be
ascertained from the authorities of the Forest Department. Population density of
different stages of borers per log or tree was determined by random samplings from
unit-area of 25 cm x 25 cm.
In all these localities, several batches of trees of C. euphyllum (girth varying from
1*38 m to 2*87 m) were felled and were cut into billets of uniform size. The number of
adult beetles active on each of these togs and the number of females observed
ovipositing thereon on the subsequent days were recorded. For each day, the egg-
nitches made by females on each logs were also counted and were marked with flags.
The infested logs were dissected every week till the entire development of the invader
species was completed. The population density of the developmental stages was also
determined. The number of emerged adults of different species was determined from
the emergence-hole counts.
Insectary studies were conducted at Port Blair using portions of the infested logs
collected from both the extensive and intensive study areas. They were kept in spacious
galvanized iron cages (1m x 50cm x 50cm) and were examined each day between
1000 and 1100 hours IST, prior to which the newly emerged adults were collected.
Insectary studies permitted observation of the characteristic features of larval galleries,
pupal chambers and exit-holes, duration of development and periods of adult
emergence. These were finally correlated with those collected from the field.
The immature stages of different species were identified following Khan and Maiti
(1983).
3. Results
3.1 Categories of the round-head borers and their succession
The round-head borers assembled in standing trees and felled logs of C. euphyllum are
listed in table 1. It is worthy of mention that this tree is the most preferred host of
round-head borers and harbours nearly 10% of the total cerambycid-fauna of these
islands. About 36 % of these borers attack unhealthy or dying trees in the forest stands.
These species are Plocaederus obesus Gahan, Pharsalia (Cycos) subgemmata
(Thomson), Acalolepta rusticator (Fabricius) and Olenecamptus bilobus (Fabricius).
Table 1. Grouping of round-head borers on the basis of their infestation to standing trees and
logs of different period lapse after felling.
Group-I Group-II
Infesting living trees and freshly felled logs Infesting recently dead trees and logs after
of 15 to 40 days after felling 40 to 80 days of felling
Plocaederus obesus Gahan, Epepeotes sp,, Clyzomedus annularis Pascoe,
Pharsalia (Cycos) subgemmata (Thomson), Coptops rufa Thomson,
Acalolepta andamanica (Breuning), Macrochenus atkinsoni Gahan and
Acalolepta rusticator (Fabricius), Marmaroglypha nicobarica Redtenbacher
Batocera rufomaculata var. andamana Thomson,
Olenecamptus bilobus (Fabricius)
Community and succession of Cerambycidae in White Dhup
437
On the basis of the community composition on the living trees and logs of different
period lapse after felling, Khan (1984) categorized the round-head borers of these
islands into four major groups. In the present study, two of these groups have been
recognized to infest the living trees and felled logs of C. euphyllum (table 1).
Figure 1 (a,b) presents the succession and assemblage of the round-head borers
belonging to each of these two groups. It clearly shows that most of the borers
belonging to group-I infest the logs within the period from about two weeks to two
months of felling and their populations in the host attain a maximum density within
this period. After that, the population declines gradually as a result of the completion of
development and mortality due to parasites, predators, competitors, diseases and other
environmental factors. The representatives of group-II, on the other hand, attack the
logs after about five weeks of felling. Their infestation continues upto about 3| months
of felling (figure la).
3.2 Succession and assemblage of species
Further observations suggest that even among the borers belonging to each of the two
groups, there is a clear sequence of succession of species infesting the logs depending
~100
[ I GROUP -I
IBB GROUP-II
i
i
MONTHS AFTER FELLING
Figure 1. Succession and assemblage of round-head borers belonging to different groups in
the logs after different period lapse of felling; (a) infestation and continuation of existence;
(b) frequency distribution.
438
T N Khan
upon the period lapse after felling and subsequent conditions. A brief account of this
phenomenon is furnished below.
3.2a Group-!: The data on succession and assemblage of the species constituting
this group are presented in figure 2 (a,b). Figure 2a presents the cumulative number of
eggs and early instar larvae harboured per unit-area of the bark and wood-surface of
the logs. Acalolepta rusticator has been recognized as the first cerambycid to infest the
logs within 15 to 30 days of felling, while Plocaederus obesus is the last one invading
after 30 to 45 days. Other species, viz Olenecamptus bilobus, Acalolepta andamanica,
Epepeotes sp., Pharsalia (Cycos) subgemmata and Batocera rufomaculata var andamana
infest the logs in succession between the two extremities (figure 2a).
Almost all the species in general exhibit a definite period of infestation. Amongst
them Plocaederus obesus has the longest period of existence inside the wood, while
Acalolepta rusticator and Epepeotes sp. have the shortest life cycle. Other species
continue their destructive activities inside the wood for a varying period from 5 to 8
months (figure 2b).
3579
MOAfTWS AFTCI? FCLLING
Figure 2. Successive infestation (a) and distribution and existence (b) of round-head borers
belonging to group-I; 1. Acalolepta rusticator, 2. Olenecamptus bilobus, 3. Acalolepta
andamanica, 4. Epepeotes sp, 5. Pharsalia (Cycos) subgemmata, 6. Batocera rufomaculata var
andamana and 7. Plocaederus obesus.
Community and succession of Cerambycidae in White Dhup
439
Figure 3. Successive infestation (a) and distribution and existence (b) of round-head
borers belonging to group-II; 1. Macrochenus atkinsoni 2. Marmaroglypha nicobarica
3. Clyzomedus annularis and 4. Coptops rufa.
3.2b Group-II: Figure 3 (a,b) shows the succession and assemblage of the borers
constituting Group-II. All these species exhibit a similar pattern of succession as shown
by those of the group-I. However, in this group the sequence of succession of species is
more pronounced. Macrochenus atkinsoni is the first invader of the group (infesting
after 40 to 55 days of felling), followed by Marmaroglypha nicobarica (stepping in after
45 to 60 days). Coptops rufa, on the other hand, is the last invading the logs of more than
two months old, being preceeded by Clyzomedus annularis (figure 3a).
Amongst these borers, Macrochenus atkinsoni and Coptops rufa exist inside the wood
for a longer period of more than 9 months (figure 3b). Clyzomedus annularis is the least
survivor (survival period 6 months).
4. Discussion
The results clearly reveal that the cerambycid-community colonizing in the felled logs
of C. euphyllum is rather poor, and this is expected in islands which support
depauperate faunal elements. Moreover, the period between felling, extraction and
storage is too short to permit development of climax community. The logs situated
inside forests, normally harbour a higher number of species with higher population
densities than in those located in non-forested areas for the same duration.
440 T N Khan
On the other hand, the existence of different round-head borers almost simul-
taneously appears to be influenced by the microhabitat differences among themselves.
Thus, Rhaphipodus (s. str.) andamanicus is dominant in and around the pith, Batocera
rufomaculata var andamana inside comparatively upper layers of the heartwood,
Pharsalia (Cycos) subgemmata inside deeper layers of the sapwood and outer
heartwood, Acalolepta andamanica inside the sapwood, while Coptops rufa is restricted
to inside the bark. Such co-existence is obviously due to availability of different food-
material in different zones of the wood. Thus, a bark or superficial wood borer feeds
mostly on the soluble sugars and simple carbohydrates, while a deep heartwood borer
feeds on the cellulose, as pointed out by Beeson and Bhatia (1939).
With regard to the succession, Acalolepta rusticator is the first to invade, while
Coptops rufa is the terminal one. The invasion of these two species is bridged up by
succession of other invaders. However, an overlapping of their infestations is not very
uncommon. Such overlapping is more pronounced among the species constituting the
group-I than in those belonging to the group-II. The higher frequency of overlapping
among the members of the group-I may, at least in part, be attributed to the higher
degree of competition for suitable oviposition-sites as should normally be expected
from a higher number of species.
Lastly, it is clear that round-head borers colonizing in the felled logs of Canarium
euphyllum utilize dying or recently dead green host for oviposition, but complete their
development at a period when the wood becomes comparatively drier. The advanced
developmental stages are, thus, associated with the exploiting efficiencies of drier wood
for their nourishment. It, therefore, appears that these borers requiring fresh tissues for
their early immature stages, can continue their growth and development in the drier
wood in the later phases of their life indicating an increase in their digestive power with
the progressive age (Graham 1925).
Acknowledgements
The author is grateful to the Chief Conservator of Forests, Andaman and Nicobar
Islands, for providing trees and felled logs for study. The work was financed by the
Department of Science and Technology, Government of India. The author is also
thankful to CSIR for the award of a fellowship.
References
Adams C C 1915 An ecological study of prairie and forest invertebrates; Bull Illinois Lab. Nat. Hist. 11
33-280
Beeson C F C and Bhatia B M 1939 On the biology of the Cerambycidae (Coleoptera); Indian Forest Rec.
(N.S.) Ent. 5 1-235
Graham S A 1925 The felled tree trunk as an ecological unit; Ecology 6 397-411
Howden H F and Vogt G B 1951 Insect communities of dead pine (Pinus virginiana Mill.); Ann. Ent. Soc. Am.
44 581-595
Khan T N 1984 Studies on some cerambycid beetles (Coleoptera) of Andaman and Nicobar Islands, India, Ph.D.
Thesis, Calcutta University
Khan T N and Maiti P K 1983 Studies on the biotaxonomy, biology and ecology of some longicorn beetle
borers (Coleoptera: Cerambycidae) of the islands of Andaman, India; Rec. zoo/. Surv. India. Occ. Paper,
No. 45 1-100
Community and succession of Cerambycidae in White Dhup 441
Maiti P K, Nandi B, Chakraborty S K and Saha N 1983 Bioecological observations on the community of the
xylophagous insects infesting the felled logs of 4Papita\ Pterocymbium tinctorium, In Insect interactions in
forest and agroecosystems, (eds) P K Sen-Sarma, Sangal and Kulshreshtha pp. 79-87
Sen-Sarma P K 1983 Host as an environment in the ecological succession of insect borers in freshly felled tree
trunks; Proc. Symp. Host Environ. (Zooi. Surv. India) pp 109-115
Shelford V E 191 3 Animal communities in temperate America as illustrated in the Chicago Region: A study in
animal ecology (Chicago: Univ. Press) 367 pp
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 443-461.
© Printed in India.
Behavioural responses in terms of feeding and reproduction in some
grasshoppers (Orthoptera: Insecta)
T N ANANTHAKRISHNAN, K DHILEEPAN and B PADMANABAN
Entomology Research Institute, Loyola College, Madras 600 034, India
MS received 2 January 1985; revised 4 March 1985
Abstract. Studies on the host preference in some acridids revealed sequentiality of feeding
behaviour aided by various sensillae, the suitability of the host plant greatly depending upon
the physical and chemical factors. Studies on acridids and pyrgomorphid species indicated that
Truxalis indica feed exclusively on grasses, Orthacris maindroni only on dicot plants and
Atractomorpha crenulata on both monocot and dicot plants. Significant variations in the
quantitative food intake on diverse host plants during post-embryonic development were
also evident. Distribution and density of diverse sensillae on the antennae, labrum, maxillary
and labial palps as well as their role in food selection are documented. The impact of antennal
and palpal ablation on host selection, the quantity of food intake as well as the influence of
diverse host plants on fecundity are discussed.
Keywords. Quantitative food utilization; sensillae; degree of preference; post embryonic
development; fecundity; grasshoppers.
1. Introduction
Food selection in * polyphagous insects is known to involve the cumulative
effects of physical, chemical and sensory factors which act independently or simul-
taneously. Short-term feeding behavioural effects on host selection appears to be
influenced by the secondary plant chemicals acting as phagostimulants and/or
deterrents and long-term physiological effects are known to involve nutrition and
antibiosis (Chapman and Bernays 1977), The role of sensillae in the maxillary and labial
palps in food selection was suggested by Blaney and Chapman (1969) and Bernays and
Chapman (1970, 1973), their numerical variations among acridids feeding on grami-
naceous and broad-leaved plants reflecting their role in food selection (Chapman and
Thomas 1978). Available information on feeding and food selection of essentially grami-
nivorous acridids relates to specific aspects concerning short term behavioural effects
pertaining to quantitative intake and utilization. Long term physiological effects
involving growth and reproduction on diverse host plants as well as the role of sensillae
ip food discrimination/avoidance, especially in dicot feeding grasshoppers, appear
meagre. For a further understanding of the food selection of grasshoppers, an attempt
has been made to study the feeding and behavioural dynamics involving feeding and
reproduction of the polyphagous pest species, Atractomorpha crenulata (Fabricius),
Orthacris maindroni Boliver (Pyrgomorphidae) and Truxalis indica Boliver (Acrididae).
2. Materials and methods
A. crenulata^ 0. maindroni and T. indica were collected from the fields in and around
Madras, reared in cages and provided with their respective host plants for
443
444 T N Ananthakrishnan, K Dhileepan and B Padmanaban
feeding. Nymphal stages were isolated and reared individually in separate cages as
well as in glass chimneys. In order to find out the exact day of moulting, the pterothorax
of the nymphs was marked with cutex. Mating pairs were isolated from the cages and
provided with transparent plastic vials (8x12 cm) filled with loose wet soil for
oviposition. The number of egg pods laid in the vials could be easily counted through
the transparent wall of the vials. The plastic vials containing egg pods were covered with
thin muslin to prevent the escape of the emerging nymphs. The emerging nymphs
were counted and reared individually in different host plants. Increase in the weight of
the nymphal stages while feeding on different host plants was recorded through the use
of a monopan balance.
To calculate the quantitative food intake and food utilization, fresh host plants were
weighed initially and kept in a flask containing water and 15 adults/nymphs were
allowed to feed for 24 hr, another set of host plants of similar weight were kept as
control simultaneously to find out the quantitative food intake by the insects. After
24 hr, both the experimental as well as the control host plants were weighed. In order to
assess the loss of weight in experimental leaves due to the feeding damage, both the
control and experimental leaves were desiccated for 7 days. The difference in the dry
weight between the control and experimental plants indicated the quantity of food
intake/number of insects. Excreta of the experimental insects were collected in
polyethylene papers during 24 hr of experimental period and weighed immediately as
well as after complete desiccation for 7 days. The difference in the quantity of food
intake and the quantity of excreta indicated the quantitative food utilization for specific
host plants.
For light microscopic observations, the labrum, labium, maxillae and antennae of the
grasshoppers were dissected in insect ringer, washed with distilled water, incubated in
1 % boiling KOH for 5 min, washed well in distilled water and mounted in glycerine.
For obtaining scanning electron microscope pictures, the labrum, labium, maxilla and
antenna were washed well in 80 % ethanol, dried and fixed on aluminium stubs using a
double adhesive tape and coated with gold using an ion coater. The coated materials
were examined in a Hitachi SEM and photographed using an attached Mamia camera.
For quantitative estimations of proteins, carbohydrates, total lipids and phenols, the
standard methods of Lowry et al (1951), Dubois et al (1956), Kok (1971) and Bray and
Thorpe (1954) respectively were followed.
3. Results
3.1 Role of sensillae in feeding behaviour
3. la Sensillae of the inner side of the labrum: Studies by Chapman and Thomas
(1978) on the distribution of sensillae on the inner side of labrum indicated the
occurrence of diverse sensillae arranged in the 'alpha' and 'beta' tract in addition to the
A-l to A-3 sensillae. Further studies on the distribution of sensillae of A. crenulata, a
dicot feeder and the T. indica, a monocot feeder indicated further variation in the
distribution and the nature of sensillae. Hence as a convenient measure the sensillae of
the innerside of the clypeo-labrum was further differentiated as SI and S2 of the inner
side of the distal margin of clypeus. In addition to the alpha and beta tracts of the
labrum another tract was also distinguished, referred to here as gamma tract, with a
Feeding and reproduction in grasshoppers
445
higher number of sensillae. All the sensillae of the gamma tract appeared to be of the
campaniform type arranged in a 4V shaped fashion. A-l sensillae were not noticed both
in light microscopic as well as in SEM studies. Further recognition of the sensillae of the
alpha tract was made into the campaniform alpha-B sensilke (S-4a) and (S-4b) sensillae.
In the beta tract all the sensillae appeared large, consisting of campaniform sensillae
(S-5) and trichoid sensillae (S-6). In the free distal margins, another group of sensillae
designated as S-7 was also recognised. In the inner side of the labrum, A-2 and A-3
sensillae alone were noticed (figure 8).
In T. indica, a similar distribution of sensillae was evident with the wide beta tract
covering the entire distal margin of the labrum. The type S-7 sensillae were
unrecognisable and numerical differences in the sensillae were also evident as compared
to that of the other species studied (figure 8).
3.1b Sensillae of maxillary and labial palps: Studies by Haskell and Schoonhoven
(1969) and Blaney (1974, 1975) indicated the presence of trichoid sensillae which are
mechanical receptors as well as another group of chemosensory trichoid sensillae.
During the post-embryonic development of A. crenulata from the nymphs to the
adults, a gradual increase in the number of sensillae at the palpal apices as well as on the
surfaces of both maxillary and labial palps was observed (figures 1 and 2). A bottom
collar is characteristic of the short trichoid apical sensillae, occurring at the apices of
both maxillary and labial palps, the entire surface of the maxillary and labial palps
bearing a number of trichoid and campaniform sensillae (figure 7).
1000
900
800
700-
b 60°"
I
9 500
t
j 400-
n
j
u
n
300-
200-
100-
O TOTAL NUMBER OF SENSILLAE
-300
-260
-220
I- 180
•140 d
in
z
-100
-60
III
Figure 1. Numerical increase of sensillae on the inner side of the labrum of A crenulata
during post-embryonic development.
446
T N Ananthakrishnan, K Dhileepan and B Padmanaban
80
60-
< 50
30
20
D LABIAL PALP APEX
• MAXILLARY PALP SURFACE
C MAXILLARY PALP APEX
• LABIAL PALP SURFACE
__
1NSTARS
Figure 2. Numerical increase of sensillae on the maxillary and labial palps of A. crenulata
during post-embryonic development.
3.1c Numerical increase of sensillae during post-embryonic development: A
consistent increase in the total number of sensillae of the labrum was evident during
post-embryonic development of A. crenulata (figure 2), the rate of increase of each type
of sensillae varying considerably. A significant increase in the sensillae types S-4b, S-6
and S-3 to the extent of about 3-4 times was observed, while others like S-2, S-4a, S-7,
S-5, A-2; A-3 and S-l showed a gradual increase as they reached the adult stage. S-2
sensiilae appeared to be numerically identical being constant during the first and
second nymphal stages, increasing in numbers during the third, retaining the same
number in the fourth and increasing again in the fifth stage nymphs.
Similarly the number of sensillae at the apices of the maxillary and labial palps also
increased during post-embryonic development, with the trichoid sensillae on both
palps being 22-27 and 24-30 in the first nymphs and 34 and 38 in the second nymphs
respectively. Further increase in the number of sensillae was evident in the other stage
also bearing 47 and 55, 57 and 72, 67 and 77 respectively in the maxillary and labial
palps of 3rd, 4th and 5th instar nymphs (figure 2).
3. Id Influence of sensory structures in the ^crimination of host plants: The
antennae, maxillary and labial palps, labrum and hypopharynx are known to play a key
role in food selection, aided by visual host location. To the control individuals, when
nine host plants were offered simultaneously, the quantitative food intake in terms of
dry weight consumed was found to be more in Ricinus communis L. and Dolichos lablab
L. and, Prosopis spicigera L and Calotropis gigantea R. Br were not consumed. One
sideantennectomised individuals showed a similar feeding range, but with a substantial
reduction in the quantity of food intake. Total antennectomy resulted -in the
quantitative food intake becoming very much reduced. Palpectomy involving labial
and maxillary palps individually or together reduced the feeding range to three plants
Feeding and reproduction in grasshoppers 447
when nine plants were offered simultaneously, with the quantity of food intake being
reduced. Labrectomy had no influence on the feeding range, except for a substantial
decrease in food intake.
3.2 Host range
Atractomorpha crenulata is a recognised cosmopolitan polyphagous pest species
feeding on diverse crop plants. Both the adults and nymphs feed on a variety of crop as
well as weed plants belonging to both dicot and monocot plants. Screening of diverse
host plants in and around the habitat of A. crenulata showed their ability to feed on the
members of family Euphorbiaceae, Solanaceae, Fabaceae, Poaceae, Amarantaceae and
Asclepidaceae, their incidence being very high (more frequent) on the dicot plants
especially by the nymphal stages. Screening of these host plants for various nymphal
instars showed that they preferred Euphorbiaceae, Fabaceae, Amarantaceae and other
dicot plants like Clerodendrum sp. However their incidence was low on monocots
especially on the members of the family Poaceae. Though A. crenulata is on record as
occurring on the members of the families Asclepiadaceae, Cucurbitaceae, Verbanaceae
they avoided these plants for feeding. The present observations indicate that crop
plants like Ricinus communis L., Solanum melongena L., S. torvum Sw., Dolichos lablab
L., Panicum maximum Jacq., Clerodendrum sp., and Achyranthes aspera L., form the
major food plants for the growth of A. crenulata. Though the nymphs have a wide host
range, jR. communis, D. lablab and A. hypogaea appear to be the preferred host plants.
The pyrgomorphid, Orthacris maindroni, in particular their nymphs fed exclusively
on dicot plants like JR. communis, D. lablab, S. melongena, S. trilobatum, C. gigantea,
Clerodendrum sp. and P. spicigera exhibiting a preference within their range of host
plants, to R. communis and weeds like Clerodendrum sp. They are polyphagous, feeding
on the members of the families of Euphorbiaceae, Fabaceae, Solanaceae, Asclepidaceae,
Vernoniaceae, Bignoniceae and Mimoseae and their occurrence on C. gigantea
appeared only more casual.
Truxalis indica feeds exclusively on monocots especially on Cynodon dactylon Pers.
(Poaceae), Cyperus rotundus L. (Cyperaceae), Carex sp.
3.3 Host preference and food plant selection
Host preference of A. crenulata on the basis of quantitative food intake indicated that
when seven preferred host plants viz R. communis, S. melongena, D. lablab, P. maximum,
S. trilobatum and Clerodendrum sp were provided, quantitative food intake was more in
R. communis, D. lablab and S. melongena. When the same host pldnts were offered
individually, quantitative food intake was high among JR. communis, S. melongena and
D. lablab, and comparatively low in the other food plants. Simultaneous offering of the
three host plants showed increased preference to R. communis and D. lablab, the former
being more preferred than the latter.
Similar variation in the quantitative food intake of different nymphs on either host
plants was also evident, with the first instar nymphs tending to feed on six out of eight
host plants offered, indicating a distinct preference towards R. communis than to
D. lablab. But the second stage nymphs showed a preference for all the six hosts offered
448
T N Ananthakrishnan, K Dhileepan and B Padmanaban
with a higher quantitative food intake in respect of R. communis and S. melongena. The
third instar nymphs showed an increase in the host range, feeding on seven of eight
hosts offered with a higher, but equal amount of food utilization on both S. melongena
and R. communis, but lesser in D. lablab. Both fourth and fifth instar nymphs fed on all
the eight host plants provided, a higher quantity of food intake being evident on /?.
communis, and very much less on S. trilobatum and Oryza saliva L (figure 3).
Individuals oiOrthacris maindroni feed on a wide spectrum of dicot plants, exhibiting
specific preference towards R. communis and to a lesser extent on D. lablab than the
other host plants. Though they feed only on dicot plants, under experimental
conditions it was possible to make them feed on monocot plants like P. maximum.
T. indica, a monocot feeder showed a specific preference to C. dactylon and to lesser
extent to other grasses and never fed on dicot plants. Though the adults of 0. maindroni
preferred R. communis, all the nymphal stages preferred to feed more on Clerodendrum
sp, the adult showing a reduced preference.
3.4 Quantitative food utilization
In addition to the amount of food intake, the quantity of food utilized and the quantity
of water excreted varied considerably while feeding on different host plants. When eight
host plants were offered to A. crenulata individually, the amount of food consumed by
15 adults, as expressed in the form of dry weight was very high in R. communis
(1,240 ing/ 15 adults), comparatively lower in D. lablab (780 mg/15 adults) and very low
900
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838 R.COMMUN1S
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1NSTARS
Figure 3. Quantitative food utilization by nymphal stages of A. crenulata.
Feeding and reproduction in grasshoppers 449
in the case of S. melongena, S. trilobatum, Clerodendurm sp. and P. maximum
(170-300 mg/15 adults). However the dry weight of food excreted was very high when
fed on R.communis (430 mg/15 adults), as compared to (100-1 3 5 mg/15 adults) on
other host plants. The amount of water consumed was also high (3,140 mg/15 adults)
on R. communis, comparatively lower in D. lablab (1,040 mg/15 adults), and very low
(286-1-664-6 mg/15 adults) in other host plants. Similarly water assimilation was also
high for both jR. communis (3- 1 2 mg/15 adults) and D. lablab (1-01 mg/15 adults) than
on other host plants (table 1).
Similar variations in the quantity of food utilization of diverse host plants were also
noticed in 0. maindroni, where the quantity of food consumed was high for
R. communis (190 mg/15 adults) than on other host plants. When the same host plants
were offered individually to O. maindroni, intake was very high in D. lablab
(550 mg/15 adults) and comparatively lower (80-330 mg/15 adults) on other host
plants like S. melongena, S. trilobatum, P. maximum, C. gigantea, Clerodendrum sp and
Bougainvilla sp. The dry weight of excreta was very high (160-175 mg/15 adults)
when fed on D. lablab, P. maximum and C. gigantea, and comparatively lower
(60-135 mg/15 adults) on S. melongena, S. trilobatum, R. communis, Clerodendrum sp
and Bougainvilla sp. Highest dry weight was assimilated when fed on D. lablab
(375 mg/15 adults), comparatively lower on Clerodendrum sp and R. communis with
195 and 138 mg/15 adults respectively. In the other host plants it was considerably
lower. Correspondingly the amount of water assimilated was equally high in
C.gigantea (1,441 mg/15 adults), D. lablab (1,3 10 mg/15 adults) and R.communis
(1,010 mg/15 adults) and in other plants it was considerably lower (table 2).
When a wide range of host plants were offered simultaneously to Truxalis indica
there was a specific preference towards Carex sp (360 mg/5 adults), Commelina sp
(200 mg/5 adults). However under continuous starvation they tend to feed on other
dicot plants like R. communis.
3.5 Life cycle
A. crenulata being a polyphagous species, the different host plants seem to influence
fecundity and the rate of post-embryonic development. Of the seven preferred host
plants provided as nymphal diet, the development was quicker on both R. communis
and A. hypogaea. On Clerodendrum sp, S. torvum and on P. maximum the duration of
development was comparatively longer, whereas no development was observed when
reared on 0. sativa (Var. I.R. 50) (figure 4, table 3).
The rate of mortality of different nymphal instars as well as the number of nymphs
attaining adult stage differ considerably on various host plants. Nymphal mortality in
all host plants tested was very high in the first nymphal stage and was lower in the
second with no mortality in the other stages. A very low nymphal mortality and a higher
rate of adult emergence were evident in JR. communis and A. hypogaea, whereas a very
high nymphal mortality and low adult emergence were observed when reared on
Clerodendrum sp and P. maximum. Interestingly mortality was 100% even in the first
instar itself when reared on 0. sativa (figure 5).
450 T N Ananthakrishnan, K Dhileepan and B Padmanaban
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Figure 4. Developmental duration of A crenulata when fed on different hosts.
3.6 Weight increase during the post-embryonic development
In addition to normal variations in the developmental rates as well as percentage of
mortality of the nymphs when reared on different host plants there were considerable
variation in the increase in the weight of the nymphs when reared on different hosts.
The first instar nymphs when developed on R. communis showed a higher weight
increase than on other hosts. From third instar nymphs onwards the weight increase
became more pronounced in female nymphs than in male nymphs. Adults emerging
from those nymphs fed on R. communis showed an increase in weight ranging from
75-90 mg in males and 220-250 mg in females, and a similar but comparatively lower
weight increase was noticed on A. hypogaea with the weight increase of 168-260 mg in
females and 65-70 mg in males respectively. Weight increase during the post-
embryonic development was very low when reared on P. maximum where the emerging
adults showed a very low weight, ranging from 60-65 mg and 1 10-115 mg in males and
females respectively. On the other host plants the weight increase was moderate,
ranging from 60-85 mg in males and 120-210 mg in the females (figure 6).
The first nymphal stage also differed considerably when developed on different host
plants. Weight increase on both R. communis and S. trilobatum was higher (20-25 mg),
and comparatively lower on A. hypogaea (15-20 mg). When developed on other host
plants like P. maximum, Clerodendrum sp and S. torvum the weight increase was very
low, ranging from 12-20 mg. Similarly the second stage nymphs also showed a higher
weight increase when reared on R. communis and A. hypogaea (30-55 mg). A very low
weight increase was observed on S. torvum (20-30 mg). In other host plants like
P. maximum, S. trilobatum and Clerodendrum sp the weight increase was intermediate
Feeding and reproduction in grasshoppers
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I II III IV V ADULT
Figure 5. Survival rate of A. crenulqta on different host plants.
ranging from 30-50 mg. The third instar nymphs reared on hosts like R. communis and
A. hypogaea showed a higher rate of weight increase with 25-40 mg/nymphs (figure 6).
3.7 Impact of diverse host plants on fecundity
In many of the phytophagous insects, it is well known that the quality of food has a
great impact on their fecundity and fertility. Many of the acridids, though
polyphagous without showing any discrimination towards specific host plant, also
indicated a significant variation in the number of egg pods laid as well as in the total
number of nymphs that emerged. The present observations based on laboratory studies
showed that the nature of host plants greatly influenced the fecundity and fertility. The
number of pods laid was easily recognizable as they were laid close to the wall of the
transparent plastic vial. Since it was not possible to count the number of eggs in each
pod, the number of young ones emerging from each pod was taken into account. The
highest number of egg pods (6/female) was laid when fed on R. communis and
A. hypogaea, whereas the lowest number of egg pods (3/female) was laid when fed on
P. maximum. Similarly the total number of nymphs emerging from all the pods was
high (182-210/female) on R. communis and A. hypogaea, whereas it was low
(96/female) on P. maximum. A moderate fecundity (120-137/female) was evident on
S. trilobatum and S. torvum.
In Orthacris maindroni, no significant variation was apparent in the incubation
period of eggs when fed on different host plants indicating that they did not influence
much on it. However the number of egg pods laid by each female in its life time varied
significantly. The highest number of pods (3/female) was laid on Clerodendrum sp and a
Feeding and reproduction in grasshoppers
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INCREASE IN TOTAL BODY WEIGHT DURING POST -
EMBRYONIC DEVELOPMENT OF ATRACTOMORPHA CRENULATA
WHEN FED ON DIFFERENT HOSTS
Figure 6. Increase in total body weight during post-embryonic development of A. crenulata
when fed on different hosts.
lesser number of pods (2/female) on both R. communis and S. trilobatum, and only one
pod was laid when fed on D. lablab. However no eggs were laid when fed with
P. maximum. The total number of nymphs emerging was high and more or less equal
(51-54/female) when fed on Clerodendrum sp and S. trilobatum and comparatively
lesser (41-43/female) on R. communis and D. lablab.
3.8 Biochemical parameters of host plants
In order to assess the impact of various biochemical parameters of the host plants on
the host preference of the grasshoppers, the following biochemical estimations were
made (table 4).
456 T N Ananthakrishnan, K Dhileepan and B Padmanaban
Total phenols as expressed in mg/g dry weight (DW) of host tissue was very high
(55 mg/g DW) in D. lablab, and comparatively low (12-3-40 mg/g DW) in other host
plants. Total protein content was very high in C. gigantea (259.3 mg/g DW) and
significantly lower in the rest of the host plants. Clerodendrum sp., showed a higher level
of carbohydrates (425 9 mg/g DW), comparatively lower in R. communis and
S. trilobatum (200-225 mg/g DW) and very low in the other host plants. A very high lipid
content was recorded in R. communis (53 mg/g DW) and lower amounts in the other
host plants ranging from 10*0-51-5 mg/g DW. Carbohydrate/protein ratio was greater
in C. gigantea (1 : 20-75), comparatively lesser in A. hypogaea, D. lablab, P. maximum,
P. spicigera and S. torvum (1 : 2-65-1 : 6-06) and very low in Clerodendrum sp.,
R. communis and S. trilobatum (1 : 0-19-1 :0-57) (table 4).
4. Discussion
Studies on the distribution and abundance of diverse sensillae on the labrum, maxillary
and labial palps, in A. crenulata (dicot-feeder), 0. maindroni (feeding on both dicot and
monocot plants) and T. indica (monocot feeder) indicated significant numerical and
distributional variations, besides recognition of further types of sensillae in the inner
side of the labrum. In all the three species discussed, A-l sensillae were not evident, but
with the 'beta' tract further divisible into the 'beta* and 'gamma' tracts. In each tract
further sub-types were recognised. As many as seven types (S-l to S-7) were evident in
labrum itself. Such a recognition of sensillae was based only on their morphology, size
and distribution and no functional significance is evident so far. Haskell and
Schoonhoven (1969), Cook (1972), Chapman and Thomas (1978) and Chapman (1982)
however showed the chemoreceptory nature of the A-l, A-2 and A-3 sensillae and
suggested others probably as mechanoreceptors.
The recognition of two types of sensillae, type-A and type-B, on the surface of the
maxillary and labial palps follows similar report by Abushama (1968) in Poecilocerus
hieroglyphicus (Klug). But later studies by Blaney (1974, 1975) and Haskell and
Schoonhoven (1969) suggested the mechanical and chemosensory nature of these
sensillae, which are known to increase as they grow into adults. Chapman and Thomas
(1978) noted that the total number of sensillae increased by 250 % for a 10-fold increase
in the surface area of the labrum. In addition to S-l to S-7 sensillae all the other A-2 and
A-3 sensillae also increased in number during post-embryonic development, a fact not
reported earlier. As no prior report exists regarding the increase of sensillae in the
maxillary and labial palps, the present observation showed an increase in the number of
sensillae during post-embryonic development varying for each type of sensillae. Trends
of an increase in sensilla number were recognised only from 3rd nymphs onwards;
such an increase can be correlated with the increase in the quantitative food intake as
well as the range of preference for their host plants. However, the role of various
sensillae located at various regions of the mouth parts in host plant selection still
remains unclear. Though Blaney and Chapman (1969) and Bernays and Chapman
(1970) indicated the role of various sensillae in food plant selection, the removal of
labrum, maxillary palps and labial palps individually or in combination did not narrow
down the host plant spectrum. However there appears to be a definite role by these
organs on the quantitative food intake, since their removal significantly reduced the
food intake. Such a reduction in the food intake is also possibly due to the stress caused
Feeding and reproduction in grasshoppers
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T N Ananthakrishnan, K Dhileepan and B Padmanaban
Figure 7. A-D. Sensillae at the palpal apices of late instars (D) portion of (B) enlarged;
E, F. collared sensillae.
by their removal of various organs like maxillary and labial palps, labrum, antennae etc.
To avoid such stress by mechanical injury, such ablated individuals were allowed for
one to two days till they are normal in their feeding behaviour. Though in the present
observations, ablations of sensillae in both maxillary and labial palps did not interfere
with the host selection, earlier studies on Locusta (Blaney and Chapman 1969; Bernays
and Chapman 1970) indicated the role of various sensillae at each palpal tip in food
selection. The 'dome-shaped sensillae' alone appear to be chemosensory, functioning as
feeding sensory structures. Though the host range or the host preference of acridids as
in Paulinea acuminata does not depend upon the relative numbers of sensillae (Bennett
1970), the number of sensillae appears considerably small in acridids with a narrow host
range than in those with a higher food range (Abushama 1968; Hummelen and Gillon
Feeding and reproduction in grasshoppers 459
1968; Bennett 1970; Gillon 1972; Perkins 1973). Present investigations showed that
the sensillae were larger in number in dicot feeders, lesser in mixed feeders and still
lesser in monocot feeders supporting the observations by Chapman and Thomas
(1978).
Detailed analysis of the host range of the three species of grasshoppers revealed their
highly poiyphagous nature, thereby exhibiting certain preference towards specific host
plants. Both A. crenulata and 0. maindroni preferred R. communis and Clerodendrum
sp., respectively, whereas the monocot feeder T. indica preferred C dactylon.
Though the adults of both species can effectively survive on monocot plants,
nymphal stages especially the first and second stage nymphs did not feed on the
monocot plants like P. maximum and 0. saliva, which is mainly due to the higher silica
content of the leaves. Similar avoidance of nymphs of Oxya nitidula towards
P. maximum due to the higher silica content was also reported by Meera (1982). In the
present observations, food preferences were estimated on the following basis:
(i) Short term feeding behavioural effects including
(a) frequent visit to specific host plants in the fields,
(b) orientation towards the preferred plants in the cages,
(c) higher quantitative food intake, and
(d) higher food utilization ability.
(ii) Long term physiological effects involving
(a) duration of post-embryonic development,
(b) weight increase of the developing nymphs on diverse host plants, and
(c) fecundity and fertility.
On the basis of the all the above parameters, R. communis appeared to be the best suited
and preferred host plant for both A. crenulata and 0. maindroni and C. dactylon for
T. indica. In addition the range of host plants of the above grasshoppers can be
successfully correlated with their relative number of sensillae at various mouthparts
like labrum, maxillary and labial palps etc. For example, 0. maindroni and A. crenulata
showing higher number of sensillae, fed on plants representing as many as 10 families,
whereas the T. indica with a comparatively lower number of sensillae fed exclusively on
members of Poaceae.
In addition the preference towards the specific host plants also varied between
nymphs and adults. The nymphs preferred leaves of R. communis as they are thin,
succulant without any trichomes or any other physical barriers. But it is well known
that the host preference of grasshoppers is influenced by physical and chemical
characteristics like nutrition or secondary chemicals (Gangwere 1972; Otte and Joern
1975, 1977; Chapman and Bernays 1977; Bernays and Chapman 1977). The quantitative
food intake and utilization were also equally high when fed on R. communis than on
other hosts. The nymphal diet also had a significant influence on the adult preference.
However the adults showed a wide spectrum of host plants than that of the nymphs,
their restriction is based upon the physical characters of the host plants.
Though the 'nymphs of A. crenulata were found to feed and develop well on a wide
range of plants, the development was quicker with a lesser percentage of mortality on
R. communis and A. hypogaea. Simultaneously the nymphs developing on R. communis
and A. hypogaea showed a higher weight increase than on other plants. In addition, the
fecundity and fertility were also equally high on the above host plants indicating that
they are the most preferred hosts. Though there appears to be no positive correlation
460
T N Ananthakrishnan, K Dhileepan and B Padmanaban
Figure 8. (A) Inner side of the labrum of Atractomorpha crenulata\ (B) Inner side of the
labrum ofTruxalis indica\ (C) Bifid sensilla in the labrum of T. indica; (D) Sensillae of the
'Beta' tract in the labrum of T. indica\ (E) Sensillum with a bottom collar in the labrum of
T. indica; (F) Hypopharynx of A. crenulata.
between biochemical parameters and host suitability or between incidence of higher
lipid content and higher carbohydrate, protein ratios among these preferred hosts
appear to be an effective factor for the higher developmental rates and increased
fecunidty of grasshoppers. Though the protein content of the hosts was known to
increase the fecundity, present observations indicate that other nutrients like lipids,
carbohydrates and carbohydrate : protein ratio may also influence the fecundity.
Acknowledgement
This work was supported by a grant from the Department of Science and Technology,
Government of India, New Delhi.
Feeding and reproduction in grasshoppers 461
References
Abushama F T 1968 Food-plant selection by Poekilocerus hieroglyphicus (Klug) (Acrididae:
Pyrgomorphinae) and some of the receptors involved; Proc. R. Entomol Soc. Lond. A43 96-104
Bennett F D 1970 Insects attacking water hyacinth in the West Indies, British Honduras and the USA;
Hyacinth Control J. 8 10-13
Bernays E A and Chapman R F 1970 Food selection by Chorthippus parallelus (Zetterstedt) (Orthoptera:
Acrididae) in the field; J. Anim. Ecol. 39 761-776
Bernays E A and Chapman R F 1973 The role of food plants in the survival and development of Chortoicetes
terminifera (Walker) under drought conditions; Aust. J. Zoo/. 21 575-592
Bernays E A and Chapman R F 1977 Deterrent chemicals as a basis of oligophagy in Locusta migrator ia (L);
Ecol Entomol 2 1-18
Blaney W M 1974 Electrophysiological responses of the terminal sensillae of the maxillary palps of Locusta
migratoria (L) to some electrolytes and non-electrolytes; J. Exp. Biol 60 275-293
Blaney W M 1975 Behavioural and electrophysiological studies of taste discrimination by the maxillary palps
of larvae of Locusta migratoria (L); J. Exp. Biol 62 555-569
Blaney R W and Chapman R F 1969 The anatomy and histology of the maxillary palp of Schistocerca
gregaria (Orthoptera: Acrididae); J. Zoo/. Lond. 158 509-535
Bray H C and Thorpe W V 1954 Analysis of phenolic compounds of interest in metabolism; Methods
Biochem. Anal. 1 27-52
Chapman R F 1982 Chemoreception: The significance of receptor numbers Adv. Insec. Physiol Vol 16
247-356
Chapman R F and Bernays E A 1977 The chemical resistance of plants to insect attack; Scl Varia. Pont. Acad.
Sci. 41
Chapman R F and Thomas J G 1978 The number and distribution of sensillae on the mouthparts of
Acridiides; Acrida 1 115 — 148
Cook A G 1972 The ultrastructure of the A A sensilla on the posterior surface of the Clypeo-labrum of Locusta
migratoria migratorioides (R and F); Z. Zellforsch. Mikrosk. Anat. 134 539-554
Dubois M, Gilles K A, Hamilton J K, Rebers P A and Smith F 1956 Colorimetric determination of sugars and
related substances; Anal Chem. 28 351-356
Gangwere S K. 1972 Host findings and feeding behaviour in the orthopteroids, especially as modified by
food availability; Rev. Univ. Madrid 21 107-158
Gillon H 1972 The effect of fire on the principal acridid species of any Ivory coast Savana; In Tall Timbers
Research Station (Florida) Proc. Ann. Tall. Timbers. Fir ecology. Conf. Tallahassee 11 419-471
Haskell P T and Schoonhoven L M 1969 The function of certain mouthpart receptors in relation to feeding in
Schistocerta gregaria and Locusta migratoria migratorioides', Entomologia Exp. Appl 12 423-440
Hummelen P and Gillon Y 1968 Etude de la nourriture des Acridiens de la savane de Lamto en cote dlvorre;
Ann. runiversite d'abidian El 199-206
Kok L T 1971 Fungal symbionts of Xyleborous spp. certain chemical components and their nutritional
significance to the ambrosia beetles Ph.D. dissertation, Univ. of Wisconsin, Madison 1 75 pp
Lowry O H, Rosebrough N J, Farr A R and Randall R J 1951 Protein measurement with folin phenol reagent;
J. Biol Chem. 193 265-275
Meera P 1982 Studies on the bio-ecology of Oxyanitidula (Walk) (Acrididae: Orthoptera; Insecta); Ph.D.
thesis, University of Madras, India pp. 211
Otte D and Joern A 1975 On feeding patterns in desert grasshoppers and the evolution of specialized diets;
Proc. Acad. Nad. Sci. Phila. 18 129-144
Otte D and Joern A 1977 On feeding patterns in desert grasshoppers and the evolution of specialized diets;
Proc. Acad. Natl Scl Phila. 128 89-126
Perkins B D 1973 Potential for water hyacinth management with biological agents in Tall Timber Research
Station (Florida) Proc. Tall Timber Conf. on Ecol. Ann. control by habitat management No. 4 Tallahassee,
Florida pp. 53-64
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 463-468.
© Printed in India.
Investigations on Heliothis armigera (Hubner) in Marathwada-
XXVIIL Key mortality factors in regular and overlapping generations
on pigeonpea
G G BILAPATE
Department of Entomology, Marathwada Agricultural University, Parbhani 431 402, India
MS received 26 November 1984; revised 9 May 1985
Abstract. The parasitisation of Heliothis armigera (Hubner) larvae by Campoletis chlorideae
Uchida and Enicospilus biconatus Townes, Townes & Gupta ( — neorufus Rao & Nikam) was
8-70 and 13*79% respectively during the first generation on pigeonpea during 1983-84 at
Parbhani. The parasitisation of H. armigera by these parasites was low in second overlapping
generation. Thus, C. chlorideae and E. biconatus were identified as key mortality factors for H.
armigera on pigeonpea.
Keywords. Key mortality factors; Heliothis armigera (Hubner); pigeonpea.
1. Introduction
Heliothis armigera (Hubner) is the most destructive pest of pigeonpea in Marathwada.
Naturally-occurring predators and parasites are more important in regulating
populations of H . armigera. Explicit instructions in insect control guides, developed
through qualitative and quantitative evaluation of natural enemies, are needed so that
the natural enemy numbers can be used more directly in decision-making (King et al
1981). Because of the high value of pigeonpea and the importance of Heliothis, there is a
need for more ecological research approach for its management.
2. Materials and methods
The sampling techniques described by Harcourt (1961) were followed with slight
modifications as it was felt desirable to sample larval stage instead of egg and pupa.
2.1 Time of sampling
Egg stage was sampled when the activity of adult moths was noticed in the field. The
first incidence of H. armigera was observed on 24 October 1983.
2.2 Laboratory culture
The known number of eggs, deposited for the first time in field were collected on the
463
464 G G Bilapate
above date for further rearing in laboratory. This laboratory culture was reared on
pigeonpea pods till the pest disappeared from the field.
2.3 Sampling of larvae
The sampling for the larvae (2 x 2 m quadrate) for regular generation in field was based
on the developmental stages of laboratory culture. When the larvae in plastic boxes
(5x5 cm) reached in I-II, III-IV sampling for the larvae was done in the field. Similarly
when the larvae in the plastic boxes passed in V-VI instars of laboratory culture, the
respective instar group was sampled in the field. In each generation 50 quadrates of
2 x 2 m were sampled randomly and the larvae were brought to the laboratory for
further rearing. The infested plants in the quadrates were carefully examined for
recording pest population in larval stage and thus the population was computed on
hectare basis.
2.4 Study of generations
To determine the distinct regular generations, an interval of five days was provided
before sampling of larvae of next generation, after the mean adult emerged from
previous generation in laboratory. This period was considered for oviposition by the
moths of previous generation. The start of overlapping generation was considered after
15 days of the start of regular generation.
2.5 Mode of observations
The larvae collected from the field were brought to the laboratory and reared on
pigeonpea pods till the adults emerged. Larval and pupal parasitism as well as, because
of unknown causes mortality were observed.
2.6 Life table
The column headings used in the life tables of the present study were similar to those
proposed by Morris and Miller (1954). The data were analysed according to the method
suggested by Harcourt (1963, 1969) and Atwal and Bains (1974). Separate budgets were
prepared to find out the key factors that influence the population trend in different
generations. The method of Varley and Gradwell ( 1 963, 1 965) was chosen for key factor
analysis.
3« Results and discussion
The results on the key mortality factors for regular generations of H. armigera are
presented in table 1. The larval mortality in I-II instar group due to Campoletis
chlorideae Uchida was 8-70 % in the first generation. The parasitisation of the larvae by
Mortality factors in pigeonpea
465
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Mortality factors in pigeonpea
467
Enicospilus biconatus Townes, Townes & Gupta (= neorufus Rao & Nikam) was
1 3-79 %. The pupal parasitism was 2-44 % by Goniophthalmus halli Mesnil. The positive
trend index (7-78) indicated that the mortality factors were ineffective in checking
further multiplication of population in the second generation. The high value of
k (0-0913) indicated that the mortality parameters in III-IV instars resulted in highest
mortality of larvae in the first regular generation. The parasitisation by C. chlorideae,
E. biconatus and Carcelia sp in the larval stage and G. halli in the pupal stage was very
low in the second generation. The total larval mortality due to C. chlorideae and
Carcelia sp was 3-26% during the third generation. The results on overlapping
generations are presented in table 2. The mortality of H. armigera larvae because of C.
Table 2. Key mortality factors for two overlapping generations of H . armigera on pigeonpea
during 1983-84 (Population/ha)
Ix
dxF
dx 100 qx Sx Logtfx)
I Overlapping generation (8-11-83 to
10-12-83)
Expected eggs
20,990
Sterility/dead
840
4-00
0-96
4-3220
Viable eggs
20,150
4-3043
0-0177
Larval instars:
MI (Nj
20,150
C. chlorideae
1,750
8-68
0-86
4-3043
Unknown
1,000
4-96
IIMV
17,400
E. biconatus
2,900
16-67
0-79
4-2405
0-0638
Virus
150
0-86
Unknown
650
3-73
V-VI
13,700
Virus
250
1-82
0-92
4-1367
0-1038
Unknown
800
5-84
Prepupa
12,650
Unknown
300
2-37
0-98
4-1021
0-0346
Pupa
12,350
G. halli
900
7-29
0-82
4-0917
0-0104
Unknown
1,250
10-12
Moths
10,200
Sex 50% females
1-00
4-0086
0-0831
Females x 2 (N3)
10,200
(Reproducing
females
= 5,100)
3-7076
0-3010
Trend index
1-19
Generation survival
0-51
II Overlapping generation
(16-12-83
to 20-1-84)
Expected eggs
24,639
Sterility/dead
739
3-00
O97
4-3916
Viable eggs
23,900
4-3784
0-0132
Larval instars:
MI (#1)
23,900
C. chlorideae
150
0-63
0-98
4-3784
Unknown
250
1-05
IIMV
23,500
E. biconatus
50
0-21
0-97
4-3711
0-0073
Unknown
600
2*55
V~VI
22,850
Unknown
500
2-19
0-98
4-3589
0-0122
Prepupa
22,350
Unknown
800
3-58
0-96
4-3493
0-0096
Pupa
21,550
G. halli
100
0-46
0-92
4-3334
0-0159
Unknown
1,500
6-96
Moths
19,950
Sex 50% females
1-00
4-2999
0-0335
Females x 2 (N3)
19,950
(Reproducing
3-9989
0-3010
females
= 9,975)
Trend index
Generation survival
Nil
0-83
468 G G Bilapate
chlorideae, E. biconatus and nuclear polyhedrosis virus was 8*68, 16-67 and 1-82%
respectively. The nil trend index in the second overlapping generation indicated that
pest population was not present in the field and only two overlapping generations were
recorded in pigeonpea. The k value of 0*0638 indicate highest contribution of mortality
in III-IV instar group in the second overlapping generation.
The parasitisation of H. armigera larvae (26-74 %) and pupae by different parasites
was comparatively greater in the first regular generation when compared to second
(1-13%) and third (3-26%) generations. Similar trend regarding the role of larval
parasites was observed in the first overlapping (28-03%) generation while the
parasitisation of H. armigera larvae was very low in the second overlapping generation.
Irrespective of high parasitisation, the population increased in second regular and
overlapping generations. The little showers of rain at the end of the first regular and at
the start of second overlapping generation could be the reason for increasing pest
population on pigeonpea. Thus, it could be concluded that C. chlorideae and
E. biconatus acted as key mortality factors in first regular generation only. Similarly
these parasites were also identified as key factor in reducing the population of larvae to
the extent of 25-35 % in the first overlapping generation. C. chlorideae alone parasitised
the H. armigera larvae to the extent of 24-92% in chickpea (Bilapate et al 1979). The
importance of this parasite in regulating the population of H . armigera has been
indicated by many researchers in India (Achan et al 1968; Bilapate 1984; Gangrade
1964; Raich 1973; Rao 1968; Raodeo et al 1974).
References
Achan P D, Mathur K C, Dharmadhikari P R and Manjunath T M 1968 Parasites of Heliothis spp. in India;
Tech. Bull Commonw. Inst. Bioi Control. 10 129-141
Atwal A S and Bains S S 1974 Applied animal ecology (Ludhiana: Kalyani publishers)
Bilapate G G 1984 Heliothis complex in India—A Review; Agric. Rev. 5(1) 13-26
Bilapate G G, Raodeo A K and Pawar V M 1979 Population dynamics of Heliothis armigera (Hubner) in
sorghum, pigeonpea and chickpea in Marathwada; Indian J. Agric. Sci. 49 560-566
Gangrade G A 1964 On the biology of Campoletis perdistinctus (Hymenoptera: Ichneumonidae) in Madhya
Pradesh, India; Ann. Ent. Soc. Am. 57 570-574
Harcourt D G 1961 Design of sampling plan for studies on the population dynamics of the diamond
backmoth, Plutella maculipennis (Curt.); Can. Entomol. 93 820-831
Harcourt D G 1963 Major mortality factors in the population dynamics of the diamond backmoth, Plutella
xylostella (Curt.); Can. Entomol Soc. Mem. 32 55-66
Harcourt D G 1969 The development and use of life tables in the study of natural insect population; Anna.
Rev. Entomol. 14 175-196
King E G, Powell J E and Smith J W 1981 Prospectus for utilization of parasites and predators for
management of Heliothis spp; Proc. Int. Workshop on Heliothis Management, 1981 ICRISAT, pp. 103-122
Morris R F and Miller C A 1954 The development of life tables for the spruce budworm; Can J. Zool. 32
283-301
Raich K V 1973 Parasitisation of gram caterpillar by an ichneumonid; PL Prot. Bull. 21 40
Rao V P 1968 Heliothis spp. and their parasites in India; PANS (A). 14 367-375
Raodeo A K, Tikar D T, Deshpande A D and Khan M I 1974 Some larval parasites of crop pests in
Marathwada region (Maharashtra). Agric. Coll Magz. Parbhani. 14 82-84
Varley G C and Gradwell G R 1963 The interpolation of insect population changes; Proc. Ceylon Ass. Sci. 18
142-156
Varley G C and Gradwell G R 1965 Interpreting winter moth population changes; Proc. Int. Congr. Ent. 12
377-378
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 469-474.
© Printed in India.
Transpiration rates and acclimation to water and temperature
of the tropical woodlice, Porcellionides pruinosus Brandt and Porcellio
laevis Latreille
G ACHUTHAN NAIR and N BALAKRISHNAN NAIR
Department of Aquatic Biology and Fisheries, University of Kerala, Trivandrum 695 007,
India
MS received 26 July 1984; revised 2 March 1985
Abstract. The transpiration rates and acclimation to water and temperature of
Porcellionides pruinosus Brandt and Porcellio laevis Latreille, the common terrestrial isopods
occurring in and around Trivandrum, India, were studied. Transpiration rates in different
temperatures (24 to 50°C) were higher in both the species during the first 1 5 minutes' exposure
when compared to 1-hour duration. Previous acclimation to different humidity and
temperature conditions affected the transpiration rates in both the species and lower rates of
transpiration occurred in isopods acclimated to dry conditions and higher temperatures.
Survival rates improved at lethal temperatures in specimens acclimated to 34°C.
Keywords. Transpiration; temperature; acclimation; isopods; Porcellionides pruinosus;
Porcellio laevis.
I. Introduction
Although terrestrial isopods are widespread in the tropical south-west coast of India,
very little is known concerning their physiological adaptations. In the present study,
Porcellionides pruinosus Brandt and Porcellio laevis Latreille, commonly found in and
around Trivandrum, were selected and their rates of transpiration under different
temperatures and the nature of temperature and humidity relations have been
examined.
Attempts were made to study the physiological adaptations of terrestrial isopods to
varying temperature and humidity conditions, the notable contributors of such studies
among others being those of Bursell (1955), Paris (1963), Warburg (1965), Edney (1968),
Cloudsley-Thompson (1969), Sutton (1969) and Dubinsky and Steinberger (1979).
2. Materials and methods
2.1 Topography
Trivandrum lies at 8°20'N lat. and 76°55'E long, in Kerala State, S.W. India. The state is
bounded on the west by the Arabian Sea and on the east by the Western Ghats. The area
has a tropical climate and the year may be divided into the hot dry pre-monsoon
(February to May), the rainy season (June to November) and the comparatively cool
469
470 G Achuthan Nair and N Balakrishnan Nair
post-monsoon (December and January) periods. The relative humidity is generally
above 70% and the maximum goes upto 90% during the monsoon. The atmospheric
temperature also varies from a mean maximum of 27-5°C during August to 33-5°C in
April and a mean minimum of 21 *6°C during January to 25-5°C in April. Rainfall varies
from month to month with the maximum during July-November and the minimum
during February-April. The region is covered with green bushes, coconut palms and
trees and the heavy litter covering the ground provide ideal conditions for the life and
propagation of isopods.
2.2 Material
Porcellionides pruinosus Brandt and Porcellio laevis Latreille, are common terrestrial
isopods found in and around Trivandrum. The former is smaller than the latter and the
mean weights of specimens used in the experiments were 4-0 mg and 14-7 mg
respectively. For transpiration studies, the largest specimens available, mostly adult
females, were selected.
23 Sites of collection and methods
The isopods were collected from 2 localities, one from the Aquarium campus near the
seashore, in sandy soils under bricks and stones in humid and shady places, and the
other from the city proper about 12 km away from the Aquarium, in loamy soil with rich
humus content and also from beneath bricks and stones in cool and shady places. A
marked difference was noticeable in the distribution of these isopods in these localities.
Over 95 % of woodlice collected from the Aquarium area were P. pruinosus while an
even higher proportion of those taken from the city proper was P. laevis. Overlapping
of these two populations was not observed in both the localities. Soil pH in the two
localities was measured with an analytical pH meter. It averaged 6-8 at the Aquarium
campus while in the city it averaged 74.
The isopods were maintained in large petri dishes containing damp filter paper with
rotting leaves lying on it at room temperature (average about 31 ± 4°C) in natural day
light and darkness.
To study the transpiration rates, the effect of acclimation in different temperature
and humidity conditions on the rates of transpiration and the effect of acclimation in
different temperatures on the lethal temperature, the methods described by Edney
(1951, 1954) and Cloudsley-Thompson (1969) were adopted. For transpiration studies
the specimens (10 each in number) which were weighed individually were exposed
separately for 15 minutes and 1 hour over phosphorous pentoxide at 24, 29, 34, 39, 45
and 50°C before the water-loss through evaporation was estimated by reweighing. The
relative humidity was kept constant at 22 % in all the temperatures tested. To calculate
the surface area of the animal, a value of K = 12 was adopted as used by
Cloudsley-Thompson (1956, 1969) for many African woodlice and it is the mean of the
figures calculated by Edney (1951) for various British woodlice.
Transpiration and acclimation studies of tropical woodlice 471
3. Results
3.1 Transpiration
Water-loss by transpiration is one of the most important physiological factors affecting
the distribution of woodlice (Edney 1954, 1968; Cloudsley-Thompson 1969). The
results on the transpiration for exposures of P. pruinosus and P. laevis for 15 minutes
and 1 hour in various temperatures are given in table 1. The transpiration rate was
higher during the first 15 minutes' exposure for both the species than for 1 hour
duration. Thus the mean water-loss for 15 minutes' exposure for P. pruinosus at 24°C
was 3-80 mg/cm2/hr which increased to 8-76 mg/cm2 /hr at 34°C and 18-89 mg/cm2/hr
at 50°C whereas the same for 1 hour exposure at 24°C was l-94mg/cm2/hr which
further increased to 3*99 mg/cm2/hr at 34°C and 9-45 mg/cm2/hr at 50°C In the case of
P. laevis the water-loss was lower for both 15 minutes and 1 hour exposures at different
temperatures when compared to that of P. pruinosus. Thus the mean water-loss for this
species for 15 minutes' exposure at 24?C was 2*01 mg/cm2/hr which gradually
increased to 3-68 mg/cm2/hr at 34°C and 8-21 mg/cm2/hr at 50°C whereas the values
for 1 hour exposure of these animals for the same temperatures were l-00mg/cm2/hr,
1-06 mg/cm2/hr and 4-26 mg/cm2/hr respectively (table 1).
3.2 Effect of acclimation to humidity and temperature on transpiration
The mean rates of transpiration from both the species previously acclimated in moist
and dry conditions and also at two different temperatures of 24°C and 34°C are given in
table 2. The transpiration is lower in those acclimated to dry conditions (relative
humidity 48%) as compared to those acclimated to moist conditions (relative
humidity 68%) and the difference in rates of transpiration between these two
conditions is quite significant (at 5 % level) in the case of P. pruinosus. Such a significant
difference, however, was not evident in the case of P. laevis (table 2).
Studies on the effect of acclimation to temperature (24 and 34°C) on transpiration
rates for both the species show that the transpiration rates are higher in both the species
Table 1. Rate of transpiration in P. pruinosus and in P. laevis.
Water loss (mg/cm2/hr)
15 min exposure 1 hr exposure
Temperature
P. pruinosus P. laevis P. pruinosus P. laevis
24
3-80 ±O07
2-01 ±004
1-94 ±003
1-00 ±0-02
29
4-15±O04
2-94 ±O06
2-91 ±0-03
1-04 ±003
34
8-76 ±0-05
3-68 ±O05
3-99 ±005
1-06 ±003
39
• 10-20 ±0-05
4-34 ±002
5-82 ±O07
2-44 ±0-04
45
14-78 ±O05
648 ±003
7-87 ±0-04
3-57 ±0-05
50
18-89±017
8-21 ±0-02
9-45 ±0-05
4-26 ±0-03
The relative humidity was kept constant at 22 %.
472
G Achuthan Nair and N Balakrishnan Nair
Table 2. Mean water loss (mg/cm2/hr) in < 10% relative humidity at room
temperatures (34 ± 1 °C) for exposure of 1 hr, in woodlice previously conditioned in
various ways (N = 16).
Species
Conditioning
t Inference
moist
dry
P. pruinosus
1-76 ±006
1-43 ±0-09
2-81 Significant at
level
5%
/><005
P. laevis
1-36±019
H5±009
O87 N.S.
24°C
34°C
P. pruinosus
1-65 ±0-20
3-45 + 051
2-98 Significant at
level
5%
P < 0-05
P. laevis
096 ±0-18
1-31 ±0-02
1-71 N.S.
N.S. — not significant.
acclimated at 34°C when compared to those acclimated to 24°C. Here also a significant
difference (at the 5% level) between the transpiration rates at 24° C and 34°C is
discernible in the case of P. pruinosus whereas such a difference was not noticed in the
case of P. laevis.
3.3 Effect of acclimation on the lethal temperatures
In preliminary tests conducted at room temperature, most individuals of P. pruinosus
survived at 41*5°C for 30 min but very few survived at 42-5°C. The lethal temperature of
P. pruinosus for an exposure of 30 minutes was, therefore, assumed to be around 42°C.
In the case of P. laevis, however, the lethal temperature was around 44°C although a
slight variation occurred on account of the dampness of the filter paper. Studies on the
survival rates in their respective lethal temperatures for 2 hr for both the species which
were previously acclimatized at two different temperatures of 24°C and 34°C showed
that survival was better for both the species acclimated at 34°C as compared to 24°C.
The results are presented in table 3. From the data it is apparent that during the initial
30 min exposure, no mortality of P. pruinosus and P. laevis acclimated at 34°C occurred
whereas a gradual reduction in the percentage survival took place in both the species
acclimated at 24°C After 90 min of exposure, however, a sudden increase in the
mortality rates for both the species acclimated at 34°C was evident and after 120 min,
the percentage of animals survived equalled, irrespective of the temperatures in which
they were acclimated (table 3).
4. Discussion
A higher rate of water-loss from the body surfaces of P. pruinosus and P. laevis during
the first 15 min of exposure to different temperatures was noted as compared to 1 hr
exposure. The cuticle of land isopods does not possess the water-proofing mechanism,
Transpiration and acclimation studies of tropical \voodlice 473
Table 3. Percentage survival of P. pruinosus and P. laevis (previously acclimated at
24°C and 34°C) in their lethal temperatures of 42°C and 44°C respectively.
Percentage survival
P. pruinosus
P. laevis
Time of exposure
Acclimated at
Acclimated at
(min)
24°C 34°C
24°C 34°C
0
100-00 100-00
100-00 100-00
15
90-91 100-00
91-67 100-00
30
72-73 100-00
83-33 100-00
45
72-73 90-91
41-67 100-00
60
63-64 90-91
33-33 83-33
75
45-45 81-83
16-67 66-67
90
36-36 81-83
16-67 41-67
105
36-36 45-45
8-33 8-33
120
36-36 36-36
8-33 8-33
an oriental layer of lipid molecules in the epicuticle so characteristic of insects and
arachnids (Lees 1947; Baement 1961). Thus the higher initial rate may be due mainly to
loss of water from layers of the cuticle external to lipid barrier, which is later followed
by shrinkage of the cuticle thus leading to a closer packing of the lipid molecules and,
therefore, to decreased permeability (Bursell 1955). This is clearly advantageous to the
animal since it acts as a regulating mechanism that reduces the rate of water-loss at a
time when conservation is most needed (Cloudsley-Thompson 1969).
Observations on the transpiration rates of P. pruinosus and P. laevis previously
acclimated to moist and dry conditions and also at two different temperatures of 24°C
and 34°C, show a significant difference in their transpiration rates in different
acclimated conditions and that the transpiration rates are higher in those conditioned
under moist conditions and at a higher temperature of 34°C. Higher cuticular
permeability at higher temperatures is a typical character of cryptozoic animals
(Lawrence 1953). The ability to loose water rapidly by transpiration certainly enables the
woodlice to withstand high temperatures for brief periods (Edney 1954). Edney (1951)
also found significantly different rates of evaporation from specimens of Armadillidium
vulgare obtained from different localities of England and concluded that the same
species showed considerable difference in their rates of evaporation resulting from
selection over a long period or an effect of acclimation within the lifetime of the
individual, or both. This seems to be true in the present study also where these animals
showed marked differences in transpiration in varying temperature and humidity
conditions.
Regarding lethal temperatures, the highest ambient temperature that land isopods
tolerate is a reflection of their genetic constitution, the period of exposure, the rate of
rise, the ambient humidity and the temperature history (acclimation) (Edney 1968).
Thus factors such as size, duration of exposure, permeability of the cuticle and
humidity, seem to play significant roles in determining the effects of high ambient
temperatures. Studies on the survival rates of P. pruinosus and P. laevis in their
respective lethal temperatures showed that the survival was better for both the species
acclimated at higher temperature. Edney (1964) also made similar observations on
474 G Achuthan Nair and N Balakrishnan Nair
Armadillidium vulgare and Porcellio laevis in California and he found that neither size
nor mortality affected the lethal temperature but that acclimation for some days in
different temperatures had a marked effect. This is true in the case of African woodlice
also (Cloudsley-Thompson 1969).
Acknowledgements
One of the authors (CAN) is grateful to CSIR, New Delhi, for the award of a fellowship.
References
Baement J W L 1961 The water relations of the insect cuticle; Bid Rev. 36 281-320
Bursell E 1955 The transpiration of terrestrial isopods; J. Exp. Biol. 32 238-255
Cloudsley-Thompson J L 1956 Studies in diurnal rhythms- VI. Bioclimatic observations in Tunisia and their
significance in relation to the physiology of the fauna especially woodlice, centipedes, scorpions and
beetles; Ann. Mag. Nat. Hist. 12 305-329
Cloudsley-Thompson J L 1969 Acclimation, water and temperature relations of the woodlice Metoponorthus
pruinosus and Periscyphis jannonei in the Sudan; J. Zool. London 158 267-276
Dubinsky Z and Steinberger Y 1979 The survival of the desert isopod Hemilepistus reaumuri (Audouin) in
relation to temperature (Isopoda: Oniscoidea); Crustaceana 36 147-154
Edney E B 1951 The evaporation of water from the woodlice and the millipede Glomeris; J. Exp. Biol. 28
91-115
Edney E B 1954 Woodiice and the land habitat; Biol. Rev. 29 185-219
Edney E B 1964 Acclimation to temperature in terrestrial isopods 1. Lethal temperature; Physiol. Zool. 37
364-377
Edney E B 1968 Transition from water to land in isopod crustaceans; Am. Zool. 8 309-326
Lawrence R F 1953 The biology of the cryptic fauna of forests (A. A. Balkema: Cape Town)
Lees A D 1947 Transpiration and the structure of the cuticle in ticks; J. Exp. Biol. 23 379-410
Paris O H 1963 The ecology of Armadillidium vulgare (Isopoda: Oniscoidea) in California grasslands: food,
enemies and weather; Ecol. Monogr. 33 1-22
Sutton S L 1969 The study of woodlice; Proc. Br. Entomol. Nat. Hist. Soc. 1 71-75
Warburg M R 1965 Water relations and internal body temperature of isopods from mesic and xeric habitats;
Physiol. Zool. 38 99-109
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 475-480.
© Printed in India.
Changes in some biochemical constituents of the fiddler crabs Uca
annulipes Latreille and U. triangularis (Milne Edwards) in response
to eyestalk removal
G V KRISHNA RAO, Y PRABHAKARA RAO and D G V PRASADA
RAO
Department of Zoology, Andhra University, Waltair 530003, India
MS received 26 April 1984; revised 5 February 1985
Abstract. Metabolic effects of eyestalk removal in the fiddler crabs Uca annulipes and
U. triangularis were studied. Bilateral extirpation of eyestalks results in hypoglycemia and a
fall and rise in the glycogen content of hepatopancreas and muscle respectively. Eyestalk
removal also caused a fall in protein and fat content of hepatopancreas and muscle
respectively. Injection of eyestalk extract restored the level of blood sugar, glycogen, protein
and fat to normal level.
Keywords. Hypoglycemia; metabolic effects; eyestalk ablation; fiddler crabs; biochemical
constituents.
1. Introduction
Among crustaceans, the presence of hyperglycemic condition was noticed after eyestalk
ablation in Callinectes by Abramowitz et al (1944), in Astacus by Kleinholz et al (1950),
in Ocypode plat y tar sis by Parvathy (1972), in Cambarus robustus by Telford (1975) and
in Parapenaeopsis hardwickii by Nagabhushanam and Kulkarni (1980). But interest-
ingly, the extirpation of eyestalks did not produce hypoglycemia in the spiny lobsters
Panulirus japonicus and P. penicellatus (Scheer and Scheer 1951) or in the prawns
Metapenaeus monoceros (Rangneker and Madhyastha 1971). In addition, no change in
the blood sugar levels was observed in the crabs Libinia emarginata and Callinectes
sapidus after eyestalk removal. The eyestalks of crustaceans also contain some other
factors that can regulate the levels of fats and proteins (Rangneker and Madhyastha
1971; Madhyastha and Rangneker 1976; Raghavaiah et al 1980). Recently Sedlmeier
and Keller (1981) investigated the effect of crustacean hyperglycemic neurohormone
(CHH) on cyclic nucleotide levels of different tissues of crayfish Orconectes limosus and
found elevation in the levels of cAMP and cGMP in all the tissues after hormone
administration.
It is evident from literature that the ablation of eyestalks causes either an increase or
decrease in any of the biochemical constituents and this is also sometimes different in
different parts of the body. Till now, there has been no convincing evidence in general of
the metabolic effects of eyestalk extracts in crustaceans. Moreover, it is apparent that
less attention has been paid to this aspect in crabs in particular when compared to other
crustaceans. Therefore, an attempt has been made to give a comparative account of the
effect of eyestalks on the levels of different biochemical constituents like carbohydrates,
fats and proteins in two species of fiddler crabs, Uca annulipes and U. triangularis.
476 G V Krishna Rao, Y Prabhakara Rao and D G V Prasada Rao
2. Material and methods
The fiddler crabs collected from a marshy area near Visakhapatnam harbour, were
maintained in the laboratory in aquaria containing sea water. The crabs were not fed
during the experimental period. After 24 hr, twenty animals of the same size (male,
intermoult) were selected for the experiment and divided into four groups (A,B,C and
D) of five each. Bilateral eyestalk ablation was performed by cutting the eyestalks at
their bases and stubs were quickly cauterised to avoid bleeding. The animals of group A
served as intact controls, while those in group D were used for sham-operation.
A single injection of eyestalk extract was given to each animal in group C. Eyestalk
extract was prepared by triturating a pair of eyestalks in 0*2 ml of sterilised sea water
(32°/00). The extract was centrifuged and the supernatant was used for injection. Each
animal received a dose of 0*03 ml which was injected into the ventral sinus by piercing
the needle of the syringe in the region between the bases of the third and fourth walking
legs. Sham-operation was performed by removing the chitinous exoskeleton around
the bases of eyestalks. Before and after eyestalk removal, animals were given cold
treatment for 30 min to avoid shocks. After eyestalk ablation and injection of eyestalk
extract, all the four groups of animals were maintained separately in glass troughs
containing sea water (32°/00) for 24 hr.
After 24 hr, blood samples from the animals of all the four groups were withdrawn by
piercing a hypodermic needle through the carapace in the pericardial region. Blood
sugar was estimated by the Folin-Wu method (1920).
At the same time hepatopancreas and muscle tissues were separated from all the
animals of the four groups and were dried separately at 1 10°C till constant weights were
obtained. Then, the dried tissue material was used for the estimation of glycogen
(Carrol et al 1956), protein (Lowry et al 1951) and fats (Folch et al 1957). Student's t test
was used to compare the values (Snedecor and Cochran 1967).
3. Results
Changes in the blood sugar level and variation in glycogen, fat and protein level of
hepatopancreas and muscle under different experimental regimens of U. annulipes and
17. triangularis are given in figures 1 to 4.
The eyestalk ablation results in a significant (P< 0-001) decline in the average
glycemic level (group B) in both the species of crabs (figure 1). Further, when the
eyestalk extract is injected into the destalked animals (group C) the average blood sugar
level is restored to its normal level. No marked alteration (P> 0*001) in the
concentration of blood sugar is noted after sham-operation (group D) (figure 1).
Bilateral eyestalk ablation also causes a significant (P< 0*001) fall in the average
value of hepatopancreatic glycogen (figure 2). Moreover, injection of eyestalk extract
into destalked animals restores the amount of glycogen to normal level and this
restored value compared well with those of the control and sham-operation groups
(figure 2).
Bilateral ablation of eyestalks causes a significant (P< 0-001) increase in the
concentration of muscle glycogen (group B) (figure 2). Injection of eyestalk extract in
the operated animals (group C) produces a partial restoration of the raised glycogen
level (figure 2).
Effects ofeyestalk removal in fiddler crabs
477
1 £.
[ ] U; ANNULIPES
\ o
[/^| Uj TRIANGULARIS
1
o
1
_. Q
rh
£
\
1
i
i
BLOOD SUGAR
O to £. &
/-7
*
*///
V/,
V/
yy,
///
B C
GROUPS
Figure 1. Changes in the blood sugar levels of U. annulipes and U. triangularis during
different experimental conditions (for groups A, B, C and D, see text).
HEPATOPANCREAS IN U. ANNULIPES
MUSCLE IN U_- ANNULIPES
HEPATOPANCREAS IN U,. TRIANGULARIS
MUSCLE IN U. TRIANGULARIS
^ 8
2
LU
e?
8 6
4-
rf
GROUPS
Figure 2. Changes in the amounts of glycogen in the hepatopancreas and muscle of U.
annulipes and U. triangularis during different experimental conditions.
Eyestalk removal also causes (group B) a significant (P< 0-001) decline in the
concentration of lipids and proteins of the hepatopancreas (figures 3 and 4). Though
there is a decline in the lipids and proteins of muscle tissue of the destalked animals
(group B), it is not statistically significant (P> 0-001). However, injection ofeyestalk
extract into the destalked animals (group C) brings about a re-establishment of the lipid
and the protein levels within the normal range (figures 3 and 4).
478
G V Krishna Rao, Y Prabhakara Rao and D G V Prasada Rao
700
6OO
500
2 25
I "[ HEPATOPANCREAS IN U_. ANNULIPES
[v!] MUSCLE IN U_. ANNULIPES
E//?| HEPATOPANCREAS IN U_. TRtANGULARlS
$jj£ MUSCLE IN U. TRIANGULARIS
I
\
GROUPS
Figure 3. Changes in the amounts of fat in the hepatopancreas and muscle of U. annulipes
and U. triangularis during different experimental conditions.
260
220
E 180
t- I4O
O
100
60
| "| HEPATOPANCREAS IN y. ANNULIPES
Pxl MUSCLE IN (J- ANNULIPES
%//\ HEPATOPANCREAS IN U.. TRIANGULARIS
$5% MUSCLE IN U. TRtANGULARlS
GROUPS
Figure 4. Changes in the amounts of protein in the hepatopancreas and muscle of U.
annulipes and U. triangularis during different experimental conditions.
4. Discussion
The observation of hypoglycemia in both the crabs after surgical removal, differ from
those on other crabs, Callinectes sapidus (Kleinholz et al 1950), Scylla serrata
(Deshmukh 1968) and Paratelphusa jacquamontii (Rangneker et al 1961) where the
eyestalk ablation resulted in hyperglycemia. But it is interesting to note that such a
Effects ofeyestalk removal in fiddler crabs 479
condition of hypoglycemia after eyestalk removal was reported earlier in only one crab
Varuna litter ata by Madhyastha and Rangneker (1976). However, similar observations
were recorded in other crustaceans, Metapenaeus sp. (Dall 1965), Panulirus japonicus
and P. penicillatus (Scheer and Scheer 1951) and Metapenaeus monoceros (Rangneker
and Madhyastha 1971).
It may also be said from the results that carbohydrate metabolism in the fiddler crabs,
Uca annulipes and U. triangularis is controlled by the interaction of two separate
factors originating from eyestalks: one might be maintaining the normal levels of blood
sugar and glycogen concentration of hepatopancreas in the intact crabs and another
might be inhibiting the synthesis of glycogen in the muscle. Therefore, eyestalk ablation
results in decrease of blood sugar and glycogen concentration in hepatopancreas and
increase in muscle glycogen concentration. Injection ofeyestalk extracts supplemented
both the factors restoring to normal range.
The reason for the depletion of glycogen leading to rapid glycogenolysis in the
hepatopancreas of destalked U. annulipes and U. triangularis may be due to the
inhibitory capacity of the enzyme phosphorylase as suggested by Fingerman (1974) and
Nagabhushanam and Kulkarni (1980). Wang and Scheer (1963) observed that the
enzyme uredinediphosphate glucoseglycogen transglucosylase (UDPG-GT) present in
muscles of crabs is under the control ofeyestalk hormones. The increase of glycogen in
the muscles of U. annulipes and U. triangularis may be ascribed to the lack of inhibition
to this enzyme.
A post operative decline in the lipids and proteins of hepatopancreas and muscle may
be due to the absence of the factors that regulate the incorporation of amino acids into
proteins and partly due to conversion of lipids and proteins to carbohydrates to
maintain a steady supply of glycogen. Similar observations were made by Raghavaiah
(1977) in Paratelphusa hydrodromus, Rangneker and Madhyastha (1971) in
Metapenaeus monoceros and Skinner (1966) in Gecarcinus later alis.
Thus the pattern of increase or decrease in different constituents is the same in both
the animals but the differences in their amounts may be attributed to the species
specificity.
Acknowledgements
The authors are thankful to the Head of the Department for facilities. GVKR and YPR are
grateful to the csm for financial assistance.
References
Abramowitz A A, Hisaw F L and Papendra D N 1944 The occurrence of diabetogenic factor in the eyestalks
of crustaceans; Biol Bull 86 1-5
Carrol N V, Longley R W and Roe J H 1956 The determination of glycogen in liver and muscle by use ol
anthrone reagent; J. Biol Chem. 220 583-593
Dall W 1 965 Studies on the physiology of shrimp, Metapenaeus sp. (Crustacea, Decapoda, Penaeidae) II,
Endocrine's and control of moulting; Aust. J. Mar. Freshwater Res. 16 1-12
Deshmukh R D 1968 Some aspects of biology of the marine crab Scylla serrata (Forskal) Ph.D. thesis.
University of Bombay, India
480 G V Krishna Rao, Y Prabhakara Rao and D G V Prasada Rao
Fingerman M 1974 Comparative endocrinology in Experimental marine biology (ed.) N Mariscal, (New York
and London: Academic Press) pp. 166-223
Folch J, Lees M and Sloane Stanley G H 1957 A simple method for the isolation and purification of total
lipids from animal tissues; J. Biol. Chem. 266 497-509
Folhi O and Wu H 1920 J. Biol. Chem. 41 367
Kleinholz L H, Havel V J and Richart R 1950 Studies in regulation of blood sugar concentration in
crustaceans II. Experimental hyperglycemia and regulatory mechanism; Biol. Bull 99 454-468
Lowry O H, Rosenbrough N J, Fair A L and Randall R J 1951 Protein measurement with folin phenol
reagent; J. Biol. Chem. 193 265-275
Madhyastha M N and Rangneker P V 1 976 Metabolic effects of eyestalk removal in the crab Varuna litterata;
Hydrobiology 48 25-31
Nagabhushanam R and Kulkarni G K 1980 Role of eyestalk hormone in the carbohydrate metabolism of a
marine penaeid prawn, Parapenaeopsis hardwickii (Miers) (Crustacea, Decapoda, Penaeidae).
Hydrobiology 74 145-150
Parvathy K 1972 Endocrine regulation of carbohydrate metabolism during the moult cycle in crustaceans. I.
Effect of eyestalk removal in Ocypode platytarsis; Mar. Biol. 14 58-62
Raghavaiah K 1977 Aspects of neuroendocrine control of nitrogen metabolism of the freshwater field crab
Paratelphusa hydrodromus (Herbst) Ph.D. thesis, Sri Venkateswara University, Tirupati
Raghavaiah K, Ramamurti R, Chandrasekharam V and Scheer B T 1980 Neuroendocrine control of nitrogen
metabolism in the Indian field crab Oziotelphusa senex senex Fabricus. I. End products and elimination;
Com/?. Biochem. Physioi B67 437-445
Rangneker P V and Madhyastha M N 1971 Effect of eyestalk ablation on the carbohydrate metabolism of the
prawn Metapenaeus monoceros (Fab.); Indian J. Exp. Biol. 9 462--464
Rangneker P V, Sabins P B and Nirmal R B 1961 The occurrence of a hypoglycemic factor in the eyestalks of
freshwater crab, Paratelphusa jacquimontii; J. Anim. Morphol Physioi. 3 137-144
Scheer B T and Scheer M A 1951 The hormonal regulation of metabolism Jn crustaceans. I. Blood sugar in
spiny lobsters; Physioi. Comp. 2 198-209
Sedlmeier D and Keller R 1981 The mode of action of the crustacean neurosecretory hyperglycemic
hormone: 1. Involvement of cyclic nucleo tides; Gen. Comp. Endocrinol. 45 82-90
Skinner D M 1966 Macromolecular changes associated with the growth of crustacean tissue; Am. Zool. 6
235-242
Snedecor G W and Cochran W G 1967 Statistical methods (Iowa: The Iowa State University Press).
Telford M 1975 Blood glucose level in crayfish III. The source of glucose and role of the eyestalk factor in
hyperglycemia of Cambarus robustus; Comp. Biochem. Physioi B51 69-73
Wang D H and Scheer B T 1963 UDPOglycogentransglucosylase and a natural inhibitor in crustacean tissue;
Comp. Biochem. Physioi. 9 263-274
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 481-488.
© Printed in India.
Fecundity of the allochthonous feeder, Rasbora daniconius (Ham.) and
of the autochthonous feeder, Puntius amphibius ( VaL)
K PREM KUMAR, P A JOHN and N K BALASUBRAMANIAN
Department of Aquatic Biology and Fisheries, University of Kerala, Trivandrum 695007,
India
MS received 4 July 1984; revised 22 February 1985
Abstract. The fecundity of an allochthonous feeder, Rasbora daniconius and an autochthon-
ous feeder, Puntius amphibius in a perennial tropical pond was assessed and found to be higher
in the latter. The fecundity of each of the species was correlated with parameters like standard
length, weight offish, length and weight of ovary. A comparison of the regression coefficients
in the relations statistically confirmed that as the length or weight of the fish and the length of
the ovary increased the rate of increase in the number of eggs was greater in P. amphibius
suggesting a better assimilation of the autochthonous food by P. amphibius than of the
allochthonous food by R. daniconius.
Keywords. Allochthonous feeder; Rasbora daniconius; fecundity; Puntius amphibius; auto-
chthonous feeder.
1. Introduction
Knowledge about the number of eggs produced by fishes is of great importance for
aquaculture. Qasim and Qayyum (1963) discussed the different ways in which
knowledge about the fecundity would be useful to the fishery biologist. Fecundity
studies have been carried out on a large number of freshwater fishes of India (Alikunhi
and Chaudhuri 1954; Qasim and Qayyum 1963; Bhatnagar 1964, 1972; Das 1964;
Parameswaran et al 1971; Saxena 1972; Selvaraj et al 1972; Varghese 1973; Sinha 1975;
Bhatt et al 1977; Jhingran 1977; Pathak and Jhingran 1977; Bisht and Upadhay 1979;
Pathani 1981). In the present paper the fecundity of an allochthonous feeder, Rasbora
daniconius and an autochthonous feeder, Puntius amphibius is compared in relation to
the type of food they consume.
2. Material and methods
The fishes were collected wild from a perennial pond during their breeding season (June
to August). Various body measurements were taken before cutting open the abdomen
and removing the ovary. On the whole 55 ripe ovaries of R. daniconius (47 and 88 mm
standard length) and 47 ovaries of P. amphibius (65 and 105 mm standard length) were
used for the study. For calculating the fecundity, the gravimetric method by which the
number of eggs in accurately weighed subsamples being multiplied by the total weights
of the ovaries was adopted. The final figure of fecundity was arrived at based on the
average of the weights and number of eggs in three subsamples of each ovary.
481
482 K Prem Kumar, P A John and N K Balasubramanian
In both the species the .correlation between the fecundity and parameters like
standard length, weight of fish, length and weight of ovary was calculated. These
relationships were studied by the method of least squares according to which either the
linear equation, 7 = a 4- b X or log Y = log a 4- b log X, a linear transformation of Y
= axb, was fitted, where 7 stood for fecundity and a and b were constants; X stood for
body measurements such as standard length (SI); total body weight (Bw); length of
ovary (O/) and ovary weight (Ow). The constants a and b were calculated in each case.
3. Results and discussion
The fecundity varied between 929 and 7398 in R. daniconius and from 3379 to 24485 in
F. amphibius. The higher fecundity of the latter clearly highlights a higher rate of egg
production in P. amphibius than in jR. daniconius.
According to table 1, the relationship between the fecundity (F) and the total body
weight, the length and weight of ovary was linear (figures 2, 3 and 4) and that between
fecundity and standard length of the fish, was curvilinear (figure 1) in R. daniconius. In
P. amphibius, the relationship between fecundity and total body weight of the fish and
length of ovary was linear (figures 6 and 7 respectively) and that between fecundity and
standard length of the fish and weight of ovary was curvilinear (figures 5 and 8
respectively) suggesting that the pattern of relationship between fecundity and other
parameters, except the weight of ovary was similar in both R. daniconius and F.
amphibius.
The correlation coefficient worked out between the fecundity and other parameters
were significant in both the species (F< 0-01).
Therefore, as the length or weight offish and the length of ovary increased, the rate of
increase in the number of eggs was greater in the autochthonous feeder, F. amphibius
than in the allochthonous feeder, R. daniconius. This suggests that the autochthonous
food in the pond, though according to the chemical analysis was less nutritive than the
allochthonous food is better made use of by F. amphibius which may result in higher egg
production also. This is natural because F. amphibius was found to have better
Table 1. The regression and correlation coefficients between the fecundity of K. daniconius
and P. amphibius and other variables of each species with equations used in each case.
Species
Variable
Equation
Regression
coefficient
Correlation
coefficient
SI
Log F = - 0-2693 -f 2-0441 log SI
2-0441
0-7138*
Bw
482-0670+0-4315 Bw
0-4315
0-7350*
R. daniconius
01
-2537-8400 4-1 8-2253 Ol
18-2253
0-6260*
Ow
166-2626 + 3-0051 Ow
3-0051
0-8866*
SI
Log F = - 1-2835 + 2-6633 log SI
2-6633
0-6904*
Bw
- 1989-5533 + 0-6023 Bw
0-6023
0-7893*
P. amphibius
01
12085-8130+518-6034 01
518-6034
0-7200*
Ow
Log F = 9-9793 + 0-8765 log Ow
0-8765
0-9581*
* Significant (P < 0-01)
Fecundity of R. daniconius and P. amphibius
483
3-8
3-6
o
o
3-2
30
"1
1-67
1-71
~~]
175
1-79 1-83
LOG- STANDARD LENGTH {mm
Figure 1. Standard length-fecundity relationship in £. daniconius.
1-87
8000-
6000-
>-4000
h-
0
2
O
2000-
_1 , — _
9000
1000
3000 5000 7000
FISH WEIGHT (mg )
11000
Figure 2. Body weight-fecundity relationship in R. daniconius.
484 K Prem Kumar, P A John and N K Balasubramanian
7000-
5000-
o
z
3000
1000
~~22~
30
34
38
LENGTH OF OVARY (mm)
Figure 3. Length of ovary-fecundity relationship in JR. daniconius.
42
8000-
6000-
4000-
z:
2000-
300 700 1100 1500
OVARY WEIGHT (mg)
Figure 4. Ovary weight-fecundity relationship in R. daniconius.
1900
2300
4.40-1
24000-
15000-
>-
H
0
Fecundity of R. daniconius and P. amphibius
1-82
1-86 1-90 1-94
LOG- STANDARD LENGTH
1-98
Figure 5. Standard length-fecundity relationship in P. amphibius.
485
2-02
6000-
10000
15000 20000
FISH WEIGHT (mg )
25000
Figure 6. Body weight-fecundity relationship in P. amphibius.
30000
486 K Prem Kumar, P A John and N K Balasubramanian
24000
15000
6000-
4-4
30 36 42
LENGTH OF OVARY (mm)
Figure 7. Length of ovary-fecundity relationship in P. amphibius.
48
52
3-2
3-5
LOG- OVARY WEIGHT (mg )
Figure 8. Ovary weight-fecundity relationship in P. amphibius.
3-8
Fecundity of R. daniconius and P. amphibius 487
conversion efficiency than jR. daniconius (Prem Kumar and John 1984). It may also be
more easy for the fish to absorb the nutrients from the organisms living in the same
medium than from the terrestrial organisms.
The curvilinear relationship between the fecundity and standard length as seen in .R.
daniconius and P. amphibius agrees with the findings of Gupta (1968) and Sinha (1975).
But, according to Qasim and Qayyum (1963) in fishes which seldom grow more than a
few inches in length there exists a linear relationship between body length and
fecundity.
A linear relationship between fecundity and fish weight similar to that in R.
daniconius and P. amphibius has been reported in Labeofimbriatus by Bhatnagar (1972),
in P. sarana by Sinha (1975) and in JR. daniconius by Nagendran et al (1981). Yuen
(1955), however, has reported a curvilinear relationship between fecundity and fish
weight in the big eye tuna.
The linear relationship between the ovary weight and fecundity as seen in R.
daniconius is a common feature which has been reported in several fishes (Pantalu 1963;
Qasim and Qayyum 1963; Bhargava 1970; Bhatt et al 1977; Nagendran et al 1981). On
the other hand, the curvilinear relationship which was found to exist between the
fecundity and ovary weight in P. amphibius has also been reported in other freshwater
fishes (Rita Kumari 1977; Babu 1981).
Acknowledgement
One of the authors (KPK) is grateful to the UGC for the award of a fellowship during
the tenure of which the present work was carried out.
References
Alikunhi K H and Chaudhuri H 1954 On the life history and bionomics of the carp minnow, Chela phulo
(Hamilton); Proc. Indian Acad. Sci. B39 76-90
Babu N 1981 Studies on the genus Amblypharyngodon Bleeker (Pisces-Teleostei-Cyprinidae) from South
India Ph.D. Thesis, University of Kerala, India 520 pp.
Bhargawa R M S 1970 The fecundity ofHeteropneustes fossilis (Bloch); J. Bombay Nat. Hist. Soc. 67 583-588
Bhatnagar G K 1964 Spawning and fecundity of Bhakhara reservoir fishes; Indian J. Fish. 11 485-502
Bhatnagar G K. 1972 Maturity, fecundity, spawning season and certain related aspects of Labeofimbriatus
(Bloch) of river Narmada near Hoshangabad; J: Inland Fish. Soc. India 4 26-37
Bhatt V S, Dalai S G and Abidi S A H 1977 Fecundity of the freshwater catfishes Mystus seenghala (Sykes),
Mystus cavasius (Ham.), Wallagonia attu (Bloch) and Heteropneustes fossilis (Bloch) from plains of
northern India; Hydrobiologia 54 219-224
Bisht J S and Upadhyay J C 1979 Studies on fecundity of a freshwater teleost, Barilius bendelisis Ham.; II All
India Seminar on Ichthyology Abst. p. 95
Das S M 1964 A study on the fecundity of some freshwater fishes of India with a note on a new concept of
comparative fecundity; Ichthyology 3 33-36
Gupta M V 1968 Observations on the fecundity of Polynemus paradiseus Linn, from the Hooghly estuarine
system; Proc. Nat. Inst. Sci. India 34 330-345
Jhingran V G 1977 Fish and fisheries of India (New Delhi: Hindustan Publishing Corporation) 954 pp
Nagendran R, Katre Shakuntala, Natarajan G N and Vasan H R K 1981 Observations on the fecundity of the
cyprinid Rasbora daniconius (Hamilton); Proc. Indian Acad. Sci. (Anim. Sci.) 90 381-388
Pantalu V R 1963 Studies on the age, growth, fecundity and spawning of Osteogeneiosus militaris (Linn.); J.
Consperm. Int. Explor. Mer. 28 295-315
488 K Prem Kumar, P A John and N K Balasubramanian
Parameswaran S, Radhakrishnan S and Selvaraj C 1971 Some observations on the biology of the carp
minnow, Osteobrama cotio (Hamilton); J. Inland Fish. Soc. India 3 103-113
Pathak S C and Jhingran A G 1977 Maturity and fecundity of Labeo calbasu (Ham.) of Loni reservoir, M. P.;
/. Inland Fish. Soc. India 9 92-93
Pathani S S 1 98 1 Fecundity of mahaseer Tor putitora (Ham.); Proc. Indian Acad. Sci. (Anim. Sci.) 90 253-260
Prem Kumar K and John P A 1984 Observations on the comparative efficiency of the allochthonous and
autochthonous food of fishes based on their biochemical analysis; An. Centra Cienc. Mar Limnol. Univ.
Nai Auton. Mexico (in press)
Qasim S Z and Qayyum A 1963 Fecundities of some freshwater fishes; Proc. Nat. Inst. Sci. India 29 373-382
Rita Kumari S D 1977 Studies on the loaches of Kerala Ph.D. thesis, University of Kerala, India 538 pp
Saxena O P 1972 Studies on the maturity and fecundity of Rita rita (Hamilton) of Ganga river system; J.
Inland Fish. Soc. India 4 169-182
Selvaraj C, Radhakrishnan S and Parameswaran S 1972 Notes on the breeding season, fecundity and life
history of a minor carp, Labeo boggut (Sykes); J. Inland Fish. Soc. India 4 87-97
Sinha M 1975 Observations on the biology of Puntius sarana (Hamilton) of Loni reservoir (Madhya
Pradesh). 2. Maturity and fecundity; J. Inland Fish. Soc. India 7 49-57
Varghese T J 1973 The fecundity of the rohu, Labeo rohita (Hamilton); Proc. Indian Acad. Sci. (Anim. Sci.)
B77 214-224
Yuen H S H 1955 Maturity and fecundity of big eye tuna in the Pacific; Spec. Sci. Rep. U.S. Fish Wildl. Ser.
Fish. 150 30 pp
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 489-501.
© Printed in India.
Development, endocrine organs and moulting in the embryos of
Dysdercus cingulatus Fabr (Heteroptera: Pyrrhocoridae)
MARIAMMA JACOB and V K K PRABHU
Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695581, India
MS received 17 December 1984
Abstract. Embryonic development of Dysdercus cingulatus is briefly described. In the
embryos, neurosecretory cells become evident in the median, lateral and ventral aspects of
protocerebrum 84 hr after egg laying. The corpora cardiaca, the corpora allata and the
prothoracic glands arise more or less simultaneously at 78 hr from the dorsolateral wall of the
stomodaeum, from the mandibular segments and from the labial segments respectively.
Secretory material appears in the brain neurosecretory cells and in the intrinsic cells of the
corpus cardiacum at 84 hr and in the nervi corpori cardiaci and aorta at 90 hr. The cells of the
prothoracic glands show signs of secretory activity at 90 hr, reaching maximum activity around
96 hr. The corpus allatum appears to be inactive in the embryo. Two embryonic moults appear
between 96 hr and 1 10 hr. Consequence of events suggests that the neurosecretory material
stimulates the prothoracic glands including embryonic moulting.
Keywords. Neurosecretory cells; corpora cardiaca; corpora allata; prothoracic glands;
secretory activity; embryonic moulting; Dysdercus cingulatus
1. Introduction
Endocrine activity during insect embryogenesis appears to have been first reported by
Jones (1956a,b) in Locusta pardalina and Locusta migratoria. Ontogeny of the
neurosecretory cells was studied by Khan and Fraser (1962) in Periplaneta americana,
and by Dorn (1972, 1975b) in Oncopeltusfasciatus. Development of corpora cardiaca
was traced in the embryos ofMelanoplus differentialis (Baden 1936), Rhodnius prolixus
(Mellanby 1936), Drosophila (Poulson 1937) and in Oncopeltusfasciatus (Dorn 1972,
1975c). Development of the corpus allatum has been worked out in Oncopeltusfasciatus
(Dorn 1972). Earlier work on the allatum includes that of Nussbaum (1889), Wheeler
(1893), Mellanby (1936) and Roonwal (1937). Development of prothoracic glands has
been studied by Toyama (1902) in the silkworm, and Darquenne (1978) in Leucophaea
maderae. Sharan and Sahni (1960) reported the presence of neurosecretory cells in the
embryos of Dysdercus cingulatus and Wells (1954) studied the development of its
prothoracic glands. The purpose of the present investigation has been to study in detail
the development of the endocrine glands in the red cotton bug Dysdercus cingulatus,
and to find out if there is any interrelationship between the endocrine activity and
embryonic moulting in this animal.
2. Materials and methods
Stock colony of Dysdercus cingulatus was reared in the laboratory as described by
Jalaja and Prabhu (1976). Pairs were kept isolated in glass chimneys on soaked cotton
48$
490 Mariamma Jacob and V KK Prabhu
seeds, and the eggs laid in clutches of 100-150 during the first gonotrophic cycle were
collected immediately after laying. Thus eggs of known age were available for the study.
2.1 Development of the embryo and the endocrines
Development of the embryo, the neurosecretory cells, the corpora cardiaca, the corpora
allata and the pro thoracic glands was studied after fixing the eggs in warm Smith's fluid.
After processing for general embryology, sections were stained in Heidenhain's iron
haematoxylin and eosin; paraffin sections were stained in Gomori's chrom alum
haematoxylin phloxin (Gomori 1941) for the study of the cerebral neuroendocrine
complex. The brain was also dissected out from four-day and five-day old embryos and
fixed in formol saline, and stained using Humberstone's performic acid victoria blue
technique (Dogra and Tandon 1964), for whole mounts.
2.2 Measurements
All measurements were taken using a calibrated ocular micrometer. In the embryo,
there were spherical, oval and pear-shaped neurosecretory cells. The diameter of the
spherical cells, the mean of the length and the breadth of the oval and pear-shaped cells
were taken and the volume of the cells calculated (see below). In the embryonic
prothoracic glands, there were spherical and oval cells. The diameter of the spherical
cells and mean of the length and the breadth of the oval cells were found. The diameter
of nuclei was also measured. The volume of spherical cells was calculated using the
formula 4/3 nrz where r was the radius, while that of oval cells was calculated using the
formula 4/3 n ab2 where a and b represented half the length and breadth respectively
(Penzlin 1971). The volume of the nuclei was also calculated using the first formula.
The length and the breadth of the corpus cardiacum were noted and the approximate
number of cells in this organ was estimated from the number of nuclei counted. The
volume of the corpus allatum was calculated according to the method of Scharrer and
Von Harnack (1960). The number of their nuclei was counted from serial sections and
the volume of the allatum per nucleus was calculated. The diameter of the corpus
allatum nuclei was also measured and the nuclear volume calculated.
3. Observation
3.1 Development of the egg
The main events during embryonic development are summarised in table 1. The eggs
which are centrolecithal, undergo first cleavage at 9 hr. The divisions are initially
synchronous, but by 16 hr become asynchronous. By 24 hr, cleavage is complete, most
of the nuclei move to the periphery, leaving behind a few remaining nuclei scattered
among yolk granules. The nuclei at the periphery undergo rapid proliferation leading
to the formation of a thin layer of blastoderm. The whole yolk is enclosed in a cellular
blastoderm by the end of 35 hr. Between 36 hr and 38 hr the cells of the blastoderm
begin to aggregate at the posterior pole towards the ventral side. The cells of the
Development, endocrine organs and moulting in Dysdercus embryos 491
Table 1. Table showing stages of embryonic development in
Dysdercus cingulatus
Number
Stages
Hours of
development
1 Oviposition and Maturation 0-8
2 Cleavage 8-24
3 Blastoderm 24-36
4 Germ Band 36-49
5 Gastrulation 49-51
6 Segmentation 51-74
(a) Differentiation of mesoderm 52-53
(b) Differentiation of neuroblast 52-55
(c) Fusion of amniotic folds 55-70
(d) Differentiation of stomodaeum
and proctodaeum 60-74
7 Blastokinesis 74-84
(a) Anatrepsis 74-78
(b) Katatrepsis 78-84
8 Definitive dorsal closure and
absorption of dorsal organ 84-90
9 Consolidation of nervous system
and Pigmentation of eye and
abdomen become apparent 90-96
10 Embryo-larval transition 96-120
(a) I moult 96-98
(b) Thoracic legs develop claws 98-108
(c) II moult 108-110
(d) Pigmentation deepened 110-120
11 Hatching 120-124
remaining portion of the blastoderm get flattened and form the extra-embryonic
blastoderm. The embryonic primordium or the germ band is formed by tubular
invagination into the yolk. Rarely two lateral, faintly marked and irregular thickenings
develop, which later fuse together from which a tubular invagination arise. By 49 hr the
germ band reaches maximum size, the blunt end extending half way along the dorsal
side, rather superficially. The thinner, inner wall of the germ band is the amnion; the
future embryo develops from the thicker outer wall; the anterior end of germ band is
comparatively broader representing the head region of the future embryo.
Gastrulation takes place between 49 and 51 hr by migration of the cells proliferating
from the longitudinal groove along the germ band. These cells form a single layer below
the original layer by 55 hr, the latter being now ectoderm. By 60 hr the germ band gets
segmented, with appendage rudiments on the head and thorax. By 65-70 hr the serosa
covering the entire egg, and the amnion covering the embryo are established.
Segmentation is distinguishable first in the thoracic region; then the cephalic region
and finally the abdominal region get segmented. In 51-52 hr old embryos, four
transverse grooves appear at the thoracic region giving rise to the three thoracic
segments, the cephalic region and the abdominal region. In close connection with the
head lobe appear three pairs of appendage rudiments: the small labral, the larger
antennal and smaller mandibular rudiments, one behind the other. Below these are the
492 Mariamma Jacob and V KK Prabhu
maxillary and the labial segments with their appendage rudiments. Rudiments of the
thoracic legs also develop on the thoracic segments. In the abdomen appear eleven
segments demarcated by transverse grooves. The head lobes are pushed in from the
surface between 65-70 hr and are covered by amnion. A fully segmented germ band
with rudiments of appendages on head and thorax was visible by 72 hr (figure 1).
The stomodaeum is in the form of a shallow depression at 60 hr, but becomes a
conspicuous tube by 74 hr. The proctodaeal invagination is also visible now;
subsequently, the cells at the tip of the proctodaeal invagination proliferate to form the
posterior enteron rudiment. The cells at the tip of the developing stomodaeum spread
over the already formed inner layer to form the anterior enteron rudiment from which
cells grow gradually to form the wall of the midgut. The inner layer which gets
separated during gastrulation constitutes the mesoderm, and this layer at anatrepsis
differentiates to somatic and splanchnic mesoderm (figure 4).
A continuous neural groove from the anterior to the posterior region is seen by 65 hr,
and neuroblasts develop from the neural ridges situated on either side of the groove.
Between 65 and 70 hr segmentally arranged ganglionic masses differentiate and by
78 hr the ventral nerve cord is completely separated from the ectoderm (figure 4).
However, the rows of neuroblasts in the mandibular segment diverge on either side of
the stomodaeum and are hence further apart in the head lobe. At 72 hr neuroblasts are
peripherally arranged; neurons are subsequently set apart from neuroblasts in the head
region, thus forming the three ganglionic masses: the proto, deuto, and the tritocer-
ebrum. A well-developed neuropile becomes evident only after rotation of the embryo.
The nervous system of the embryo just after blastokinesis consists of the brain, the
three thoracic ganglia and 1 1 abdominal ganglia. By 90 hr the abdominal ganglia get
consolidated to form a single ganglionic mass attached to the last thoracic ganglion.
Anatrepsis, the folding of the appendages to the ventral side, and shortening of the
germ band as a whole, constituting the first phase of blastokinesis is over by 78 hr
(figure 2). The second phase or rotation of the embryo, constituting katatrepsis (figure
3) is over by 84 hr; the provisional dorsal closure is replaced by permanent embryonic
ectoderm by 90 hr. The dorsal organ is now absorbed by the yolk.
3.2 Endocrine organs
3.2a Neurosecretory cells: After blastokinesis, at 84 hr of development when the
brain develops neuropile, the neurosecretory cells become evident, when many of them
already contain quite a lot of neurosecretory material (table 2) stainable blue with
Gomori's haematoxylin. The cells may be either oval, spherical or pear (figure 8)
shaped. They are distributed in the median, lateral and ventral aspects of the
protocerebrum (figure 9). The data on these cells during embryonic development are
shown in table 2. The secretory material gradually decreases in quantity from around
94 hr. Cell size as well as nuclear size of the neurosecretory cells, which may be taken as
an index of their activity, also decreases from 96 hr onwards or slightly earlier. Later on
fifth day towards hatching, the number of recognizable neurosecretory cells are very
few as many of them have emptied their secretory content; the cells as well as the nuclei
also dwindle substantially in size, indicating very low activity.
Development, endocrine organs and moulting in Dysdercus embryos 493
• 03mm
aJb
pg
Figures 1-5. Camera lucida sketches. (1) 72 hr old embryo showing fully segmented germ
band with developed appendages, dorsal view. (2) 78 hr old embryo showing anatrepsis. Note
that the appendages are withdrawn from the surface of the yolk and folded on the ventral
surface of the embryo, ventral view. (3) 82 hr old embryo showing advanced katatrepsis, where
the head reached almost at the anterior pole of the egg, ventral view. (4) Cross-section through
germ band showing differentiation of mesoderm and nerve cord. (5) Frontal section through
embryo at 78 hr. Origin of corpora cardiaca, corpora allata, and prothoracic glands have been
shown (Abbreviations: ab — abdomen; ad — appendage; am — amnion; amc — amniotic cavity;
an — antenna; ca — corpus allatum; cc — corpus cardiacum; cs — contracted serosa; dc —
deiitocerebrum; ec — ectoderm; h — head; la — labial appendage; mna — mandibular appendage;
mxa — maxillary appendage; nb — neuroblast; nc — nerve cord; ne — neuron; oe — oesophagus;
pc — protocerebrum; pg — prothoracic gland; s — serosa; sg — salivary gland; sm — somatic
mesoderm; spm — splanchnic mesoderm; ta — thoracic appendage; tc — tritocerebrum; tl —
thoracic legs; y — yolk).
3.2b The corpora cardiaca: Rudiments of the corpora cardiaca make their appear-
ance at 78 hr during anatrepsis (figure 5). The corpora cardiaca originate as a string of
cells on either side from the dorsolateral walls of the stomodaeum (figure 10). After
blastokinesis they appear triangular, placed behind the brain on either side of
oesophagus, below the aorta. The cells of the cardiaca are confined to the posterior
portion of the organs whereas the anterior portion is composed of nerve fibres. The
494
Mariamma Jacob and V KK Prabhu
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Development, endocrine organs and moulting in Dysdercus embryos 495
to
>-ao
Figures 6-9. Microphotographs. (6) Sagittal section through 98 hr old embryo showing first
embryonic moult. See the separating first cuticle and the depositing second cuticle x 700. (7)
Sagittal section through 108 hr old embryo showing second moult. Original cuticle has already
been removed along with chorion and hence not visible in section. Separation of second cuticle
and deposition of third cuticle are shown x 700. (8) Frontal section through brain of four-day-
old embryo showing lateral pear shaped neurosecretory cell x 700. (9) Whole mount of brain
of four-day-old embryo showing median, lateral and ventral neurosecretory cells x 400 (ao —
aorta; br — brain; Ic — first cuticle; lie — second cuticle, IIIc — third cuticle; ec — ectoderm;
Insc — lateral neurosecretory cells; m — muscle; mnsc — median neurosecretory cells; nsc —
neurosecretory cells; vnsc — ventral neurosecretory cells; y — yolk)
corpus cardiacum is about 30-35 jan long, 20-25 /im broad and 20 /xm thick. No
change in size is observed in the cardiaca during further embryonic development. There
are altogether 25-30 cells in a cardiacum. Of these only five to six cells, which occupy the
posterior portion, are stainable blue by Gomori's method and thes^ constitute the
intrinsic secretory cells of the cardiacum. The remaining cells, the chromophobe cells,
which do not contain any stainable material, surround the nerve fibres; the nerve fibres
hence occupy the central region of the cardiacum.
496 Mariamma Jacob and V K K Prabhu
3.2c Stainability of the brain-cardiacum tract: As already reported above, at 84 hr,
the brain neurosecretory cells and five to six cells of the corpus cardiacum are stainable
blue by Gomori's method. At this time the tract from the brain or the nerve fibres of the
cardiacum is not stainable. Evidently, transportation of secretory material to car-
diacum has not yet started. However at 90 hr both the tracts become stainable
indicating initiation of transportation. At 102 hr, though in the brain neurosecretory
cells there is decrease in their neurosecretory content and many of these cells are
scarcely recognizable, the connecting nerve tracts are stainable due to transportation of
material. At 120 hr only a few neurosecretory cells in the brain are distinguishable and
they show very little neurosecretory material. The secretory material decreases in the
connecting tract and the nerve fibres in the cardiacum. By now the Stainability of the
cardiacum also decreases. Apparently, most of the material from the nerve tract and
cardiacum has now been released.
3.2d Neurosecretory material and the aorta: Till 84 hr, there is very little evidence
for the presence of neurosecretory material in the aorta wall. But there is gradual
accumulation of secretory material in the aorta thereafter and by 96 hr, the aorta
contained as much secretory material as in the nervi corpori cardiaci (figure 11).
However, by 120 hr all the secretory material in the aorta appears to have been released
as there is hardly any secretory product in the aorta now.
3.2e Corpora allata: The corpora allata develop simultaneously with corpora
cardiaca as paired invaginations from the mandibular segments at 78 hr during
anatrepsis (figure 5) and migrate in the anterodorsal direction. They finally come to be
situated behind the corpora cardiaca attached to the oesophagus below the aorta
(figure 10). Each corpus allatum has now about 35 cells which are closely packed. The
corpora cardiaca and the corpora allata are seen in close association after blastokinesis.
The corpora allata do not appear to undergo any change with regard to size during
embryonic development (table 3). Their nuclei however undergo a further increase to
over 50 in number.
3.2f Prothoracic glands: The primordia of the prothoracic glands make their
appearance at the time of anatrepsis (figure 5). They originate as a small pair of
invaginations from the labial segments along with a larger pair of invaginations (the
future salivary glands). After blastokinesis the salivary gland rudiments extend
backwards into the thorax and the prothoracic gland rudiments are also drawn along
with it. There are 90-100 cells in the prothoracic glands of a four-day-old embryo.
Prothoracic gland is a ribbon of cells which are loosly embedded in their connective
tissue. During 78-84 hr, the glands consist of oval as well as spherical cells, the majority
being oval. The changes undergone by the cells are shown in table 4 (figure 12). Around
96 hr the cell size and nuclear size have increased to about 8 times. By 120 hr both the
cells as well as the nuclei have again become smaller, shrinking to their original size,
indicating an apparant cessation of activity.
3.3 Embryonic moults
By 96-98 hr the first cuticle appears to separate from epidermis and a new one is
Development, endocrine organs and moulting in Dysdercus embryos 497
Figures 10-12. Microphotographs. (10) Frontal section through 78 hr old embryo showing
the development of corpora cardiaca and corpora allata x 700. (11) Frontal section through
brain of four-day-old embryo showing close association of corpora allata and corpora cardiaca
and .the presence of secretory material in the aorta and nervi corpori cardiaci x 700. (12)
Sagittal section through 84 hr old embryo showing prothoracic gland cells x 1 000 (ao — aorta;
br — brain; ca — corpora allata; cc — corpora cardiaca; n — nucleus; ncc — nervi corpori cardiaci;
oe — oesophagus; ov — oval cells; sp — spherical cells; st — stomodaeum).
Table 3. Changes in corpus allatum of Dysdercus cingulatus during embryonic development
(data represent mean values of cells from six embryos at each hour)
Development
Volume of
Number of
Mean cell
Nuclear
Nuclear
hour
Ca jum3
nuclei
volume
size
volume
84
3401
53
64-16
2-33
6-61
96
3924
60
65-40
2-33
6-61
120
3640
58
62-75
2-33
6-61
observed attached to the epidermis, constituting the first embryonic moult (figure 6).
Similarly, at 108-1 10 hr a thicker cuticle is observed close to the epidermis, the second
cuticle having separated from it, constituting the second moult (figure 7). During the
first moult muscle fibers are not well differentiated whereas during the second moult
thick muscle fibers are seen.
498 Mariamma Jacob and V K K Prabhu
Table 4. Changes in cell size and nuclear size of prothoracic glands in the embryos of
Dysdercus cingulatus during development (data represent mean values of cells from six
embryos at each hour)
Development
(hours)
Cell diameter
(urn)
Cell
volume
/im3
Nuclear
diameter
jj.ni
Nuclear
volume
/im3
Spherical cells
Oval cells
84
4-67
53-32
2-33
6-61
1 = 5-8
b = 4-28
55-60
2-11
4-91
96
9-32
—
423-88
4-61
51-29
120
5-16
—
71-93
2-16
5-27
1 = 5-61
b = 4-80
67-64
2-20
5-57
4. Discussion
The pattern of embryonic development in Dysdercus cingulatus follows the general
heteropteran pattern (Anderson 1972). The blastoderm in this animal forms a median
thickening, though very rarely two lateral thickenings are also observed, when they are
rather irregular and faintly marked on the surface of the embryo, as in Oncopeltus
fasciatus (Butt 1949) but unlike Blissus leucopterus hirtus (Choban and Gupta
1972), Rhodnius prolixus (Mellanby 1935) and Pyrrhocoris apterus (Seidel 1924). The
developing germ band lay close to the ventral surface of the eggs of Dysdercus
cingulatus as in Pyrrhocoris apterus (Seidel 1924) and in Oncopeltus fasciatus (Butt
1949). However, the germ band of Rhodnius prolixus lay superficially on the dorsal side
of the egg (Mellanby 1935) whereas in Blissus leucopterus hirtus (Choban and Gupta
1972) it is neither on the dorsal side nor on the ventral side, but it took a position in
between.
In Dysdercus cingulatus, the present studies revealed that masses of cells proliferated
from the embryonic layer, -migrating into the yolk. Subsequently, they arranged
themselves as a layer inner to the original layer, forming the mesoderm as in Blissus
(Choban and Gupta 1972). In Locusta, Roonwal (1936) proposed a multiphased
gastrulation. In Pyrrhocoris gastrulation was by sinking of a ridge of cells without a
lumen into the yolk (Seidel 1924), and consisted not only of invagination but also
delamination of the inner layer corresponding to the entomesoblast whose cells
differentiated from the ectoblast (Matolin 1973). In Rhodnius prolixus however the
central part of the embryonic rudiment invaginated, which overgrew to the lateral
portions (Mellanby 1935).
In the head region, six segments got differentiated during embryonic development in
Dysdercus cingulatus, as in Oncopeltus fasciatus (Butt 1949) and in Blissus (Choban and
Gupta 1972), with five pairs of appendage rudiments viz. labral, antennal, mandibular,
maxillary and labial. No appendage rudiment was observed on the first abdominal
segment in Dysdercus cingulatus as in Blissus (Choban and Gupta 1972) or in
Oncopeltus (Butt 1949) though Dorn (1972) and Matolin (1973) observed a pleuro-
podium in Oncopeltus fasciatus and Pyrrhocoris apterus respectively. A prominent
pleuropodium was also present in Belostoma and Ranatra (Hussey 1926).
Development, endocrine organs and moulting in Dysdercus embryos 499
In Dysdercus cingulatus, the anterior and posterior enteron rudiments developed
independently of the inner layer by proliferation of ectoderm cells from the tip of the
stomodaeal and proctodaeal invaginations, unlike in Pyrrhocoris (Seidel 1924) and in
Rhodnius prolixus (Mellanby 1935) in which the enteron arose from masses of cells
from the anterior and posterior ends of the inner layer.
Neurosecretory cells have been reported earlier also in the embryos of many insects
(Khan and Eraser 1962; Dorn 1972; Jones 1956a), including Dysdercus cingulatus
(Sharan and Sahni 1960; Mariamma Jacob and Prabhu 1979), but the present studies
give details of the secretory activity of the cells and their relationship with other
endocrine glands during development, as well as moulting. The corpora cardiaca in the
embryos of Dysdercus arose from the stomodaeum as in other insects (Mellanby 1936;
Baden 1936; Pflugfelder 1937; Poulson 1937; Roonwal 1937; Dorn 1972). The allatum
in the embryo of Dysdercus cingulatus arose from mandibular segments as ectodermal
invaginations, though they appeared to arise from mandibular and maxillary segments
in Carausius morosus (Leuzinger et al 1926), and in Locusta migratoria (Roonwal 1937)
and from maxillary segments in Oncopeltus fasciatus (Dorn 1972), Rhodnius prolixus
(Mellanby 1936). The present studies in Dysdercus cingulatus showed that the allatum is
apparently inactive during embryonic development of the animal, as it did not show any
cyclic changes in the embryo. This is in step with the findings of Jones (1956a) and
Sharan and Sahni (1960) though in Periplaneta americana and Oncopeltus fasciatus the
corpus allatum showed fluctuation in size suggesting secretory activity (Khan and
Fraser 1962; Dorn 1972, 1975a). The present studies show that in Dysdercus cingulatus
also prothoracic glands arose from labial segments as already reported in insects (Wells
1954; Dorn 1972; Darquenne 1978).
From the present studies, it is clear that there is secretory cycle in the brain
neurosecretory cells in the embryos of Dysdercus cingulatus. There were about 14
neurosecretory cells in the brain; their activity decreased from 84 hr to 120 hr while the
size of the cell and of nuclei and the secretory content were taken as the criteria. When
stainability of the cells, the nerve tract and the cardiacum are considered, there was clear
accumulation by 84 hr followed by a release of material which is complete by 120 hr
when the cells, the tract and the aorta, all become devoid of secretory material,
indicating that the release has already been taken place by 120 hr. It may be noted that
though in the adult Dysdercus cingulatus the neurosecretory tract from the A-cells does
not enter the corpus cardiacum, but bypasses it to enter the aorta (Jalaja and Prabhu
1977), in the embryo the tract passes through the cardiacum. This is comparable to the
condition in Oncopeltus fasciatus (Dorn 1975b, 1977). The prothoracic gland cells
tremendously increase in size from 84 hr to 96 hr when their nuclei also reach maximum
size indicating very high activity around this period, followed by gradual inactivity. It
would appear that the release of neurosecretory material stimulates prothoracic gland
activity which release its products around 96 hr and thereafter. Two embryonic moults
in Dysdercus cingulatus observed during 96-98 hr and 108-1 10 hr in the present study
are apparently connected with the release of moulting hormone by the prothoracic
glands during their secretory cycle reported above. The corpus allatum however does
not appear to play any role in the embryo. Possibly the maternal juvenile hormone
found in embryos (Gilbert and Schneiderman 1961) serves the purpose, whereas the
embryo appears to be capable of producing its own ecdysone required for embryonic
moults.
500 Mariamma Jacob and V KK Prabhu
Acknowledgements
One of the authors (MJ) is grateful to C.S.I.R., New Delhi, for a fellowship.
References
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S J Counce and C H Waddington (London, New York: Academic Press) 95-163
Baden V 1936 Embryology of the nervous system in the grasshopper Melanoplus differ entialis (Acrididae:
Orthoptera); J. Morphol 60 1959
Butt F H 1949 Embryology of the milkweed bug Oncopeltusfasciatus; Cornell Univ. Agr. Exp. Sta. Mem. 283
1-43
Choban R C and Gupta A P 1972 Meiosis and early embryology of Blissus leucopterus hirtus Montandon
(Heteroptera: Lygaeidae); Int. J. Insect Morphol. Embryol. 1 301-314
Darquenne J 1978 Developpement embryonnaire des glandes prothoraciques chez Leucophaea maderae
(Fabr) (Dictyoptera: Blaberidae); Int. J. Insect Morphol Embryol. 7 215-220
Dogra G S and Tandon B K 1964 Adaptation of certain histological techniques for in situ demonstration of
the neuroendocrine system of insects and other animals; Q. J. Microsc. Sci. 105 445-466
Dorn A 1972 Die endocrinen Driisen im Embryo von Oncopeltusfasciatus Dallas (Insecta: Heteroptera).
Morphogenese, Funktionsaufnahme, Beeinflussung des Gewebewachstums und Beziehungen zu den
embryonalen Hautungen; Z. Morphol Tiere 71 52-104
Dorn A 1975a Struktur und Funktion des embryonalen Corpus allatum von Oncopeltusfasciatus Dallas
(Insecta: Heteroptera); Verh. dtsch. zool Ges. 67 85-89
Dorn A 1975b Elektronmikroskopische studien uber des neurosekretorische system von Oncopeltusfasciatus
Dallas (Insecta: Heteroptera) wahrend der Embryonalentwicklung; Cytobiologie 10 227-234
Don A 1975c Elektronmikroskopische studien uber differenzierung und funktionsaufnahme der Corpora
cardiaca in Embryo von Oncopeltusfasciatus Dallas (Insecta: Heteroptera); Cytobiologie 10 235-248
Dorn A 1977 Hormonal control of Egg maturation and embryonic development in insects; in Advances in
invertebrate reproduction (eds) K G Adiyodi and R G Adiyodi (Karivellur: Peralam-Kenoth) pp. 451-481
Gilbert L I and Schneidennan H A 1961 The content of juvenile hormone and lipid in Lepidoptera. Sexual
difference and developmental changes; Gen. Comp. Endocrinol 1 453-472
Gomori G 1941 Observations with differential stains on human islets of Langerhans; Am. J. Pathol 17
395-406
Hussey P B 1926 Studies on the pleuropodia of Belostoma flumineum Say and Ranatrafusca Paliscot de
Beauvois; Entomol Am. 7 1-81
Jalaja M and Prabhu V K K 1976 Effect of the chemosterilants apholate and metepa on the ovaries of the red
cotton bug Dysdercus cingulatus Fabr. (Heteroptera: Pyrrhocoridae); Entomon I 43-53
Jalaja M and Prabhu V K K 1977 Endocrine control of vitellogenesis in the red cotton bug Dysdercus
cingulatus Fabr. (Heteroptera: Pyrrhocoridae); Entomon 2 17-29
Jones B M 1956a Endocrine activity during insect embryogenesis. Function of the ventral head glands in
Locust embryos Locusta pardalina and Locusta migratoria; J. Exp. Biol 33 174-185
Jones B M 1956b Endocrine activity during insect embryogenesis. Control of events in development
following the embryonic moult (Locusta migratoria and Locusta pardalina); J. Exp. Biol 33 685-696
Khan T R and Fraser A 1962 Neurosecretion in the embryos and late stages of the cockroach Periplaneta
americana (L); Mem. Soc. Endocrinol 12 349-369
Leuzinger H, Wiesmann R and Lehman F E 1926 Zur Kenntnis der anatomie und Entwicklungsgeschichte
der stabhae schrecke Carausius morosus; Gustav Fischer Jena 11 1-44
Mariamma Jacob and Prabhu V K K 1979 Effects of two juvenile hormone analogues on embryonic
neuroendocrine system of Dysdercus cingulatus (Heteroptera: Pyrrhocoridae); Entomon 4 205-208
Matolin S 1973 The embryonic development of Pyrrhocoris apterus (L) (Heteroptera: Pyrrhocoridae); Ada
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embryonen; Biol Zbl 9 516-522
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Penzlin H 1971 The effect of Neurohormone D upon the nuclear volume of the prothoracic gland in
Periplaneta americana L. (Blatteria); in Insect endocrines (eds) VIA Novak and K Slama (Academic
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Z. Wss. Zooi 149 477-512
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Roonwal M L 1936 Studies on the embryology of the African migratory locust Locusta migratoria
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175-244
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 503-508.
© Printed in India.
Seasonal variations and the role of neurosecretory hormones on the
androgenic gland of the prawn Macrobrachium lamerrii
R SAROJINI and G GYANANATH*
Department of Zoology, Marathwada University, Aurangabad 431 004, India
* Department of Biosciences, Sri Sathya Sai Institute, Prasanthinilayam 515 134, Andhra
Pradesh, India
Abstract. The androgenic gland of M . lamerrii is situated nearlhe seminal vesicle and along
the vas deferens of the male prawns. Histologically it is made up of cords of cells which are
arranged loosely. Histochemical analysis of the androgenic gland cells showed the presence of
cystine/cysteins, protein bound amino acid groups. Androgenic gland of M . lamerrii shows
signs of increased secretory activity during the sexually active phase of the male. In adult
prawns, eyestalk ablation results in the hypertrophy of androgenic gland. Brain and thoracic
ganglion extracts also showed enhanced secretory activity of androgenic gland and cor-
responding gonadal activity in the male prawns.
Keywords. Androgenic gland; eyestalk ablation; Macrobrachium lamerrii; eyestalk extract;
brain extract; thoracic ganglion.
1. Introduction
The androgenic gland which secretes androgenic hormones has been established as the
endocrine gland which is responsible for the differentiation of the primary, secondary
sexual characters in the malacostracan crustaceans (Charniaux-Cotton 1960, 1964).
Seasonal variations in the androgenic gland activity has been reported in the
crustaceans (Hoffman 1968, 1969). The neuroendocrine control of androgenic gland by
eyestalk ablation and eyestalk extract injections was experimented in crabs (Demeusy
and Veillet 1958; Rangnekar et al 1971). The above literature shows that the freshwater
prawns were neglected in this aspect which prompted us to note the seasonal cyclicity of
androgenic gland and the role of neurosecretory hormones on the androgenic gland of
the freshwater prawn, Macrobrachium lamerrii.
2. Material and methods
The androgenic glands (AG) used in the present study were obtained from intermoult
(C) prawns. The AG were dissected out and kept in crustacean saline for morphological
observations. For histological preparations, the AG were fixed in Bouins fluid and
sectioned at 8 /an, stained in Gomori's chrome alum hematoxylin phloxine (CHP). The
eyestalk ablation, eyestalk extract and brain, and thoracic ganglion extract injections
were done as described by Diwan and Nagabhushanam (1974).
Histochemical nature of the androgenic gland was studied by treating the tissues in
Susa, carnoy and alcoholic Bouins fixative for 24 hr. The tissues were dehydrated,
paraffin embedded and sections (7-8 /x in thickness) were treated for histochemical tests
503
504
R Sarojini and G Gyananath
Table 1. Results of histochemical tests on the androgenic gland of M. lamerrii.
Tests
Cytoplasm Nucleus
For proteins
Mercuric bromophenol blue 4-4- +
Millons reaction (Bensley and Gersh modification) — —
Millons reaction (Baker modification) — —
Ninhydrin-Schiff method + +
Ferric cyanide method for -SH groups — —
Aldehyde fuchsin 4- ±
For carbohydrates
Best's carmine — —
Performic acid (Schiff method (PFAS)) - -
Periodic acid-Schiff method (PAS) ± ±
For lipids
Sudan black B ± ±
— = negative; ± = doubtful; 4- = positive; + + = intensity moderate.
as given in table 1. The histology of the testis was done by fixing the tissue in Bouin's
fluid and staining the sections with Hani's haematoxylin eosin. The number of
testicular follicles were counted.
3. Observations and results
3.1 Histology and histochemistry of AG
In the male M. lamerrii the AG was located near the terminal part of the seminal vesicle
and extended over the vas deferens to which it is superficially attached (figure 1). The
diameter of the AG measured at the seminal vesicle was 75 /an and near the vas deferens
it is in the form of a diffused structure.
In whole mounts the AG looked transparent and the size varied with the reproductive
stage of the animal. (21*8-905 /im). In histological preparations, the cords of the cells
VSD
Figure 1. Gross morphology of androgenic gland. (AG-androgenic gland, sv-seminal vesicle,
vso-vas deferens.)
Seasonal variations on the androgenic gland of M. lamerrii 505
are loosely arranged, the cytoplasm in the cells scanty and the nuclei stained dark blue-
black.
The histochemical tests revealed that the AG cells are positive to bromophenol blue
and ferric ferricyanide and Millon's reaction failed to detect SH groups and tyrosine,
but aldehyde fuchsin and ninhydrin tests gave positive results. The performic acid
Schiff and Best's carmine tests gave negative reports.
In routine histological preparations, the gland cells appear almost identical and the
cell boundaries can be hardly made out in light microscopy when the gland is active, the
cells contain large amounts of CH positive granules. During inactive stages, signs of
nuclear pycnosis becomes distinct.
3.2 Seasonal variations in the AC
The AG remained in a regressed state all through, May, June and July (table 1). Pycnotic
and shrunken nuclei were observed throughout the gland. The gland cells very rarely
showed the presence of fine basophilic granules. The AG started hypertrophy by August
and September. The nuclei were large and the cells were multinucleolated and fine
basophilie granules were observed scattered evenly in the cytoplasm. Nuclear pycnosis
was rare in the cells. By October, November and December the hypertrophy of the AG
increased steadily and during February the hypertrophied AG showed regional
differences in appearance and histology also. Some areas in the gland showed
basophilic granules in the cytoplasm and enlarged nuclei. The AG of March animals was
largely comparable to those of February except that in some areas, the cells showed
accumulations of granules into masses. By April the cytoplasmic granular bodies
increased in size to form larger and larger units and nuclear pycnosis was increasingly
evident. By May and June the AGas a whole atrophied significantly and the cytoplasmic
granules became scarce and the cells started degenerating.
3.3 Seasonal variations in the testicular follicles
The testis showed cyclic changes and this was evident by the variation in the number of
testicular follicles. The number of testicular follicles were highest during February. No
testicular follicles appeared in May and June. From November onwards these showed a
steep incline reaching maximum in February. By end of April the testis did not show
any activity.
3.4 Effect of neurosecretory hormones on AG
Eyestalk ablation caused hypertrophy after 15 days of operation. This was evident by
the increase in the cell diameters and also the nuclear diameters. In the eyestalk ablated
individuals, the cell and nuclear diameters showed an enhancement from 0-98 + 0-29 \JL
to 2-16 ± OlOju and 047 + 0-02^ to 1-53 ± 0-07^ respectively. When the eyestalk
extract injection was given to the eyestalk ablated animals, there was a significant
decrease in the cell and nuclear diameters (table 2). Central nervous tissue extracts when
injected into normal and eyestalk ablated individuals brought about enhancement in
506
R Sarojini and G Gyananath
Table 2. Seasonal variation of androgenic gland activity.
Month
Size of AG
± SD(JU)
Secretory
activity
Of AG
No. of
testicular
follicles ± SD
May
5-2 ±1-2
No activity
No follicles
June
4-9 + 0-9
No activity
No follicles
July
6-4 ±0-7
No activity
3-2 ±0-1
August
7-3 ±0-2
Cells active
3-4 ±0-2
September
10-4 ±0-6
No. of cells increased
5-0 ±0-4
October
* 15-3 ±0-8
Cells compactly arranged
7-6 ±0-5*
November
*20-0±0-5
Cytoplasm developed
12-8 ±0-7*
December
*25-0±0-9
Hypertrophy
19-8 ±0-7*
January
*29-6±0-l
Hypertrophy
21 -2 ±0-4*
February
*45-9±0-6
Hypertrophy
39-2 + 0-4*
March
*34-2±0-7
Regression started
16-4 ±0-2*
April
*3<M±(M
Regression started
14-2 ±0-6*
* P < 0-05.
the above parameters. However, this enhancement was more in the individuals who had
eyestalk ablation than normal ones.
4. Discussion
The location and gross morphology of the AG of M . lamerrii is comparable to that of
other decapod crustaceans (Charniaux-Cotton et al 1966). The AG showed its peak
activity during February to March and it remained inactive from May to July. Gain in
size of the gland, abundance of basophilic granules in the cytoplasm, increase in the size
of nuclei and multinucleolated nature are some of the parameters taken into account to
consider signs of increased secretory activity of the AG. All these cytological events in the
gland cells occur when the male is in the sexually active phase. Seasonal changes in AG
activity have been reported earlier in the crayfish, Orconectes nais (Carpenter and
Deroos 1970). In the present study when the secretory activity of AG is correlated with
the number of testicular follicles there appears to be a direct relationship (table 1).
Secretory activity of the AG in crustaceans is maintained by the influence of
neurosecretory hormones (Charniaux-Cotton 1960, for review; Payen et al 1971).
Eyestalk ablation brought about hypertrophy of AG in the marine crab, Scylla serrata
(Rangnekar et al 1971). Inhibitory gonadotropins are produced in the neurosecretory
complex of the eyestalks which inhibit testis development and AG. From the available
literature it can be said that inhibitory action of gonadotropins is at first at AG level and
further changes occur in the spermatogenic activity of crustaceans. Brain and thoracic
ganglion extracts enhanced the spermatogenic activity and corresponding increase in
androgenic gland activity. A stimulatory gonadotropin released from the photo-
cerebrum was reported in Orchestia gammarella (Bounenfant 1967) and Paratelphusa
hydrodromous (Adiyodi and Adiyodi 1974). Amato and Payen (1978) have suggested
two types of specific neurohormones, one controlling the growth of the AG and the other
Seasonal variations on the androgenic gland of M. lamerrii
Table 3. Effect of eyestalk ablation and injection of central
nervous tissue extracts on the androgenic gland activity.
507
Treatment
Cell diameter Nuclear diameter
± so (ju) ± so (n)
Normal (control)
5-98 ±0-21
2-51 ±0-50
Normal + eyestalk
extract
5-71 ±0-10
2-50 ±0-10
Normal + brain
extract
* 12-07 ±0-3
4-37 ±0-30
Normal + thoracic
ganglion extract
* 12-87 ±<M
4-23 ±0-06
Eyestalk ablated
*12-16±0-1
4-53 ±0-70
Eyestalk ablated +
eyestalk extract
7-01 ±0-5
2-52 ±0-60
Eyestalk ablated +
brain extract
* 13-56 ±04
1-57 ±0-22
Eyestalk ablated +
thoracic ganglion
extract.
* 13-69 ±0-2
1-73+0-10
*P < 0-05.
synthesis of male hormone. Further work on these lines is needed to establish the above
facts in the freshwater prawn, M . lamerrii.
Acknowledgements
The authors are grateful to Dr R Nagabhushanam, for facilities. One of the authors
(GG) is thankful to the UGC for financial assistance.
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Charniaux-Cotton H 1964 Endocrinologie et genetique du sexe chez les crustaces superienrs; Ann.
Endocrinol. 25 36-42
Charniaux-Cotton H, Zerbib C and Meusy J J 1966 Monographic de la glande androgene des crustaces
superieurs, Crustaceana 10 113-136
Demeusy N and Veilet A 1958 Influence de 1'ablation des pedoncules oculaires sur la glande androgene de
Carcinus maeas\ C. R. Acad Sci. Paris. 246 1104-1107
508 R Sarojini and G Gyananath
Diwan A D and Nagabhushanam R 1974 Reproductive cycle and histochemical changes in the gonad of the
freshwater crab, Barytelphusa cunicularis; Indian J. Fish. 21 164-176
Hoffman D L 1968 Seasonal eyestalk inhibition of the androgenic glands of a protandric shrimp; Nature
(London) 218 170-172
Hoffman D L 1 969 The development of the androgenic glands of a protandric shrimp; Biol Bull. 137 286-296
Payen G J P, Costlow Jr and Charniaux-Cotton H 1971 Comparative study of the ultrastructures of the
androgenic glands of normal crabs and crabs undergoing bilateral eyestalk removal during larval life or
after puberty in the species, Rhithropanopeus harrisii and Callinectes sapidus; Gen. Comp. Endocrinol. 17
526-542
Rangnekar P V, Madhyastha M N and Latey A N 1971 Hormonal control of reproduction in the male crab,
Scylla serrata; J. Anim. Morphol Physiol. 18 17-19
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 509-515.
© Printed in India.
Mermithid nematodes as parasites of Heliothis spp. and other crop
pests in Andhra Pradesh, India
V S BHATNAGAR*, C S PAWAR, D R JADHAV and J C DAVIES**
Cropping Entomology, International Crops Research Institute for the Semi-Arid Tropics,
Patancheru P.O., Andhra Pradesh 502324, India
*Present address: FAO/CILSS IPM Project, B.P. 281, Kaolack, Senegal
**Overseas Development Administration, Tropical Development and Research Institute,
College House, Wright's Lane, London W8 5SJ
MS received 3 December 1984
Abstract. Insect pests were collected from cultivated and wild plant species to study their
parasites in Andhra Pradesh, India. Besides insects, nematodes emerged as parasites. While
Hexamermis spp. were common in most lepidoptera, Ovomermis albicans (Siebold) was
recovered from Heliothis spp. The nematodes were active, even more than insect parasites,
during early monsoon. They were more active on light-soils than on heavy-soils. Against
Heliothis armigera (Hubner) in particular, their incidence was more on "low-growing" crops
like Arachis hypogaea (L.), and Lycopersicon esculentum (L.), and weeds. The nematode
Pentatomimermis sp. was recorded from the bug Nezara viridula L.
Keywords. Mermithids; Ovomermis albicans; Hexamermis spp; Pentatomimermis sp.;
Heliothis spp.
1. Introduction
The mermithid nematodes, in general, are known to infect a wide range of insects in
15 different orders (Nickle 1972). Ramakrishnan and Kumar (1976) reported the
association of species of Mermis, Agamermis, Hexamermis, and Geomermis with 40
insect species in India. In this paper, observations on mermithids as parasites of some
important insect pests on dry-land crops and their role in regulating the pests
populations are described.
2. Material and methods
Insects were collected (1975-83) in their available stages from Medak, Rangareddy
and Mahaboobnagar districts of Andhra Pradesh, India and reared in glass vials
(9 x 2-5 cm) in the laboratory on the same natural hosts to study critically for nematode
and insect parasites. The nematodes, when emerged, were preserved by the method
suggested by IA Rubtsov (personal communication). The rates of nematode parasitism
recorded over years in different months on different crops were calculated on the basis
of total larvae in the samples which showed the nematodes.
50Q
510 V S Bhatnagar et al
3. Results and discussion
3.1 Nematode species and insect hosts
The mermithid nematode species identified from different insect hosts were as
follows
Ovomermis albicans (Siebold) : Heliothis armgera (Hubner)
: H. assulta Guenee
: H. peltigera SchifF
Hexamermis spp : Achaea Janata L.
: Chilo partellus Swinhoe
: Cydia critica Meyr.
: C. ptychora Meyr.
: Lampides boeticus L.
: Marasmia suspicalis Walker
: Menochilus sexmaculatus F.
: Mythimna separata Walker
: Scirpophaga incertulas Walker
: Spodoptera exigua Hubner
: 5. litura F.
Pentatommermis sp. : Nezara viridula L.
0. albicans was recovered from the larvae of all the three Heliothis species found in
India. Hexamermis spp. were recovered from many insects including a coleopteran
M. sexmaculatus which predates on eggs and larvae of some insect pests.
Pentatomimermis sp. emerged from AT. viridula. The nematodes also emerged from the
adults of H . armigera, N. viridula and S. incertulas.
The nematodes recorded on Heliothis and N. viridula are new records for these are
not listed by Poinar (1975, 1979) in his reviews on entomophagus nematodes.
3.2 Nematode parasitism in relation to season and host crops
The rates of nematode parasitism recorded in different insects on cultivated
and uncultivated host plants in different months are given in table 1. Although the
flection of insects was from almost all months of year, the nematode parasitism was
611 t0 DCCember Whh Peak aCtivity generally durin*
Wefe m°re aCdVe °n Ught~S°ils <alfisols) than on heavy-soils
cajan (L.) Millsp which are grown on both types of soils. A. hypogaea
Z"SICOn eSC?entUm (L) m n°rmally gr°Wn ^-soils, and weed? are
most common on these soils
and ?tome 9ynandra (L-} Briq" and relativ^ less °n
d H T^ hete that H- pehigera is more Predominant on
and H. assulta on D. metel (Bhatnagar and Davies 1978).
Mermithid nematodes as parasites
511
Table 1. Occurrence of nematode parasitism ( %) in insect pests on cultivated and wild host
plants in Andhra Pradesh, India (1975-83).
InsecJ Plant species
Month
Light soils
(Alfisols)
Heavy soils
(Vertisols)
Ovomermis albicans (Siebold)
Heliothis Arachis hypogaea (L.)., Groundnut
Jun.
3-8 (424)2
—
armigera
Jul.
20-5 (677)4
—
Aug.
30-0 (1035)7
—
Sep.
39-4 (279)3
—
Cajanus cajan (L.) Millsp.,
Jul.
—
0-2 (444)1
Pigeonpea
Sep.
—
1-4 (418)1
Oct.
2-4 (817)3
—
Nov.
1-0 (960)3
—
Cicer arietinum (L.)., Chickpea
Aug.
1-3 (382)1
—
Oct.
—
0-2 (429)1
Helianthus annus L., Sunflower
Aug.
—
0-4 (237)1
Ipomoea batatas (L.) Lam., Sweet
Sep.
— -
16-6 (6)1
potato
Lycopersicon esculentum (L.).-,
Jul.
52-0 (50)1
—
Tomato
Aug.
42-5 (153)1
—
Sorghum bicolor (L.) Moench.,
Aug.
0-7 (400)1
2-9 (35)1
Sorghum
Sep.
1-0 (798)3
0-5 (206)1
Vigna aureus (Roxb.) Hepper.,
Aug.
18-2 (II)1
—
Mungbean
V. radiata (L.) Wilczek., Black gram
Aug.
—
3-3 (150)1
Zea mays L., Maize
Aug.
31-2 (16)1
1-0 (1122)2
Sep.
8-3 (132)1
—
Acalypha indie a L.*
Aug.
5-9 (17)1
—
Cleome gynandra (L.) Briq.*
Jun.
1-1 (94)1
—
Jul.
57-8 (1404)2
—
Aug.
33-5 (176)1
—
Gomphrena celosioides Mart.*
Jun.
4-3 (138)1
—
Jul.
61-9 (698)1
—
Aug.
18-0 (1626)2
__
Sep.
1-0 (400)1
— -
Leucas aspera L*
Aug.
6-3 (16)1
—
Tridax procumbens L.*
Jul.
50-0 (2)1
—
H. assulta** Datura metal L.*
Jul.
4-0 (297)2
—
Aug.
0-4 (526)2
—
H. peltigera** Acanthospermum hispidum DC*
Jun.
1-3 (80)1
—
Jul.
43-1 (788)4
—
Aug.
46-1 (475)4
—
Sep.
19-0 (79)1
—
Nov.
4-2 (24)1
—
Hexamermis Spp.
Achaea
Janata Ricinus communis L., Castor
Aug.
25-7 (140)2
—
Chilo Sorghum bicolor (L.) Moench.,
Jul.
20-6 (475)5
—
partellus Sorghum
Aug.
9-0 (575)4
2-3 (220)2
Dec.
15-4 (13)1
—
Cydia Cajanus cajan (L.) Millsp.,
Aug.
—
3-0 (162)2
critica Pigeonpea
Sep.
3-3 (180)1
1-2 (240)1
C. ptychora „ „
Aug.
50-0 (6)1
—
512 V S Bhatnagar et al
Tabkl. (Contd.)
Insect
Plant species
Month
Light soils
(Alfisols)
Heavy soils
(Vertisols)
Lampides
boeticus
Vigna radiata (L.) Wilczek., Black
gram
Jul.
Aug.
'
20-0 (45)1
6-0 (50)1
Marasmia
Sorghum bicolor (L.) Moench.,
Oct.
34 (89)1
___
suspicalis
Menochilus
Sorghum
Aug.
6-7 (330)2
5-3 (342)3
sexmaculatus
Mythimna
separata
»
Jul.
Aug.
50-0 (100)1
24-5 (233)2
25-9 (309)2
Spodoptera
Cicer arietinum (L.)., Chickpea
Jul.
Oct.
59-0 (52)1
1-0 (100)1
Spodoptera
Arachis hypogaea (L.)., Groundnut
Aug.
2-0 (50)1
—
litura
Sep.
12-5 (24)1
—
Pentatomimermis sp.
Naiara
viridula
Pennisetum americanum (L.) Leeke.,
Oct.
—
3-1 (96)1
Millet
* : Weeds.
** : 90-95 °0 in total Heliothis larvae on the weed.
Figures in parentheses give the total collection in samples showing parasitism over years. Superscripts 1 -7
refer to the number of years of data available.
3.3 Nematode and insect parasitism
The overall level of nematode parasitism recorded in Heliothis spp. irrespective of host
plants on alfisols at ICRISAT Center are shown in figure 1. It was higher than insect
parasitism in the early part of the season and ceased subsequently to almost nil by
October. Amongst Heliothis spp., H. peltigera was most vulnerable, and H. assulta the
least. In August 1979, Heliothis collection from one of the light-soil grazing areas, about
45 km from ICRISAT Center and uncultivated for at least 15 years, showed as high as
924 % (n = 305) nematode parasitism on A. hispidum, but no parasitism due to insects.
The weed was growing as 18 plants/m2 and had as many as 74 Heliothis larvae/ 100
plants.
The multiple parasitism, involving the nematodes and insects, was rare. Nematodes
were recorded only twice in association with hymenopterans Campoletis chlorideae
Uchida and Microchelonus curvimaculatus Cameron in Heliothis larvae collected in
August on A. hispidum.
3.4 Nematode activity and distribution in the soil
Nematodes were common on lepidoptera than on other insects. One to four nematodes
emerged from a majority of Heliothis larvae, but as many as 63 nematodes were
recorded from a larva of H. peltigera collected on A. hispidum (table 2). Usually one
nematode emerged from a larva of H . assulta. The nematodes that emerged from
Mermithid nematodes as parasites
513
ID
t^S
oo i/> o
—• CM
514
V S Bhatnagar et al
Table 2. Number of nematodes emerged from Heliothis larvae (1976-77)
Number of larvae
No. of
Nematodes
Heliothis
armigera
Heliothis
peltigera
Heliothis
assulta
Jul
Aug. Sep. Jul. Aug. Sep.
Jul.
Aug. Sep.
01-05
36
15
3
53
143
14
2
0
1
06-10
3
3
0
64
22
1
0
0
0
11-15
3
1
0
62
4
0
0
0
0
16-20
0
0
0
60
1
0
0
0
0
21-25
0
0
0
19
0
0
0
0
0
26-30
0
0
0
9
0
0
0
0
0
31-35
0
0
0
2
0
0
0
0
0
50-60
0
0
0
2
0
0
0
0
0
>60
0
0
0
1
0
0
0
0
0
Cumulative
average of
3-6
3-5
2-3
13-9
3-4
2-0
1-5
0-0
1-0
nematodes/
larva
Heliothis, measured 3-22 cm in length. The nematode number declined from July to
September.
Nematode activity appeared to be stimulated by the arrival of the premonsoon
showers in June, and varied seasonally remaining often localised. In soil samples from
1-30 cm depth, collected only in alfisols, more nematodes were recovered from
20-30 cm, and more so in July when the monsoon is normally well set. Nematodes
frequently had a patchy distribution and varied in population.
Heliothis larvae parasitised by nematode were creamy yellow and sluggish and ate
little. They survived for one to two days when juvenile nematodes emerged, but died
soon with the emergence of adults. Emergence was observed both during the day and
night. The nematodes emerged usually from the abdominal region of insects.
Glaser et al (1942) recorded Neoaplectana chresima Steiner as a natural endoparasite
of Heliothis spp. including H. armigera from the USA. Poinar (1979) considered N.
chresima as a strain of N. carpocapsae Weiser. It should be noted here that H. armigera
is present only in the old world and the report from USA could be because of the
taxonomic confusion during that time (Hardwick 1965; Nye 1982). In India, Achan et al
(1968) identified the nematode parasitic on H. armigera as Hexamermis sp., and specific
identification was considered difficult for want of adult nematodes. However, in the
present study it was possible because of recovery of sufficient number of adult
nematodes. The nematode has been identified as Ovomermis albicans (Siebold).
Achan et al (1968) considered the nematode on H. armigera to be specific on
L. esculentum. However, the present investigation shows that the nematode is parasitic
on all the three Heliothis species in India, and is associated with many host plants.
Achan et al (1968) recorded a maximum of six juvenile nematodes from a larva of H.
armigera, but our record is of 14 juveniles from a larva of H. armigera, and of as many
as 63 juveniles from a larva of H. peltigera.
Laumond et al (1979) reported the infectivity of N. carpocapsae to N. viridula in the
Mermithid nematodes as parasites 5 1 5
laboratory, but there is no record of its natural occurrence on this bug, at least from
India. Hexamermis sp. has been identified in general from pentatomid bugs (Gokulpure
1970). The nematode Pentatomimermis sp. is a new record on N. viridula.
4. Conclusions
The occurrence of nematode parasites early in the season and their higher incidence on
alfisols, and on "low growing" crops like groundnut, tomato and weeds are important
findings of this study. This should help plant protectionists to plan effective utilisation
of the total parasitic fauna of the pest in nature, and particularly in integrated
management of pests like H. armigera which attack many crops in succession. The soil
application of insecticides to control a few insect pests in some crops needs to be
investigated for their effects on entomophagus nematodes.
In view of the importance of mermithid nematodes as parasites on insect pests, a
clearcut information is required on individual species in relation to a given insect host
to consider their utility in pest management. Further a gathering of base data with large
samples from farmers' fields is essential to know how beneficial these mermithids could
be in suppressing the pest in nature.
Acknowledgements
The assistance of Prof. I A Rubtsov of the Academy of Science, USSR in identifying the
nematodes is gratefully acknowledged. The pest scouts of the Cropping Systems
Entomology are also acknowledged.
References
Achan P D, Mathur K C, Dharmadhikari P R and Manjunath T M 1968 Parasite ofHeliothis spp. in India.
Tech. Bull. No. 10, Commonwealth Institute of Biological Control, Bangalore, India, pp. 120-149
Bhatnagar V S and Davies J C 1978 Factors affecting populations of gram pod borer, Heliothis armigera
(Hubner) (Lepidoptera: Noctuidae) in the period 1974-77 at Patancheru, Andhra Pradesh; Bull. Entomol.
19 52-64
Glaser R W, McCoy E E and Girth H B 1942 The biology and culture of Neoaplectana chresima, a new
nematode parasitic in insects; J. ParasitoL 28 123-129
Gokulpure R S 1970 Some hosts of a juvenile mermithid of the genus Hexamermis Steiner; Indian J. Entomol.
32 387-389
Hardwick D F 1965 The corn earworm complex; Mem. Entomol. Soc. Canada 40 1-127
Laumond C, Mauleon H and Kermarrec A 1979 Donnees nouvelles sur le spectre d'hotes et le parasitisme du
nematode entomophage Neoaplectana carpocapse; Entomophaga 24 13-20
Nickle W R 1972 A contribution to our knowledge of Mermithidae (Noctuidae); J. Nematoi 4 122-146
Nye I W B 1982 The nomenclature of Heliothis and associated taxa (Lepidoptera: Noctuidae): Past and
present. Proc. Int. Workshop on Heliothis Management. ICRISAT, Patancheru, A.P., India pp. 3-8
Poinar G O Jr (ed.) 1975 Entomogenous nematodes. A manual and host list of insect-nematode associations.
(Leiden: Brill) 31 7 pp
Poinar G O Jr (ed.) 1979 Nematodes groups. Nematodes for biological control of insects (Florida, California:
CRC Press) 11 -198
Ramakrishnan N and Kumar S 1976 Biological control of insects by pathogens and nematodes; Pesticides
pp. 32-47
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 517-521.
© Printed in India.
Wing microsculpturing in the termite genus Amitermes (Teraiitidae,
Amitermitinae)
M L ROONWAL and N S RATHORE
Desert Regional Station, Zoological Survey of India, Jodhpur 342006, India
MS received 22 October 1984; revised 19 March 1985
Abstract. Wing microsculpturing has been described in the genus Amitermes. It occurs on
the upper and lower wing surfaces and is composed of a single row of small (3-5 /im long),
thorny papillae at the anterior margin and numerous micrasters all over the wing membrane
(size 6-8 /zm x 5-7 /on; density 9200-9600/mm2). The micrasters are with 5-8 arms and of
the complex type (types V-X). No arrowheads are present. The position of Amitermes is
discussed in the general scheme of termite microsculpturing. Comparison is also made with the
condition in the Zoraptera, Embioptera and the Blattoidea.
Keywords. Wing microsculpturing; Amitermes belli; termites; isoptera; Amitermitinae.
1. Introduction
The occurrence of an elaborate and dense pattern of cuticular microstructures of
various shapes and sizes has been established in recent years in a long series of studies
(1967-1983) by Roonwal and co-workers; for a summary see Roonwal (1983a, b).
Over 80 genera and 250 species belonging to all the major families and subfamilies of
termites (Isoptera) have been studied in this respect, including the subfamily
Amitermitinae (Roonwal 1981; Roonwal and Rathore 1977, 1982; Roonwal and Verma
1980, 1983; Roonwal et al 1974). The genus Amitermes Silvestri (Amitennitinae,
Termitidae), however, remained unstudied due to dearth of material. Recently, we were
able to procure winged examples of a member of this genus (A belli), and the present
account fills the lacuna in our knowledge of wing microsculpturing in this subfamily.
2. Material and methods
Winged forms, along with soldiers and workers, of Amitermes belli were collected from
Sariska Forest, 18km south-east of Alwar (Alwar District, Rajasthan, India, ca 27-30 N
lat., 76-30 E long.), ex rotten date palm trunk; N. S. Rathore Coll., 18 June 1983. It is a
soil dwelling, wet and semi-arid zone species occurring in Western India and Pakistan.
Its ecology and distribution are described by Roonwal (1976) and Roonwal and Bose
(1964). Sex ratios (in colony): Alates, 45 males (57%), 34 females (43%).
Wings were mounted in glycerine as well as the quick-drying D.P.X. mounting
medium (BDH/Glaxo); both gave good results.
517
518 ML Roonwal and N S Rathore
3. Results
3.1 Genus Amitermes Silvestri
This is a large, widespread genus of which the winged forms of a single species, A. belli,
were alone available for study.
3.2 Amitermes belli (Desneux)
(Termes belli Desneux, 1906, Annales Soc, Ent. Beige, Brussels, 49 (12); p. 352.)
Wings (figure 1) small; with scale, forewings 8-5 x 2-0 mm, hindwings 8-2 x 2-1 mm.
Membrane transparent, pale smoky; anterior veins brown, rest paler. Hairs (length
50-1 10 /an) fairly numerous on anterior margin and a little below it; similar but smaller
ones on posterior margin; on membrane a few small ones here and there, somewhat
longer (45-135 /xm) and more numerous on basal scales.
3.3 Microsculpturing (figures 2 and 3)
Microsculpturing consisting of papillae and micrasters; is found on both upper and
lower surfaces of the wing.
3.3a Papillae: A single row of small (length 3-5 /on), pointed, thorny papillae on
anterior margin; none on posterior margin and membrane.
3.3b Micrasters: Present in considerable density all over membrane right up to
margins; lumpy and distorted on basal scales and on adjacent portions. Generally of the
complex, thick, many-armed, type (types V~X of Roonwal et al 1974), with 5-8 arms,
mostly 6-7; with asteroid and odd shapes. Sizes 6-8 jim x 5-7 /xm. Density (per mm2) in
the middle of wing is fairly uniform and is as follows: Dorsal surface: Forewing 9600,
hindwing 9280. Ventral surface: forewing 9200, hindwing 9600.
Sc+R Rs M Cu
3 mm
Figure 1. Amitermes belli (Sariska, Rajasthan). Right wings, to show venation. A. Forewing.
B. Hindwing. Cu., cubitus; M, media; Rs., radial sector; Sc. + R., subcosta + radius.
Wing microsculpturing in termites
519
** **,
* JL ,
^ *t ^
^>Jt
^ * *
•**V
k * ,
~* ^
* *
,
*
m.:
50
Mm
B
* * ' *
m.
D
#•
Figure 2. Amitermes belli (Sariska, Rajasthan). Portions of wing surfaces to show micro-
sculpturing. A. Portion of anterior margin of right forewing, in dorsal view, to show
micrasters. B. Part of middle of wing membrane of right forewing, in dorsal view. C. Same, of
hindwing. D. Same, of ventral view of forewing. E. Same of hindwing. F. Micrasters from
dorsal surface of forewing, enlarged and rearranged, h., hairs; m., micrasters; p., papillae.
4. Discussion
Amitermes conforms to the amitermitine pattern as discussed by Roonwal (1 98 3b, chart
1) and falls in major group B, i.e. "Higher Group I (with micrasters but without rods)",
and in the second amitermitine category, of advanced genera, e.g., Eremotermes,
Microcerotermes, etc., which have complex, many-armed micrasters. The papillae too
are pointed and thorny: as. is characteristic of the Amitermitinae. Similarly, hairs are
common on the wing margins, but rare on the membrane. All these similarities
emphasise the relative uniformity of the subfamily Amitermitinae.
Termite-like wing microsculpturing is absent in the allied orders Zoraptera
(Roonwal 1983b) and Embioptera (Roonwal and Rathore 1984) where only hairs or
hair-like structures are present. In the cockroaches, (Blattoidea), however, microscul-
pturing is present but is less elaborate (Roohwalind Rathore 1983).
520
M L Roonwal and N S Rathore
UPP1^ <&t < tarn; *w j^.* ^ **** s^Ji ^* ^ « ^ *^
% * I .*.> .^V:";. 5 iy;'^: >-*r-
^i.% ^^*5^V^ * ?£>.:-*• •*
T *» >-!2 J^
'
r r
50XJ
C
B. Middle of wing membrane.
clea^ ones,
- -
ofte ,•
£ed '" Curves and cirdes- The
AcknonJedgements
References
^^
tennite, A study in desert
Wing microsculpturing in termites 521
Roonwal M L 1977 Microsculpturing on termite wings (Isoptera), and its significance; Proc. Zool. Soc.
Calcutta 28 31 -39
Roonwal M L 1919 Termite life and termite control in tropical South Asia. (Jodhpur: Scientific Publishers.)
Roonwal M L 1981 Evolution and systematic significance of wing micro-sculpturing in termites (Isoptera).
XL Some hitherto unstudied genera and species in five families; Proc. Indian Nati Sci. Acad. B47 467-473
Roonwal M L 1983a Universal occurrence of external cuticular, microscopic papillae and allied structures as
an essential character of termites (Isoptera, Insecta), and redefinition of the order; Zool Anz. 211 1 37-144
Roonwal M L 1983b Evolution and systematic significance of wing microsculpturing in termites. XIII. Order
Isoptera; Proc. Indian Natl. Sci. Acad. B49 359-391
Roonwal M L and Bose G 1964 Termite fauna of Rajasthan, India. Zoologica, 40 (3) (Heft 113): I-VI + 1-58
pp. 5 pis.
Roonwal M L and Rathore N S 1977 Third study of evolution and systematic significance of wing micro-
sculpturing in termites: Micrasters in some Rhinotermitidae and Termitidae; Zool. An?. 198 298-312
Roonwal M L and Rathore N S 1982 Evolution and systematic significance of wing microsculpturing in
termites (Isoptera). XII. Sculpturing on wing scales; Proc. Indian Natl. Sci. Acad. B48 322-343
Roonwal M L and Rathore N S 1 983 Wing microsculpturing in the small house cockroach, Supella longipalpa
(Dictyoptera, Blattidae); Proc. Indian Acad. Sci. (Anim. Sci.) 92 333-342
Roonwal M L and Rathore N S 1984 Wing microsculpturing in some Embioptera (Insecta), with a new type
of structure, and ecological and taxonomic considerations; Indian J. Entomol. 45 323-334
Roonwal M L and Verma S C 1980 Evolution and systematic significance of wing microsculpturing in
termites (Isoptera). VIII. Subfamily Amitermitinae of family Termitidae; Proc. Indian Natl. Sci. Acad.
B46 250-263
Roonwal M L and Verma S C 1983 New data on wing microsculpturing in termites (Kalotermitidae,
Rhinotermitidae and Termitidae); Ann. Ent. I 27-34
Roonwal M L, Verma S C and Rathore N S 1974 On a new systematic character in termites, the micrasters;
Zeit. Zool. Syst. Evolutions-forsch. 12 55-76
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 523-526.
© Printed in India.
Effect of two graded doses of x-irradiatiora on the rat adrenal gland and
its protection by S-phenetyl formamidino 4(N-ethyl isothioamide)
morpholine dihydrochloride
P K CHATURVEDI and S S HASAN
Department of Zoology, Garhwal University Campus, Pauri-Garhwal 246001, India
MS received 10 December 1984; revised 17 May 1985
Abstract. In the present study the amount of protection offered by the use of S-phenetyl
formamidino 4(N-ethyl isothioamide) morpholine dihydrochloride to adrenal gland was
investigated on albino rats against two graded doses of x-rays (1500R each). Total body
x-irradiation brought about the hypertrophy and degranulation of adrenal cortical and
medullary cells. The extent of hypertrophy and degranulation increased after the 2nd exposure
to x-rays. Treatment with S-phenetyl formamidino 4(N-ethyl isothioamide) morpholine
dihydrochloride prior to each dose of irradiation precluded the radiation changes caused in the
adrenal cortex and medulla of the rats.
Keywords. X-rays; S-phenetyl formamidino 4(N-ethyl isothioamide) morpholine
dihydrochloride; adrenal gland.
1. Introduction
Earlier studies have demonstrated that ionizing radiation decreases production of
corticoids from the adrenal gland (Nabors et al 1974; Nabors 1962; Berliner et al 1962;
Stevens et al 1963). It is known that adrenal glands respond appreciably to irradiation,
and the response is generally dose-dependent (Dougherty and White 1946; French et al
1955). Hasan et al (1977) reported degranulation and hypertrophy of adrenal cortex
and medullary cells at various intervals of post-irradiation. Studies conducted hitherto
on the adrenal gland in relation to radiation dealt with a single dose of irradiation.
Similarly there seems to be paucity of information on the chemical protection of
adrenal cortex and medulla against two graded doses of x-irradiation (Bacq et al 1 955;
Sarkar et al 1978). Thus in the present study the protection offered to the adrenal by S-
phenetyl formamidino 4(N-ethyl isothioamide) morpholine dihydrochloride — a
known chemical radio protector (Hasan et al 1983) against two graded doses of x-
irradiation was studied on the albino rats.
2. Materials and methods
One hundred albino rats of porton strain weighing 100 ± 10 g were used in this study.
Before the commencement of the experiment rats were acclimatized to laboratory
conditions for about a fortnight. During acclimatization and experimentation, rats
were maintained on balanced laboratory diet procured from the Hindustan Levers
Limited (Bombay) arid water ad libitum. After acclimatization, the rats were divided
into four groups each containing equal number of animals.
523
524
P K Chaturvedi and S S Hasan
Group I: rats receiving physiological saline and serving as control for groups II
and IV.
Group II: rats were exposed to two graded doses of x-rays (3000R), 1500R at one
week interval.
Group III: rats receiving twice 30mg/kg of the antiradiation compound and
exposed to two graded doses of x-rays (3000R), 1 500R at one- week
interval.
Group IV: rats receiving twice 30 mg/kg of antiradiation compound at the time
when group III rats were administered the drug to serve as control for
group III.
S-phenetyl formamidino 4(N-ethyl isothioamide) morpholine dihydrochloride
(kindly supplied by Dr S N Pandey, Department of Pharmaceutics, Institute of
Technology, Banaras Hindu University, Varanasi) was used as antiradiation compound
(Hasan el al 1983).
Before preparing the injectable solution the compound was activised with the use of
acetone. The injectable solution was prepared in 0-9 % physiological saline and the pH
was adjusted to 9-0. Half ml of the injectable solution containing 3 mg of the compound
was administered to rats intraperitoneally 30 minutes before each exposure to x-ray
Figure 1 . Sections of adrenal cortex of normal control and experimental rats, haematoxylin
and eosin ( x 160) A. Normal control showing sparsely granulated cells in all the three layers
and cortex with rich supply of blood vessels. B. 7 days after second exposure to x-ray
treatment showing granulated cells, nuclei with granular nucleoplasm and cortex with rich
supply of blood vessels. C. 7 days after second dose of drug treatment showing granulated
cells and nuclei with agranular nucleoplasm, cortical cells separated by sinusoids in some
places. D. 7 days after second injection of the drug/second exposure to x-rays showing
degranulated cells in zona glomerulosa and granulated cells in zona fasciculata and zona
reticularis, nuclei with granular nucleoplasm, cortex with rich supply of blood vessels.
X-irradiation and protection of adrenal gland
525
Figure 2. Sections of adrenal medulla of normal control and experimental rats, haemat-
oxylin and eosin ( x 160). E. Normal control showing granulated medullary cells. F. 7 days
after second exposure to x-ray treatment showing chromaffin granules bordering the nuclei,
nuclei with granular nucleoplasm, dilated blood vessels. G. 7 days after second dose of drug
treatment showing chromaffin granules filled medullary cells and nuclei with agranular
nucleoplasm. H. 7 days after second dose of drug/second exposure to x-rays showing scantly
granulated chromaffin cells and rich supply of blood vessels.
source. The vehicle carrier (0-9 % NaCl solution) of the drug was injected likewise to
rats of group I to serve as control. The whole body of each rat of groups (II) and (III)
was exposed twice to an x-ray source at one- week interval each time for a dose of 1 500R
(80 kV; 200 MAS; time 1 sec; distance 80cm). The rats of the control as well as the
experimental groups were housed under identical animal husbandry conditions.
The LD50 of the drug was found to exceed 0-5g/kg body weight.
The first sacrifice from each group was made at the end of the 7th day after the first
exposure to 1500R and the later sacrifices were performed at intervals of 7,14 and 28
days after the second exposure to 1500R. At the time of sacrifice adrenal of each rat was
dissected out and fixed in Bouin's fluid for histological examination. Thick paraffin
sections (5/i) were cut and stained with haematoxylin and eosin.
3. Results and discussion
In the present study the animals exposed to x-rays showed hypertrophy and
degranulation of cortical and medullary cells and the extent of degranulation increased
526 P K Chaturvedi and S S Hasan
after the second dose of irradiation (figures IB and 2F). The hypertrophy and depletion
of granular contents from the cells indicate an increase in the cellular activity of cortex
and medulla in comparison with normal control animals whose adreno-cortical and
medullary cells were granulated; besides, there was little mobilization of granular
contents in the cells (figures 1A and 2E). In the cortex of the irradiated animals
pretreated with the drug S-phenetyl formamidino 4(N-ethyl isothioamide) morpholine
dihydrochloride, the cells are arranged in whorls and have a densely staining
cytoplasm. Throughout the cortex and the medulla runs a rich vascular bed of sinusoids
(figures ID and 2H), whereas cortical and medullary cells of the rats treated with the
antiradiation compound alone appeared to show the tightly packed cells particularly in
the cortex were separated by wide vacant spaces which were devoid of vascular bed of
sinusoids (figures 1C and 2G). Similarly there was no mobilization of granular mass in
the wide medullary spaces of the animals treated with the antiradiation compound
alone (figure 2G). This suggested the low profile of cellular activity in the cortex and
medulla of drug-treated animals. Bacq (1965) opines that transformation of DNAfrom a
metabolically active into resting state plays a major role in the radiation protection.
Thus this study infers that the radiation injury induced in this resting state of nuclei of
adreno-cortical and medullary cells could be repaired with the treatment of the drug
prior to each dose of radiation than in the non-treated irradiated animals.
References
Bacq Z M 1965 Chemical protection against ionizing radiation (Springfeld: Charless C Thomas).
Bacq Z M, Cuypers Y, Evrand E and Soctens R 1955 Action de la cysteamine sur la resistance du rat a la
depression barometrique; C. R. Soc. Biol. Paris 149 2014-2017
Berliner D L, Stevens W and Berliner M L 1962 The effect of ionizing radiation on the biosynthesis and bio-
transformation of corticosteroids. In: The effects of ionizing radiation on the reproductive system. (Oxford:
Pergamon Press).
Dougherty T F and White A 1946 Pituitary-adrenal cortical control of lymphocyte structure and function as
revealed by experimental irradiation. Endocrinology 39 370-385
French A B, Migeon C J, Samuels L T and Bowers J Z 1955 Effects of whole-body x-irradiation on
17-hydroxy corticosteroid levels, leucocytes and volume of packed red ceils in the rhesus monkey; Am. J.
Physiol. 182 469-476
Hasan S S, Chaturvedi P K and Pandey S N 1983 Effect of two graded doses of whole-body x-irradiation and
radioprotection by the use of S-phenetyl formamidino 4(N-ethyl isothioamide) morpholine dihydro-
chloride; Nucl. Med. 22 237-245 :
Hasan S S, Sarkar F H, Sharma T R, Prasad G C, Pant G C and Udupa K N 1977 Response of adrenal gland
to whole-body 60Co irradiation; Indian J. Exp. Biol 15 513-516 ;
Nabors C I Jr 1962 A model system for the study of radiation effects on adrenal steroidogenesis; Radiat. Res.
U 557
Nabors C J Jr, Hinckley J S and Fowkes K L 1974 Steroid 11-^-hydroxy dehydrogenase activity in beagles
bearing 241; Radiat. Res. 59 212 (Abstr)
Sarkar F H, Hasan S S, Pandey S N, Prasad G C and Udupa K N 1978 Effect of a chemical radioprotector
administered before irradiation on thyroid, testes, and pituitary; Indian J. Med. Res. 67 616-621
Stevens W, Berliner D L and Dougherty T F 1963 The effect of x-irradiation on the conjugation of steroids;
Radiat. Res. 20 510-518
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 527-531.
© Printed in India.
Effects of DDT and malathion on tissue succinic dehydrogenase activity
(SDH) and lactic dehydrogeease isoeozymes (LDH) of Sarotherodon
mossambicus (Peters)
K RAMALINGAM
Department of Natural Sciences, Arignar Anna Government Arts College, Cheyyar 604 407,
India
MS received 25 June 1984; revised 6 March 1985
Abstract. Liver and muscle succinic dehydrogenase enzyme activity in Sarotherpdon
mdssambicus subjected to sublethal concentrations of DDT and malathion declined signifi-
cantly when compared to the control. The isoenzyme patterns of serum, liver and muscle in the
fishes exposed to toxicants showed marked variations from that of the control. The variations
in LDH isoenzyme patterns attribute alteration in the oxidative capacity of the tissues. The
histological changes in the liver of the experimental group also revealed the harmful effects of
DDT and malathion. The results suggest an alteration in the tissue metabolism towards an
anaerobic type.
Keywords. Organochlorine; organophosphorous; isoenzymes; oxidative metabolism.
1. Introduction
The discernible effects of toxicants on the histological profile of specific tissues in fish as
demonstrated by several investigators include necrobiotic changes in the liver cells,
tubular damage of the kidneys and lamellar abnormalities of gills (Baker 1969; Gardner
and Yevish 1970; Skidmore 1970). Taking a cue from the above studies and also of their
own on Salmo'gairdneri, Bilinski and Jonas (1972) inferred that such changes in tissues
are the resultant effects of oxygen deficiency in them. As the activity of oxidative
enzymes is known to imply the tissue oxygen levels, studies on enzyme systems in fishes
exposed to toxicants would be of interest. However investigations on such enzymes are
meagre. In the present study succinic dehydrogenase activity in liver and muscle, lactic
dehydrogenase isoenzymes of serum, liver and muscle and the liver histology were
determined in Sarotherodon mossambicus exposed to a sublethal dose of two pesticides.
2. Material and methods
Specimens of 5. mossambicus (1 5-20 g) were obtained from the local ponds maintained
by the State Fisheries, Tamil Nadu and acclimated to laboratory conditions for 1 5 days
as suggested by Chavin and Young (1970). The fishes were fed daily with cooked rice
mixed with dried prawn powder ad libitum. A set of 10 fishes each was exposed to 100 1
of dechlorinated water containing DDT (chlorinated compound) and malathion
(organophosphorous compound) at concentrations of 0-01 and 0-95 ppm respectively.
The above sublethal doses were derived as reported previously (Ramalingam and
Ramalingam 1982). A time course study at intervals of 24 hr, 7 days and 15 days was
527
528
K Ramalingam
conducted with controls run simultaneously. Every 24 hr, water was changed in both
control and experiment. The toxicants were also added to water along with renewal and
thus exposure to DDT and malathion was repeated. At the end of each interval, fishes
were dissected and the tissues, liver and muscle were removed for analyses. Before
taking the tissues, blood collected by severing the tail was allowed to clot and the serum
formed after the clot retraction was used for the LDH isoenzyme analysis. The time of
sampling was confined to 9-10 hr. The tissue SDH activity was estimated by the method
of Kun and Abood (1949). For LDH isoenzymes in serum, liver and muscle, disc
electrophoresis as detailed by Smith (1968) was followed. The LDH fractions in the
polyacrylamide gel were numbered according to their electrophoretic mobility,
considering the fast moving fraction which is discernible next to the bromophenol blue
marker end as No.l. The patterns of LDH fractions of control vs experiment are
indicated in figures 1-6. Analysis of variance was applied to study the significance of SDH
values within the groups at the three intervals. Student t-test was made to determine the
significance of values between control group and experiment.
T ,
Ja
1= 1=
Figures 1 and 2. Serum LDH isoenzymes pattern. Control vs experiment. (1. DDT, 2.
Malathion).
CONfROL 2« HOURS ? OAYS « DAYS -.•*,» > '
Figures 3 and 4. Liver LDH isoenzymes pattern. Control vs experiment . (3. DDT, 4. Malathion).
it:!MM
Figures 5 and 6.- Muscle LDH isoenzymes pattern. Control vs experiment. (5. DDT, 6.
Malathion).
Liver and muscle SDH enzyme activity in S. mossambicus 529
3. Results and discussion
The SDH activity of the tissues of control and experiment is illustrated in table 1. The
mean values of SDH activity range from 51-91 ± 1-49 to 86-10 + 7-63 ^g rrc
reduced/100 mg wet wt/hr in the liver and 8-05 ± 0-85 to 12-00 ± 0*98 jig TTC
reduced/ 1 00 mg wet wt/hr in the muscle of the control. The above variations in the
control could be due to the fluctuations present in the blood and tissue total
carbohydrate and its other intermediary metabolites of Kreb's cycle in this species
(Ramalingam 1982). Similar variations in the metabolites of the Kreb's cycle enzymes
have also been reported in several species of normal unstressed fishes (Chavin and
Young 1970).
The SDH values in DDT and malathion exposed fish tissues are significantly lower
compared to control at all the intervals (P = 0-05). This suggests an impairment in the
aerobic capacity of the tissues. Similar to the present study lower activity of oxidative
enzymes by chlorinated compounds (Janicki and Kinter 1971; Timothy et al 1974;
Sreeramulu Chetty et al 1978) as well as by organophosphorous compounds
(Sivaprasada Rao and Ramana Rao 1979; Ranganatha and Ramamurthi 1978) has been
observed in other fishes and vertebrates also. They also indicated the depression
of cellular oxidation. The accumulation of lactic acid in the liver and muscle of
S. mossambicus and also the dimunition of whole animal respiration at the correspond-
ing intervals when the SDH activity was lowered (Ramalingam 1980) also supports the
suggestion that DDT and malathion cause impairment in the aerobic capacity of the
tissues. The results also reveal variations in the level of the activity of SDH within the test
groups namely DDT and malathion exposed fishes (ANOVA). Such variations may be due
to the action of detoxifying mechanisms by the tissue microsomal enzymes (Janardhan
et al 1972), which also occur after exposure to the toxicants.
Analysis of lactic dehydrogenase isoenzymes in the present study reveal that samples
from individual fish as well as the pooled sample show identical isoenzyme pattern in
the control. This suggests that there is no genetic variability in the species. However, the
LDH isoenzyme pattern was altered, as a sequel to the changes in the SDH activity in the
test groups. In the serum of DDT and malathion exposed fishes, an increase in the slow
moving fractions was noticed (figures 1 and 2). Bostrom and Johansson (1972) reported
that the fast moving aerobic fraction no. 1 of LDH is most affected by PCP, a chlorinated
compound. Quantitative studies using organophosphate also revealed an anaerobic
mode of LDH activity in T. mossambica (Ranganatha and Ramamurthy 1978)
corroborating the appearance of slow moving fractions in the present study. In
mammals treated with pesticides, the slow moving LDH fractions were attributed to the
Table 1. SDH activity (mean ± SD in /zg reduced TTC/ 100 mg wet wt/hr) in liver and muscle.
24 hr
7 days
15 days
Liver
Muscle
Liver
Muscle
Liver
Muscle
Control
DDT
Malathion
51-91
40-71
42-30
± 1-49
± 3-25*
± 3-96*
8-05 ±
5-11 ±
4-24 ±
0-85
0-32*
0-11*
58-11 ±
20-25 ±
14-86 ±
3-78
3-57*
3-85*
8-07
3-79
4-40
±0-80
± 0-52*
± 0-35*
86-10 ± 7-63
31-12 ± 1-50*
29-03 ± 1-76*
12-00
5-95
3-97
±0-98
± 0-20*
±0-20*
* Significant (/> = 0-05)
530
K Ramalingam
Figure 7. A. Section of the liver of normal fish (control). B. DDT treated fish liver section
(arrow indicates periportal necrosis). C. Malathion treated fish liver section (arrow indicates
vacuolation and fatty degeneration).
necrotic changes in the hepatic cells and the consequent release of isoenzymes into
circulation (Weime and Van Maercke 1961; Zimmerman et al 1971; Truhaut et al 1973).
The liver histology in the present study also reveal vacuolation, fatty changes and
degeneration in the fishes exposed to DDT and malathion (figures 7A,B and C).
Concurrent to the increase in the LDH fractions in serum, a decrease in the total number
of fractions was noticed in liver and muscle. Other changes in these tissues are the
appearance of additional new slow moving anaerobic fractions, change in the intensity
to staining of the above fractions and the disappearance of some of the fast moving
fractions. Similar changes have been noticed in the tissues of rats and rabbits
administered with other toxicants (Zimmerman et al 1971; Truhaut et al 1973) and also
in Carassius auratus subjected to thermal stress (Smit et al 1974). In the light of the
above studies and also of the changes noticed in the metabolites of this species, the
changes observed in SDH activity and LDH isoenzymes in the present study are suggestive
of alteration of the metabolic pathway more towards the anaerobic side during EDTand
malathion intoxication.
References
Baker J P P 1969 Histological and electron microscopical observations on copper poisoning in the winter
flounder Pseudopleuronectes americanus; J. Fish. Res. Bd. Can. 26 2785-2793
Liver and muscle SDH enzyme activity in S. mossambicus 531
Bilinski E and Jonas E E 1972 Oxidation of lactate to carbondioxide by rainbow trout Salmo gairdneri tissues;
J. Fish. Res. Bd. Can. 29 1467-1471
Bostrom S L and Johansson R G 1972 Effects of pentachloro phenol on enzymes involved in energy
metabolism in the liver of the eel; Comp. Biochem. Physiol. 41 359-369
Chavin W and Young J E 1970 Factors in the determination of normal serum glucose levels of goldfish
Carassius auratus; Comp. Biochem. Physiol. 33 629-653
Gardner G R and Yevish P P 1970 Histological and haematological responses of an estuarine teleost to
cadmium; J. Fish. Res. Bd. Can. 27 2185-2196
Janardhan A, Yadgiri B, Reddy E M and Naidu N V 1972 Effects of DDT pretreatment on malathion toxicity
in chickens; Indian J. Exp. Biol. 17 315-316
Janicki R H and Kinter W B 1971 DDT inhibition of Na+, K+ and Mg+ + ATP ase in the intestinal mucosal
and gills of marine teleosts; Nature New Biol. 233 148-149
Kun E and Abood L G 1949 Colorimetric estimation of succinic dehydrogenase by rrc; Science 109 144-146
Ramalingam K 1980 Studies on the effects of sublethal concentrations of a few toxicants on biochemistry,
physiology and histology ofTilapia mossambica (Peters) Ph.D Thesis, University of Madras, India
Ramalingam K 1982 Biochemical composition of blood, liver and muscle of Sarotherodon mossambicus
(Peters); Natl. Acad. Sci. Lett. 5 35-36
Ramalingam K and Ramalingam K 1982 Effects of sublethal levels of DDT, malathion and mercury on tissue
proteins of Sarotherodon mossambicus (Peters); Proc. Indian Acad. Sci. (Anim. Sci.) 91 501-505
Ranganatha K P and Ramamurthi R 1 978 Effect of sumithion (fenitrothion) on some selected enzyme system
in the fish Tilapia mossambica (Peters); Indian J. Exp. Biol. 16 809-810
Sivaprasada Rao K and Ramana Rao K V 1979 Effects of sublethal concentrations of methyl parathion on
selected oxidative enzymes and organic constituents in the tissues of the freshwater fish Tilapia
mossambica (Peters); Curr. Sci. 48 526-528
Skidmore J F 1970 Respiration and osmoregulation in rainbow trout with gills damaged by zinc sulphate;
Wat. Res. 6217-230
Smit H, Vandenberg R J, Kijni I and Hartog D 1974 Some experiments on thermal acclimation in the goldfish
Carassius auratus; Neth. J. Zool. 24 32-49
Smith I 1968 Acrylamide gel disc electrophoresis section I. Technique of disc electrophoresis. In
Chromatographic and electrophoretic technique Vol 2 365—389
Sreeramulu Chetty C, Rajendra L, Indira K and Swami K S 1978 In vitro effects of organochlorine pesticide
DDT on catalytic potential of SDH in gastronemius muscle of frog Rana hexadactyla; Curr. Sci. 47 842-844
Timothy P, Leedem R, Campbell D and Johnson D W 1974 Osmoregulatory responses to DDT and varying
salinities in Salmo gairdneri i. Gill Na+, K+ ATP ase; Comp. Biochem. Physiol. 49 197-205
Truhaut R, Nguyen Phulich, Ngoc Tram, Le Quang Thuan et Heline Dutertre catella 1 973 Etude de quelques
activites enzymatiques seriques et de quelques constituents biochemiques sanguins au cours des
intoxications subaigues avec le tetra chloro 1112 ethane cheoz le lapin; J. Eur. Toxicol. 2 81-84
Wieme R J and Van Maercke Y 1961 The fifth electrophoretically slowest serum lactic dehydrogenase as an
index of liver injury; Ann. N. Y. Acad. Sci. 94 898-91 1
Zimmerman H J, Kendler J and Koff R S 1971 Intraperitoneal halothane administration: Evidence of hepatic
and muscle injury; Proc. Soc. Exp. Biol. Med. 138 678-682
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 533-545.
© Printed in India.
infrastructure of the eggs of Reduviidae: L Eggs of Piratinae
(Insecta — Heteroptera)
E T HARIDASS
Entomology Research Institute, Loyola College, Madras 600034, India
MS received 25 February 1985; revised 28 May 1985
Abstract. Eggs of Piratinae are unique among reduviids in possessing stellate chorionic
filaments that remain exposed even after the insertion of the egg into the ground. These eggs
have developed many structures for supplying ambient oxygen to the developing embryo
inside them. The ultrastructure of the chorion, the operculum and the aeromicropylar system
of the eggs of seven species of piratinae bugs are reported.
Keywords. Reduviidae; Piratinae, egg; chorion; operculum.
1. Introduction
Reduviid bugs are known to colonise varied habitats, depositing their eggs in a variety
of situations, scattering them loosely, inserting or burying them in the soil, or attaching
them to barks or twigs or leaves of trees and shrubs or even to the under surfaces of
rocks and stones. These peculiar eggs have attracted the attention of several workers
including Readio (1926), Southwood (1956), Miller (1953, 1971), Cobben (1968) and
Hinton (1969, 1981). Though the eggs of a number of subfamilies are studied in detail,
those of Piratinae, considered unique among Reduviidae, have not been given sufficient
emphasis and hence an attempt has been made to study the ultrastructure of seven
species collected from southern India.
2. Materials and methods
Eggs of the following species were studied.
(a) Pirates affinis Serville (e) Ectomocoris cordiger Stal
(b) Pirates mundulus Stal (f) Sirthenia flavipes Stal
(c) Ectomocoris tibialis Distant (g) Catamiarus brevipennis Serville
(d) Ectomocoris ochropterus Stal
Eggs were collected from laboratory cultures after oviposition in the soil and also
from dead gravid females with swollen abdomen that were treated with 5 % KOH for few
hours. Due to the hard nature of the egg shells, embedding with paraffin wax was not
successful, hence hand sections of liquid nitrogen-frozen eggs were made using sharp
razor blade. Such sections obtained from ovarian and oviposited eggs, as well as from
empty shells were dehydrated in alcohol, cleared in clove oil or xylene and mounted in
canada balsam or euparol. Longitudinally split entire egg shells were similarly mounted
for counting aeropyles and micropyles. Ultratome (LKB Nova) was used to cut epoxy
533
534 E T Haridass
resin-embedded eggs for ultrathin sections for observations of chorionic details. The
surface areas and volumes of the eggs (the shape of which can be approximated
reasonably to that of a cylinder mounted on a semi ellipsoid of revolution, the bottom
one fourth of the total height being the height of the ellipsoidal part) were calculated
/ / e2 e4 e6 \ lib2
^Surface area: II ab^l -_-— -— ^
4b llllab2
where e2 = 1 — ?; volume: — — —
a *rO
The approximate number of the follicular pits was counted using an occular grid under
a microscope for a given square area and these values were used to arrive at the total
follicular number of the chorion of different eggs. Alcohol preserved eggs, both ovarian
and oviposited, were sonic cleaned, gold coated (EIKO I B.2 ion coater) for 2-4 minutes
and scanned with an electron microscope (Hitachi — 450 A) using 10 and 15kV
emission currents.
3. Observation
3. 1 Oviposition
All piratine bugs examined insert their eggs in loose soil. After suitable site selection, the
gravid female assumes a slanting posture with raised head and thorax and with the
abdominal apex making a twisting side to side, as well as downward, thrusting
movements. The plate-like ovipositors guide the eggs for vertical insertion into the soil.
The posterior pole of the egg is always directed away from the head of the female and
after oviposition the apical parts of the egg alone are exposed outside the soil. The
female covers up the egg with sand grains and particles of debris using her hind legs.
The entire oviposition of a single egg lasts 3-4 minutes and in a day a maximum of 9
eggs are laid and females exhibit 3-^4 oviposition cycles. The eggs are abandoned, there
being no parental care in any of the species observed and all of them displayed a similar
kind of oviposition behaviour.
3.2 Structure of the egg
3.2a Structure, colour and size: Eggs of piratine species are more or less of an
uniform shape, cylindrical apically, the basal fourth of the total height being ellipsoidal
with a cap-like point at the centre of the basal end. Freshly laid eggs have distinct
concave and convex surfaces, corresponding to the ventral and dorsal sides of the fully
developed embryo inside the egg. After the commencement of embryogenesis the eggs
increase in size, altering their dorsal and ventral curvatures, tending to make them more
spherical. The anterior end of the egg is characterised by an operculum surrounded by
chorionic filaments. Generally the eggs are light yellowish or cream-coloured, more due
to the large quantity of yolk inside. Eggs of Pirates mundulus, however, are reddish
brown. The chorionic filaments resemble the eggs in colouration, except in Pirates
affinis and Ectomocoris cordiger where these are brownish black. The sizes of the
Eggs of Piratinae
Table!. Eggs of some Piratinae
535
Egg* Surface area
Length Width Chorionic filament (in sq. mm)
Number of (in mm) Length Width Volume**
Species Aeropyle Micropyle (a) (b) (in //) (in c. mm)
Pirates affinis
270
26-4
2-6
1-29
1280-4
37-14
12-54
3-12
Pirates mundulus
243
18-0
1-34
0-62
341-44
13-92
3-49
0-38
Sirthenia flavipes
169
19-3
1-96
O85
43747
32-48
6-34
1-02
Catamiarus brevipennis
369
15-6
2-73
1-34
320-1
9-28
13-55
3-64
Ectomocoris tibialis
240
16-7
1-98
0-95
160-05
11-6
7-32
1-29
Ectomocoris ochropterus
246
13-3
2-12
0-92
192-06
6-96
7-31
1-31
Ectomocoris cordiger
175
25-0
1-37
0-73
288-09
13-92
4-34
0-53
* Values are for the average of 10 eggs. The shape of the egg can be approximated to that of a cylinder
mounted on a semi-ellipsoid of revolution, the bottom one fourth of the total height being the height of the
ellipsoid part.
+ + Surface area = nab{ 1- — • -^ — )+ , where e = 1 =- **Volume = 11
V 24 160 468 / a a2 48
different eggs, along with the various measurements of their parts are tabulated
(table 1).
3.2b Body of the egg: The main body of piratine eggs is always inserted into the soil
and the surfaces of these are characterised by numerous polygonal follicular areas. In
Ectomocoris tibialis, E. ochropterus, such areas have deep central pits (figure 1, E,F; 4,
G,H; 5G). In Pirates mundulus, Catamiarus brevipennis, Sirthenia flavipes and
Ectomocoris cordiger the polygonal areas are flat with rounded or elongated tubercles
marking their boundaries (figure 1 B-D,G; 4A, C,E and F). In Pirates affinis the
follicular pits are prominent, being shallow anteriorly, but increasing in depth
progressively towards the posterior, giving the appearance of overlapping tiles (figure 1;
3B). The posterior-most part of all eggs examined, has a central cap-like projection,
the surface of which is always beset with deep pits extending well into the interior
(figure 3D,H).
The chorion of the eggs shows only two layers, the exo- and endochorion. Very close
to the collar, the exochorion is thinner than the endochorion, but towards the posterior
part of the egg, the former is thicker than the latter (figure 2A, D). The boundaries of the
prominent follicular pits and tubercle-bounded follicular areas arise as extensions from
the exochorion possessing thicker exochorion than the central areas. The innermost
part of the endochorion shows an aerostatic layer; the net-work of air spaces of this is
separated from the lumen of the egg by a very thin layer with numerous rounded, blunt
E T Haridass
100fji
Figure 1. Eggs of Piratinae. A: Entire egg, operculum and follicular pits of Pirates affinis.
Al: L.S. of the anterior half of the egg. B: Entire egg, operculum and follicular pits of Pirates
mundulus. Bl: L.S. of the anterior half of the egg. C: Entire egg, operculum and follicular pits
ofCatamiarus brevipennis. Cl: L.S. of the anterior half of the. egg. D: Entire egg, operculum
and follicular pits of Sirtheniaflavipes. Dl: L.S. of the anteriof half of the egg. E: Entire egg,
operculum and follicular pits of Ectomocoris tibialis. El: L.S. pf the anterior half of the egg.
F: Entire egg, operculum and follicular pits of £. ochropterus< Fl: L.S. of the anterior half of
the egg. G: Entire egg, Operculum and Follicular pits of E.'cordiger. Bar scale: 1000
entire egg and operculum; 250 n for L.S. of the egg; 100 /z for follicular pits.
tubercles on the outer surfaces abutting the rest of the endochorion (figure 3G; 4J). The
basal specialised prominence of the egg with deep follicular pits shows numerous pore
canals extending into the endochorion and opening finally; into the aerostatic layer
(figure 2D; 3H). ( \
3.2c Collar: The anterior rim of the cylindrical egg has a prominent collar consisting
of two distinct regions: (i) an outer collar rim, composed mostly of exochorion,
projecting away from the central axis and enclosing a ring-lite spermatic groove on its
inner side; (ii) an inner T shaped sealing bar, formed wholly of endochorion, the basal-
free edge of it is slightly turned upwards and projecting into the lumen of the egg like a
ring (figure 1 A1-G1; 2A; 3C, E). This wedge of sealing bar encloses on its upper side a
circular, shallow, but wide groove with numerous vertical columns of alternating ridges
Eggs of Piratinae
537
ai
100 jj
Figure 2. Egg of Pirates affinis. A: L.S. of the anterior lateral part of the operculum and
collar region of the chorion. Arrows indicate points of breakage at the time of eclosion.
B: Outer surface view of the collar region of chorion. C: Inner surface view of the collar region
of chorion. D: L.S. of the basal part of chorion.
Abbreviations, a, aeropyle, ai, aeropylar inner opening, al, aerostatic layer, as, air space, cc,
chorionic collar, cf, chorionic filament, enc, endochorion, exc, exochorion, fp, follicular pit, m,
micropyle, me, micropylar external opening, mi, micropylar inner opening, o, operculum, of,
opercular filament, pc, pore canal, sb, sealing bar, sbr, sealing bar ridge, sbf, sealing bar furrow,
sg, spermatic groove.
and furrows on its inner wall (figure 3F). The blunt circular basal margin of the
operculum with similar vertical columns of ridges and furrows (figure 2C; 5A-D & H),
flush with the ridges and furrows of the sealing bar, enabling the operculum to fit
exactly into the groove of the sealing bar (figure 2A; 3C, E). The aerostatic inner layer of
the chorion takes its origin from the distal parts of the wedge of sealing bar, from below
the groove which receives the margins of the operculum (figure 2A).
The anterior apices of the collar side of the sealing bar, inner to the spermatic groove,
give rise to numerous upwardly projecting endochorionic extensions, each of which
corresponds to one furrow and two half ridges of the sealing bar. At about the middle
level of the operculum, between 2 and 5 of these- extensios merge into one and
immediately above this fusion they join again with similar extensions arising from the
outer walls of opercular rim. The chorionic filaments thus formed are contributed by
538
E T Haridass
"t f Tt **T"*rS
\Y,?fr//<7/
Figure 3. Scanning electron micrographs of the egg of Pirates qffinis. A? Anterior and
opercular surface view of the egg (scale: 250 /i). B: External aeropylar openings on the
chbrionic .filaments (scale: 38 ju). C: L.S. through anterior part of the collar region and
operculiTOt$cak: 300 /i). D: Follicular pits at the basal part of the egg (scale: 60 /i). E: L.S.
through the .anterior part of the collar region and operculum of an ovarian egg (scale: 130 /i).
F: Ridges and furrows of the sealing bar on the inner surfaces of the chorionic collar. Arrow
points to the outer micropylar opening at the free edge of the sealing bar (scale: 72 /*).
G: Outer view of the aerostatic inner layer after separation from the endochorion (scale: 5 /4
H: Pore canals (arrows) from the basal follicular pits (scale: 42 M)-
Eggs of Piratinae 539
both operculum and collar. These filaments finally project further up and radiate away
from the central egg-axis (figure 1; 2 A; 3 A, C). While the opercular extensions of the
chorionic filaments may or may not be coloured, those from the sealing bar are always
colourless. During eclosion the opercular connections with chorionic filaments get
snapped like rings at two places, one at the innermost part of the wedge of the sealing
bar below the operculum, from where the aerostatic layer originates and the other at the
junction where the opercular extensions join with those of the collar (figures 5 A— D &
H). Hence after the escape of young nymphs, the chorionic filaments of empty eggs
appear to arise only from the inner aspects of the collar rim, inner to the spermatic
groove.
In freshly laid eggs the chorionic filaments converge over the operculum and project
towards the central axis of the eggs, but 20-30 minutes after oviposition, they open out
and project away giving the eggs a star-like appearance characteristic of piratinae
(figure 3A; 4E & F).
There is considerable variation among the different species examined in so far as the
length and structure of the chorionic filaments are concerned. While these filaments in
E. tibialis, E. ochropterus and C. brevipennis are short and extend only upto the level of
the opercular tip (figure 1C, E & F; 4C, G, & H), those of E. cordiger, S.flavipes, and
P. mundulus are longer and extend well beyond the operculum (figure 1B,D & G; 4A,E
and F). The maximum development of the filaments is seen in P. affinis, the blackish-
brown filaments projecting around the periphery of the collar (figure 1A; 3A, C). In
S.flavipes the collar region is peculiar in that the collar rim is united at regular intervals
with the outer basal parts of the chorionic filaments all around, enclosing the spermatic
groove completely, except for regularly placed openings at the apical outer surfaces
(figure 2E; 5E).
3.2d Aeropyles and micropyles: The apical parts of the chorionic filaments, extend-
ing to about a fourth to one half of their length from the apex, show numerous
irregularly shaped and linearly arranged external aeropylar openings. These openings
in most of the species become progressively reduced in size and fused towards the bases
of the filaments (figure 3B; 4B). In S. flavipes however, these external openings are
distinct and extend to about three fourths of the filaments (figure 5E). The external
aeropyle openings lead to many irregular air spaces that extend towards the collar and
in the endochorion of the sealing bar, the air conduits join into regularly arranged
vertical channels, the aeropyles. The aeropyles are placed, in the centre of the vertical
furrows of the sealing bar, bounded on either side by its ridges, ultimately opening into
the inner aerostatic layer of the endochorion through distinct apertures, the inner
aeropylar openings (figure 2C). These openings, placed between the bases of the ridges
of the sealing bar, demarcate the lower limits of the collar region. The inner aerostatic
layer distributed throughout the interior of the body, commences from the lower wedge
of the sealing bar where the inner aeropylar openings finally open (figure 2A).
The micropyles, easily identified by their funnel-shaped external openings, originate
from the spermatic groove, in the inner wall of the collar rim (figure 2A; 51). They
extend down along the chorion, running almost parallel to the sealing bar and at the
level of the inner aeropylar openings, make a 'IT turn and run through the wedge of
sealing bar to open finally into the lumen of the egg prior to the line of weakness
between the collar and the opercular base. When viewed from the apical side of the egg,
140
E T Haridass
Figure 4. Scanning electron micrographs of the eggs of Piratinae. A: Pirates mundulus:
Anterior half of the egg with operculum (scale: 125/4 B: Chorionic filaments with external
aeropylar openings of the same (scale: 26 /*). C: Catamiarus brevipennis: Collar rim and
chorioriic filaments (scale: 97 /*). D: Sirtheniaflavipes: Anterior half of the egg with operculum
(scale: 250 /i). E: Enlarged view of the same (scale: 172 //). F: Ectomocoris cordiger: Anterior
half of the egg with operculum (scale: 162 /i). G: Ectomocoris tibialis: Anterior half of the egg
with operculum (scale: 95 /i). H: Ectomocoris ochropterus: Anterior half of the egg with
operculum (scale: 215 /*).
Eggs of Piratinae
541
Figure 5. Scanning electron micrographs of the eggs of Piratinae. A: Operculum of
Ectomocoris tibialis (scale: 118 u). B: Operculum of Pirates mundulus (scale: 86 u),
C:. Operculum of Ectomocoris ochropterus (scale: 154/j). D: Operculum of Catamiarus
brevipennis (scale: 137 u). E: Sirthenia flavipes: Collar and chorionic filaments (scale: 75 u).
F: Spongy area at the operculur disc of the same (scale: 57 u). G: Enlarged view of the
follicular pits of Ectomocoris tibialis (scale: 17 u). H: Operculum of Pirates affinis (scale: 267 /*).
I: Micropyles of Pirates mundulus at the collar region (Photomicrograph: scale: 376 u}. J: L.S.
of chorion of Pirates affinis showing the aerostatic layer (arrows; scale: 14 u).
542 E T Haridass
the bent portion of the micropyles are arranged in a clockwise direction. The number of
micropyles and aeropyles, along with their dimensions is tabulated (table 1).
3.2e Operculum: The operculum of piratine eggs is disc-like and fits in the collar
through a very distinct sealing bar. The exo-endochorionic differentiation, seen clearly
in the body of the egg, is absent in the operculum and it is wholly composed of
endochorion. While the posterior part of the operculum is simple and dome-shaped, the
anterior surface shows variations in different species. In E. tibialis, E. ochropterus,
E. cordiger, C. brevipennis and P. affinis (figures 1, 2, 4 & 5), the upper surface of the
operculum has a central concave disc of shallow area consisting of air containing
spongy network, the height of which gradually increases towards the opercular
periphery and ultimately project as small, but numerous filaments. The spongy network
is supported by slanting rings of endochorionic extensions. In addition, the centre of
the disc may show additional smaller supports. In E. tibialis and E. ochropterus a thin
highly porous veil-like extension connects the extremities of the extensions to the upper
middle of the operculum (figure 1, El, Fl; 4G, H; 5A, C). In E. cordiger, instead of the
shallow central depression, the operculum has the central area elevated like a button
with the spongy network restricted to this elevated part (figure 1G; 4F). In P. mundulus
the entire upper surface is in the form of an upturned air-filled cup having tubercular
projections (figure IB 1, 5B). In the last two species the central part of the opercular
disc has a vertical endochorionic column with the apex flared giving an 4X' or ' Y' shape
in cross-section. The most peculiar operculum is that of S.flavipes where the central
disc of the upper surface is produced into a vertical pillar supporting in its extreme an
inverted air-filled saucer-shaped disc with spongy area confined to the centre (figure 1,
Dl; 5F). The pillar-like support has numerous pore canals running through it.
The periphery of the operculum is nearly 4-5 times as thick as its central area and
shows large air-filled spaces supported by struts or pillars. The height of such struts of
pillars, as well as the sizes of the air spaces progressively get reduced towards the central
area of the operculum (figure 2). Through the basal wall of the air-filled cavities,
especially from the central shallow area, run numerous pore canals through the
endochorion and open into the lumen of the egg. The aerostatic inner layer visible
throughout the body is distinctly absent in the endochorion of the operculum. During
eclosion the operculum breaks free from the sealing bar of the collar at two places
(figure 2A) and is removed like a lid from the body of the egg without causing any
damage to the latter.
4. Discussion
A detailed study of the eggs of several terrestrial heteroptera enabled Southwood (1956)
to distinguish Cimicomorpha from Pentatomomorpha on the basis of operculum,
sealing bar and aero-micropylar systems. In spite of the general similarities with other
families, the characters of the reduviid eggs were recognised as unique in several aspects
(Readio 1926; Miller 1953; Southwood 1956). Among the reduviids the eggs of
Piratinae and Harpactorinae stand out as mbst characteristic. Though there are several
descriptions pertaining to the eggs of Harpactorinae, not much attention has been given
Eggs ofPiratinae 543
to the eggs of Piratinae. Readio (1926) was the first to mention the chorionic filaments
in Piratinae. While describing the biology of many southern Rhodesian reduviids,
Miller (1953) provided information on the eggs of some Piratinae. Even in the classical
work of Cobben (1968) and Hinton (1981) there is hardly any mention about this
subfamily.
The cylindrical shape of the piratine eggs with a pointed and semi-ellipsoidal basal
region enables their easy insertion into the ground. This type of vertical deposition of
eggs in the soil, leaving only the apical parts exposed, is unique to Piratinae (Haridass
1974) and such a method does not fit into the ovipositional schemes of insects provided
by South wood (1956).
The structure of the shell or chorion of insect eggs is a product of the follicular cells
and this has been a subject of controversy for long. The architectural as well as the
chemical diversity of the chorion have been shown to be highly variable and there has
been neither strict correspondence between layers of eggs of different insects nor any
homology with the terms exo- and endochorion (Cobben 1968; Hinton 1969). In fact,
Pantel (1913), Hinton (1963), and Hartley (1964) suggested that these layers be merely
called outer and inner chorionic layers. Generally the most accepted terminology for
the outer and inner layers of chorion are exochorion and endochorion, respectively. All
other layers secreted outside the eggs, either by the oviduct or by the female accessory
glands, are called extrachorion (Hartley 1961) or as suprachorion (Cobben 1968), as
against the layer/s secreted inner to the chorion by the developing embryo as
subchorion (Beament 1949). Eggs of all the piratine species examined revealed only two
distinct layers, the exo- and endochorion, throughout their body, but varying in
thickness in those eggs with marked follicular pits or with tubercle-bounded follicular
areas. The seven different proteinaceous layers reported for Rhodnius eggs (Beament
1946) have not been observed presently and the existence of such layers has also been
doubted (Cobben 1968; Hinton 1969). Unlike the body, the operculum of these eggs is
made up of only the endochorion. The true operculum common in Cimicomorpha, is
defined as one which is formed only of endochorion, with sealing bar and micropyles
(Southwood 1956). The innermost layer of the shells in all piratine species examined is a
continuous aerostatic layer separated from the rest of the endochorion by blunt
tubercular struts enclosing air spaces. This layer appears to be common in many
reduviids (Southwood 1956; Cobben and Henstra 1968; Cobben 1968; Salkeld 1972;
Hinton 1981) and it is also believed to have evolved independently along different lines
among various groups of insects (Cobben 1978).
Like other terrestrial insects, Piratinae lay eggs in relatively dry environment and
hence face the requirement of extensive surface area for obtaining oxygen from the
ambient air, as well as the danger of losing considerable water through such surfaces. In
spite of all these adverse factors, the piratine eggs have evolved efficient respiratory
system that has satisfied their oxygen needs without losing too much water. The surface
area/ volume ratios of the eggs being high (table 1) the contrasting problems of oxygen
uptake and water loss are very great for the small-sized eggs and so the existence of
complicated respiratory system in them appears very relevant.
The air containing aerostatic inner layer in piratine eggs is connected with the
ambient air. The numerous external aeropylar openings of the chorionic filaments
establish a pathway for air passage between ambient oxygen and the inner aerostatic
layer through a network of air channels in the filaments and in the collar region. In
addition, the oxygen available inside the ground is also taken to the aerostatic layer
544 E T Haridass
from the deepest part of the egg lying buried, through a system of pore canals of the
specialised basal part. The absence of a distinct aerostatic layer in the operculum is
compensated by the development of network of spongy areas and large air-filled spaces
in this part of the egg and with a system of well-developed pore canals, the air spaces are
connected to the lumen of the egg enabling the apical parts of the developing embryos
to get their share of the ambient oxygen.
Terrestrial habitats of Piratinae are often liable to sudden flooding, particularly
during rainy seasons, and to get submerged in water for periods longer than the
embryonic development is a natural and regular problem for these eggs. Hence the
adaptations of these terrestrial eggs for respiration water are as complex as those of
aquatic eggs. Eggs which can submerge in water always support a thin layer of air all
around them and this physical gill has been termed as plastron (Brocher 1912 a,b). Such
plastrons can be formed by an aggregation and/or enlargement of aeropyles as well as
by a close network of air containing areas of the chorion. The importance of plastron is
well documented for Coleoptera (Thrope and Crisp 1949) and the instances of«plastron
respiration among terrestrial insects are believed to outnumber those of aquatic insects
(Hinton 1981). Plastron enables the egg to remain submerged indefinitely and to obtain
oxygen from ambient water as well as to provide direct air routes in dry conditions.
Terrestrial insects, having a small ratio of the total aeropylar area per mg of egg to effect
an efficient plastron respiration, are known to lay eggs in habitats that are usually
wetted by rain and in many such insects, like the minds, the aeropyles are restricted to
the anterior end of the eggs on elevated tubercles or stalks (Hinton 1962; Cobben 1968).
The elevation of aeropyles on such respiratory horns helps the eggs to utilize
atmospheric air when the egg is covered by a thicker layer of water. In piratine eggs the
operculum is provided with a complicated system of air-containing network and the
general surface of the chorion is never smooth; they always have prominent follicular
pits or tubercle bounded follicular areas. These structures of the chorion and the
operculum, the irregular prominences of the collar and the external aeropylar openings
of the chorionic filaments, all act as hydrofuge. When dipped in water these areas resist
wetting and always hold a thin layer of air all around them. Though not so well
developed as in other terrestrial eggs reported so far, such an air layer still serves as
efficient plastron for those eggs.
As in other Cimicomorpha, the eggs of Reduviidae are fully formed before
fertilization and it is essential that the eggs are provided with openings in the chorion
for the entry of sperms for fertilization. Leuckart (1855) was the first to describe these in
insects eggs -and such canals are termed as micropyles. Complete forms of true
aeropyles and micropyles are found only in Reduviidae (Southwood 1956) and in
piratine eggs they are typical like those described in many other Reduviidae (Beament
1947; Southwood 1956; Cobben 1968; Salkeld 1972; Haridas 1974; Hinton 1981) arising
from the inner aspects of the spermatic groove in the collar and opening into the lumen
of the egg through the endochorion near the sealing bar. When viewed from the apical
side, the inner bent portions of these sperm canals are arranged in a clock-wise
direction, as has been reported for other reduviid eggs (Cobben 1968). Though the
maximum of micropyles reported for Reduviidae is only 15 (Cobben 1968) the species
presently examined possessed a higher number, the maximum for a single egg being 31
(table 1). This number is reduced in eggs laid at the fag end of the ovipositional cycle and
as Beament (1947) suggested, this reduced number of micropyles could be one of the
reasons for the higher number of unfertilized eggs laid by older females.
Eggs of Piratinae 545
Acknowledgement
The author wishes to express his sincere thanks to Prof. T N Ananthakrishnan for
guidance and advice, as well as for his useful criticisms of the manuscript. This work was
supported by a grant from the Department of Science and Technology.
References
Beament J W L 1946 The formation and structure of chorion of the egg of an hemipteran, Rhodnius prolixus;
Q. J, Micr. Sci. 87 393-439
Beament J W L 1947 The formation and structure of Micropylar complex in the egg shell of Rhodnius
prolixus Stal (Het-Reduviidae); J. Exp. Biol 23 213-233
Beament J W L 1949 The penetration of Insect shell II. The properties and permeability of subchorial
membranes during development of Rhodnius prolixus Stal; Bull. Exp. Res. 39 467-488
Brocher F 1912a Reserches sur is respiration des insects aquaticum adultes: Les haemonia. Ann. Biol.
Lucerta 5 5-26
Brocher F 1912b Reserches sur la respiration des insectes aquaticum adultes: Les elmides. Ann. Biol. Lucerta.
5 136-179
Cobben R H 1968 Evolutionary trends in Heteroptera Part I: Eggs, architecture' of the shell, gross
embryology and eclosion, Centre for Agric. Pub. & Documentation, Waganingen 475 pp.
Cobben R H 1978 Evolutionary trends in Heteroptera Part II: Mouthpart structure and feeding strategies,
Mededlingen Landbouwhoschool, Waganingen 407 pp.
Cobben R H and Henstra S 1968 The egg of an assasin bug (Rhinocoris sp) from Ivory Coast. JOEL NEWS
632pp
Haridass E T 1974 Biological and ethological studies on some South Indian Reduviids (Hemiptera-Reduviidae);
Ph.D. Thesis, University of Madras, Madras, India
Hartley J C 1961 The shell of acridid eggs; Q. J. Micr. ScL 102 249-255
Hartley J C 1 964 The structure of the eggs of the British Tettigoniidae. (Orthoptera); Proc. R. Entomol. Soc. ,
London 39 111-117
Hinton H E 1962 The structure of the shell and respiratory system of the eggs ofHelopekis and related genera
(Hemiptera-Miridae); Proc. Zool. Sci., London 139 482-488
Hinton H E 1963 The respiratory system of the egg shell of the blow-fly, Calliphora erythorocephala Meigen
as seen with electron microscope; J. Insect Physiol. 9 121-129
Hinton H E 1969 Respiratory systems of insect egg shells; Annu. Rev. Entomol. 14 343-368
Hinton H E 1981 Biology of insect eggs, Vol I, II, III. (Oxford, New York. Pergamon Press) 1125 pp
Leuckart R 1855 Ueber die micropyle and dan feinern Bau der Schalenhant bei den Insekteneiern; Arch. Anat.
Physiol Wiss. Med. 1855 90-253
Miller N C E 1953 Notes on the biology of the Reduviidae of Southern Rhodesia; Trans. Zool. Soc. London 27
541-656
Miller N C E 1971 The biology of Heteroptera (2nd edition), (London: E. W. Classey Ltd.) 206 pp
Pantel J 1913 Recherches sur les Dipteres a larves entombies II. Les enveloppes de 1'oeuf les formations aue'
en dependent, les degats indirects du paratisme; Cellule 26 1-289
Readio P A 1926 Studies on the eggs of some Reduviidae; (Heteroptera); Univ. Kansas. Sci. Bull. 16 157-179
Salkeld E H 1972 The chorionic architecture ofZelus exsanguis (Hemiptera — Reduviidae); Can. Entomol. 104
434-442
Southwood T R E 1956 The structure of the eggs of the terrestrial Heteroptera and its relationship to the
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 547-556.
© Printed in India.
Host selection and food utilization of the red pumpkin beetle,
Raphidopalpa foveicollis (Lucas) (Chrysomelidae: Coleoptera)
K RAMAN and R S ANNADURAI
Entomology Research Institute, Loyola College, Madras 600034, India
MS received 2 January 1985; revised 22 February 1985
Abstract. An analysis of the host plant relationships with respect to the red pumpkin beetle,
Raphidopalpa foveicollis Lucas is presented based on the role of receptors involved in host
selection, the quantitative food utilization on different cucurbitaceous host plants and the
biochemical parameters involved in food plant selection. Orientation of the beetles towards
the host plants appeared to be profoundly affected when the receptors present on the antennae
and mouthparts were ablated or coated. Though significant differences were observed with
regard to the quantity of food ingested among different host plants, ingestion of food was
higher for mature leaves and flowers compared to young and senescent leaves. Accordingly,
mature leaves and flowers showed high nitrogen and proteins, low sugars, moderately high
phenols and narrow C/N ratio compared to other plant parts. The chemosensory receptors
present on the antennae and mouthparts were also studied using scanning electron
microscope.
Keywords. Host selection; food utilization; Raphidopalpa foveicollis.
1. Introduction
An individual plant is heterogenous to any phytophagous insect and since the plant
parts vary in their nutritional value, the food utilization on different parts of the same
plant appears to be important in order to determine the pest status of the concerned
species. The red pumpkin beetle, Raphidopalpa foveicollis Lucas feeds on a variety of
cucurbitaceous crops causing economic losses. Observations by Sihna and Krishna
(1971) and Grewal and Sandhu (1982) have shown the relative preference of the beetle
towards different parts of the cucurbits, Lagenaria vulgaris Ser and Cucumis melo L. In
view of the fact that the information on the comparative utilization of the different
plant parts viz flowers, young, mature and senescent leaves is meagre, an attempt has
been made to study the relative preference and quantitative food utilization of some
economically important cucurbits and a weed host by R. foveicollis in terms of some
important chemical parameters. In addition, the role of sensory structures in host
selection by R. foveicollis was also investigated using scanning electron microscope
(SEM).
2. Material and methods
Adult individuals of mixed age and sex groups, collected from the college campus were
used in this investigation. The different cucurbitaceous plants viz Luffa cylindrica
547
548 K Raman and R S Annadurai
Roem, Luffa acutangula Roxb, Trichosanthes anguina L, Cucurbita maxima Duch,
Benincasa cerifera Savi, Momordica charantia L and Mukia scabrella Am were collected
from the college farm.
2.1 Role of receptors in host selection
The role of receptors in host selection was studied by ablation of antennae, painting the
eyes with nail polish, and coating the mouthparts with nail polish. In each of these tests
ten adults of R.foveicollis were subjected to various treatments and another ten adults
were used as control. The beetles were released into a glass trough containing the
preferred host plant at the centre and covered by a glass plate. The number of beetles
(control and experimental) orienting towards the host plant was monitored for 15 min.
Each set of experiment was repeated three times.
2.2 SEM studies
To get a detailed picture of the sensilla on the antennae and mouthparts SEM studies
were made. The mouthparts and antennae were detached from etherized specimens,
dried in a dust-free desiccator for 1-2 days, fixed to aluminium stubs using double
adhesive tapes and then coated with gold for 2-3 min in a standard ion-coater.
Micrographs were made using a Hitachi scanning electron microscope, table top model
S 415 A under 15 kV emission current.
2.3 Quantitative food utilization
To study the quantitative food utilization by R.foveicollis, leaf discs (7-5 cm dia) or an
entire leaf were placed inside a petri dish over a moist filter paper of the same
dimension. The initial weight of the leaf disc and the filter paper was determined. The
test insects were fed with water for 2-3 hr by providing a moist cotton to clear their guts
before the start of the experiment. A pair of beetles (male and female) were released into
the dish and covered by another petri plate. A control was also maintained without the
test insects. Twentyfour hours after the release of the insect, the leaf discs and filter
paper were removed and reweighed. Before reweighing the filter paper, the sides of the
petri dishes were carefully wiped with it to collect any excrement left on the dishes. After
correcting for the weight loss due to transpiration by the leaf, the comparative food
utilization on different host plants was assessed by determining the quantity of food
ingested/24 hr and the coefficient of digestibility (Waldbauer 1968). Each set of
experiment was repeated three times.
2.4 Biochemical analysis
The different cucurbitaceous host plant parts viz flowers (1-day old), young (3-5 days
old), mature (5-10 days old), and senescent leaves (more than 10 days old) were analysed
for various biochemical components. The total nitrogen was estimated (Humphries
Host selection and food utilization in R. foveicollis 549
1956) and multiplied by 6-25 to estimate the total protein content. In addition, the total
carbohydrates (Dubois et al 1956) and phenols (Bray and Thorpe 1954) were also
estimated. The C/N ratio was computed for the different host plants.
3. Results
3.1 Role of receptors in host selection
It was observed that orientation towards the host plant by R. foveicollis was profoundly
affected when subjected to various tests such as antennal ablation, blinding of eyes and
coating of mouthparts. Antennectomized and blinded beetles took a longer time in
moving near the host plant. The mean numbers of antennectomized beetles that
located the host plant and involved in active feeding were 2-0 and 0-67 respectively.
When eyes were blinded, the mean number of beetles which located the host plant was
only 0-33 and they did not involve in active feeding. However, in control, the number of
beetles involved in host location and active feeding was 7-00 and 5-00 respectively. The
beetles, whose mouthparts have been suitably coated, did not show any positive
response to initiate feeding even after locating the host plant (table 1).
3.2 SEM studies
Figure 1 depicts the sensory structures involved in perceiving the odours/stimulus
emanating from the host plants and are located on the antennae and mouthparts of
R. foveicollis, involving (1) sensilla trichoidea: four long sensory hairs at the apex of
each antennae, functioning as olfactory chemoreceptors, (2) sensilla basiconica: short
sensillae distributed all along the antenna and designated as trichoid sensillae involved
in olfaction, and (3) sensilla chaetica: bristles with blunt tips or sometimes with sharp
pointed ends serve as mechanoreceptors. Ablation of antennae in R. foveicollis deprived
the insect of the ability to perceive stimuli and interfered with the normal orientation
towards the host plant. The outer and inner face of the labrum bears sparsely
distributed trichoid sensillae. While the mandibles did not bear any sensory hairs, on
Table 1 . Effect of antennectomy , blinding of eyes and coating of maxillae on the host
plant location and feeding by R. foveicollis.
Mean number of beetles orienting towards
the host plant
Treatments Host location t value Active feeding t value
Antennectomized
2-00
6-10*
0-67
6-69*
Eyes blinded
0-33
10-30**
o-oo
12-20**
Maxilla coated
0-33
10-30**
0-00
12-20**
Control
7-00
5-00
Data represent mean of three replicates. *Significant at 5 % level, **significant at 1 %
level.
550
K Raman and R S Annadurai
Figure 1. A. Antenna showing four long Sensilla trichoidea at the tip. B. Distribution of
sensilla basiconica (short sensillae) and Sensilla chaetica (bristles with blunt tips) on the
antenna. C. Stipes of maxilla showing the campaniform sensilla. D. Enlarged view of the
campaniform sensilla on the stipes. E. Labial palp. F. Labial palp showing the presence of
two types of sensillae at the tip.
the maxilla, the stipes carried large number of sensory hairs (figure 1). The maxillary
palp did not carry any sensilla but the labial tips showed the presence of two types of
sensillae, one group with sharp and pointed ends and the other with blunt ends. These
sensillae appear to be the campaniform sensillum and act as gustatory chemoreceptors.
Host selection and food utilization in R. foveicollis
3.3 Quantitative food utilization
551
The quantity of food ingested by R. foveicollis varied considerably among different
cucurbitaceous host plant parts (table 2). On young leaves, maximum consumption was
observed on B. cerifera (0-367 mg) followed by T. anguina (0-362 mg), L. cylindrica
(0-342 mg), L. acutangula (0-259 mg) and C. maxima (0-256 mg). The food ingested was
the least on the weed host M. scabrella (0-199 mg). However, the quantity of food
ingested on mature and senescent leaves was the highest for T. anguina (0-510 and
0-427 mg) as against 0-469, 0-335 mg; 0-385, 0*339 mg; 0-350, 0-307 mg; 0-295, 0-272 mg
and 0-188, 0-1 78 nag on L. cylindrica, B. cerifera, L. acutangula, C. maxima and
M. scabrella respectively. With regard to the flowers, the highest consumption was
observed for C. maxima (0-395 mg) compared to other host plants. Thus an overall
assessment of the pattern of food ingestion by R. foveicollis showed that apart from the
Table 2. Quantitative food utilization by R. foveicollis on certain cucurbitaceous
hosts
Plant parts
Host plants
Quantity of
food ingested
(mg)
Coefficient of
digestability(%)
L. cylindrica
0-342 ±0000*
92-950 ±0562
r. anguina
0362 ± 0-038
93-770 ±1-490
C. maxima
0256 ±0033
89-456±2-919
Young leaves
L. acutangula
O259±O008
89-575± 1-219
B. cerifera
0367±O027
94-005 ±1-041
M. charantia
— — —
— — —
M. scabrella
O199±0001
85-929 ±1-489
L. cylindrica
0469±O005
94-570±0271
T. anguina
0510±0001
96-275 ±0873
C. maxima
O295±O027
9 1-985 ±1-983
Mature leaves
L. acutangula
O350±0039
94-000 ±0481
B. cerifera
0385 ±0007
94-805 ±1-292
M. charantia
— — —
— — —
M. scabrella
01 88 ±0036
86-705 ±0708
L. cylindrica
0335 ±0004
9O290±0572
T. anguina
0427±O065
95-316±0053
C. maxima
O272±O065
88-971 ±1-021
Senescent leaves
L. acutangula
0307±O053
90228 ±0553
B. cerifera
0339 ±0022
93-805±0512
M. charantia
— — —
— — —
M. scabrella
0178 ±0016
86-516±0038
L. cylindrica
0347 ±0049*
84-725 ±O802
T. anguina
0355 ±0010
85-070 ±0140
C. maxima
O395±O066
87-848 ±0539
Flowers
L. acutangula
0349 ±0022
85-673±0138
B. cerifera
O372±O071
85-022±0412
M. charantia
— — —
— — —
M . scabrella
O165±O040
65-455 ±0-412
* Indicate mean + S.E.
552 K Raman and R S Annadurai
preference among different cucurbitaceous host plants, the preference was more for
mature leaves and flowers compared to young and senescent leaves. Observations on
the feeding behaviour of the beetle under field conditions also revealed a similar picture.
The non-utilization of the cucurbitaceous host, M. charantia is of interest since the
beetles were not found feeding on this plant in the field as well as under laboratory
conditions. The computation of the coefficient of digestibility of various plant parts
also showed a direct proportionality with the quantity of food ingested. Moreover, the
digestibility was comparatively higher when R.foveicollis was fed on mature leaves as
against other host plant parts.
3.4 Biochemical analysis
Data on the biochemical analysis of different cucurbitaceous host plants are furnished
in table 3. With regard to water content, it was found to be higher in young leaves and
flowers compared to mature and senescent leaves, but significant differences were not
observed among different host plants, excepting in the case of senescent leaves. The
estimation of nitrogen and protein content showed significant differences between
different parts of the plant as well as among different host plants. While the nitrogen
and protein content was appreciably high for B. cerifera and L. cylindrica, on other host
plants they were comparatively lower. It was maximum in mature leaves compared to
young and senescent leaves and moderately high in flowers.
Estimation of carbohydrates showed that it was lower in L. acutangula, T. anguina
and M. scabrella when compared to C. maxima. Variation was evident between the
different parts of the plant such as mature and senescent leaves exhibiting lower
carbohydrates as compared to young leaves and flowers. Similarly, the phenol content
in M. scabrella and T. anguina was less when compared to other host plants and young
leaves showed higher phenolic contents compared to other parts. Computation of C/N
ratio also indicated considerable differences among different cucurbitaceous host
plants as well as between plant parts. The ratio was narrow for mature leaves followed
by senescent leaves, flowers and young leaves. However, it was high in M. charantia
compared to other host plants.
3.5 Host plant preference in terms of food utilization and biochemical parameters
An assessment of the host plant preference of R.foveicollis in terms of the quantity of
food ingested and various biochemical components also revealed some interesting
results. With regard to the utilization of flowers, the preference among different plants
is as follows: C. maxima > B. cerifera > T. anguina > L. acutangula > L. cylindrica > M,
scabrella. Although the percentage of nitrogen and protein was maximum in L.
cylindrica, the quantity of food ingested was comparatively lower when compared to
C. maxima, probably due to the increased carbohydrates and phenols. A similar trend
was also evident for L. acutangula. Moreover, the low nitrogen and protein in
C. maxima have been compensated by high food intake by the beetles.
The presence of increased amounts of nitrogen and protein, low phenols and
moderate carbohydrate in the young leaves of B. cerifera resulted in an increased
quantity of food intake by R.foveicollis. On the contrary, the high concentration of
Host selection and food utilization in R. foveicollis
Table 3. Chemical composition of certain cucurbitaceous host plants
553
Plant Moisture Nitrogen
Host plants parts (%) (%)
Proteins Carbohydrates Phenols C/N
( %) (mg/g fresh wt) (mg/g fresh wt) ratio
Y
83-28
5-32
33-25
2-38
2-60
0-45
L. cylindrica
M
S
77-76
62-73
6-44
4-79
40-25
29-94
1-25
1-08
2-53
2-19
0-19
0-23
F
80-43
5-95
37-19
2-13
2-60
0-36
Y
84-39
3-64
22-75
1-13
1-85
0-31
M
84-00
5-18
32-38
1-00
1-63
019
T. anguina
S
76-84
3-15
19-69
0-93
1-80
029
F
83-10
3-41
21-81
2-00
2-10
059
Y
82-24
1-61
1006
2-60
2-00
1-61
M
72-94
3-15
19-69
1-48
1-83
047
C. maxima
S
68-32
2-10
16-63
1-13
1-35
054
F
8010
2-66
16-63
1-18
1-35
044
Y
78-14
2-17
13-56
1-63
2-60
O75
L. acutangula
M
S
73-75
66-00
2-94
2-01
18-38
18-81
1-13
0-85
2-53
2-30
038
028
F
79-75
4-69
29-31
1-73
1-56
037
Y
84-00
6-09
38-06
2-23
3-03
037
M
79-26
6-58
41-13
2-13
2-60
032
B. cerifera
S
77-62
4-34
27-13
2-23
2-18
051
F
8000
2-38
14-88
1-75
2-18
074
Y
82-34
1-96
12-25
2-39
2-61
1-22
M
78-84
2-17
13-56
1-64
2-00
O75
M. charantia
S
73.92
1-26
7-88
1-08
1-08
085
F
81-42
1-61
10-06
2-28
1-75
1-42
Y
84-00
1-68
10-50
1-25
1-50
074
M. scabrella*
M
S
78-77
76-63
1-75
1-96
10-94
12-25
0-95
0-93
1-55
1-55
054
047
F
82-50
1-12
7-00
1-10
2-15
098
"Indicates cucurbitaceous weed host. Y: Young leaves; M: Mature leaves; S: Senescent leaves; F: Flowers
phenols and carbohydrates in L. cylindrica reduced the food consumption. The food
intake in L. acutangula, C. maxima and M. scabrella was sufficiently low presumably
due to low nitrogen and protein. Thus the preference for young leaves indicate the
following sequence: B. cerifera > T. anguina > L. cylindrica > L. acutangula > C.
maxima > M. scabrella.
The pattern of food consumption on mature and senescent leaves also indicate a
striking relationship between leaf nitrogen, protein, carbohydrate and total phenolic
contents. High food intake was evident in the presence of increased nitrogen and
protein, low carbohydrate and phenols, as in the case of mature leaves of T. anguina
followed by L. cylindrica and B. cerifera respectively. In the case of low nitrogen and
low phenols, compensation was achieved by a relatively high food intake as seen in the
senescent leaves of T. anguina.
554 K Raman and R S Annadurai
4. Discussion
Chemoreceptors of insects form a complicated and subtle sensory system that enables
them to differentiate between many natural stimuli of fairly great diversity
(Schoonhoven 1977). In the present study three types of sensory hairs were observed on
the antennae of R.foveicollis viz sensilla trichoidea, sensilla basiconica and sensilla
chaetica. In addition, the sensillum present on the stipes of the maxillae and tip of the
labial palps appears to be gustatory receptors. Schneider (1964) opined that the sensilla
present on the antennae acts as olfactory and mechanoreceptory chemoreceptors. In
acridids, the campaniform and trichoid sensilla located on the maxilla and labium were .
found to act as gustatory chemoreceptors (Chapman and Thomas 1978). Thus the f
differences in sensitivity of chemoreceptors to different host plant odours enable the
insect to orient towards its food plant and the delay in location of host plants and the
failure to initiate feeding by R.foveicollis could be attributed to the inability to perceive
the plant odours as a result of antennal ablation and coating of the sensory areas
located on the mouthparts.
It is well known that both primary and secondary plant chemicals are indispensable
in the regulation of feeding behaviour of phytophagous insects. While primary plant
chemicals serve as essential phagostimulants to elicit initial feeding response and to
maintain continuous feeding on the host plant, many secondary plant chemicals exhibit
repellent, deterrant and toxic effects, and their presence provides the basis of insect
resistance in the majority of plants (Hsiao 1974). The greater preference of R.foveicollis
towards leaves and flowers was attributed to some chemical stimuli present in these
regions (Sinha and Krishna 1971). In the present investigation, the differences in the
relative preference and quantitative food utilization of R.foveicollis among different
cucurbitaceous host plants as well as between different plant parts appear to be due to
variations in their nutritive qualities, M. charantia being an exception. Mature leaves
and flowers showed high nitrogen and proteins, low carbohydrates, moderately high
phenols and narrow C/N ratio compared to other plant parts. The importance of
organic nitrogen (McNeill and Southwood 1978), sugars and proteins (Beck 1956), and
phenols (Hartfield et al 1982), in the host plant selection by phytophagous insects is very
well established. Moreover, a lower C/N ratio is also associated with greater
susceptibility of the host plant to insect attack (Jayaraj 1967). With increasing plant age,
the total soluble nitrogen decreased appreciably thereby interfering with the develop-
ment of the aphid, Myzus persicae Sulz on Brussels sprout plants (van Emden and
Bashford 1971). Marian and Pandian (1980) showed that the consumption, assimi-
lation and net-conversion of leaf proteins by the Danaus chrysippus L larvae fed on
senescent leaf of Calotropis gigantea R. Br. were significantly less than those fed on
normal leaf. However, in the present study it was observed that a definite relationship
existed between the quantity of food ingested and the different biochemical parameters
such as the nitrogen, protein, carbohydrate and phenolic contents of different plant
parts. The presence of increased nitrogen and protein, low carbohydrate and phenol in
the host plant resulted in increased food consumption by R.foveicollis. Similarly, the
food intake was also relatively high in the case of host plants with low nitrogen and low
phenols. This emphasises the fact that R.foveicollis exhibited a compensatory
behavioural and physiological response i.e. altering the food consumption and
utilization in response to variation in the nutritional suitability of their food (Slansky
1982).
Host selection and food utilization in R. foveicollis 555
Chandaravadana and Pal (1983) reported the presence of a triterpenoid feeding
eterrant from M. charantia which could also be the reason for the non-utilization of
bis plant by R. foveicollis. In addition, Sinha and Krishna (1970) showed that for an
ptimum level of the alkaloid, cucurbitacin E (a feeding incitant widely distributed in
ucurbitaceous plants) was essential to stimulate feeding in R. foveicollis. While a lower
oncentration of this compound will not initiate the beetle feeding, higher concentra-
ons can also act as a feeding deterrant. Thus the differential feeding behaviour of
[.foveicollis on different parts of the cucurbitaceous plants could also be due to the
istribution and varied concentrations of such secondary substances. It could therefore
e concluded that the qualitative and quantitative differences in the primary and
jcondary chemicals coupled with the discriminating powers of the various chemosen-
Dry receptors (Vis$er 1983) located in the antennae and mouthparts determine the
election of food plants/plant parts by R. foveicollis.
Lcknowledgement
'he authors are grateful to Prof. T N Ananthakrishnan, for valuable suggestions and
ritical perusal of the manuscript.
References
eck S D 1956 A biomodal response to dietary sugars by an insect; Biol Bull. 10 219-228
ray H C and Thorpe W V 1954 Analysis of phenolic compounds of interest in metabolism; Methods
Biochem. Anal. 1 27-52
handravadana M V and Pal A B 1983 Triterpenoid feeding deterrent of Raphidopalpa foveicollis L from
Momordica charantia; Curr. Sci. 52 87-88
hapman R F and Thomas G 1978 The number and distribution of sensilla of the mouthparts of
Acrididoidea; Extrait. fAcrida 7 115-148
>ubois M, Gilles K A, Hamilton J K, Robers P A and Smith F 1 956 Colorimetric determination of sugars and
related substances; Anal. Chem. 28 351-356
rrewal S S and Sandhu G S 1982 The feeding preference of red pumpkin beetle for different leaves of
cucurbits; Sci. Cul 48 389-390
[artfield L D, Frazier J L and Ferreira J 1982 Gustatory discrimination of sugars, amino acids and selected
allelochemicals by the tarnished plant bug, Lygus lineolaris; Physiol Entomol. 1 15-23
[siao T H 1974 Chemical influence on feeding behaviour of Leptinotarsa beetles; in Experimental analysis of
insect behaviour (ed.) L B Browne (Berlin: Springer- Verlag) pp. 237-248
[umphries E C 1956 Mineral compounds and ash analysis; in Modern methods of plant analysis (eds) K Peach
and M V Tracey (Berlin: Springer- Verlag) Vol. 1 pp. 542
lyaraj S 1967 Effect of leafhopper infestation on the metabolism of carbohydrates and nitrogen in castor
varieties in relation to their resistance to Empoasca flavescens F. (Homoptera: Jassidae); Indian J. Exp.
Biol. 5 156-162
farian M P and Pandian T J 1980 Effects of feeding senescent leaf of Calotropis gigantea on food utilization
in the Monarch butterfly, Danaus chrysippus; Entomon 5 257—264
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relationship; Symp. Phyto-Chem. Soc., Reading 1977
:hneider D 1964 Insect antennae; Annu. Rev. Entomol 9 103-123
choonhoven L M 1977 Insect chemosensory responses to plant and animal hosts; in Chemical control of
insect behaviour- Theory and application (eds) H H Shorey and J R McKelvey Jr. (New York: John Wiley)
pp. 1-5
556 K Raman and R S Annadurai
Sinha A K and Krishna S S 1970 Further studies on the feeding behaviour of Aulacophora foveicollis on
Cucurbitacin; J. Econ. EntomoL 30 333-334
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Chrysomelidae) on Lagenaria vulgaris Ser. (Cucurbitaceae); Bull. EntomoL Soc. Nigeria 3 60-63
Slansky Jr F 1982 Toward a nutritional ecology of insects, in Proc. 5th Int. Symp. Insect-Plant Relationships,
Wageningen, 1982 pp. 253-259
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relation to plant age and leaf amino acids; EntomoL Exp. Appl. 14 349-360
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© Printed in India.
Distribution of earthworms in Madras
SULTAN A ISMAIL and V A MURTHY*
Department of Zoology, The New College, Madras 600014, India
* Department of Zoology, Loyola College, Madras 600034, 'India
Abstract. Distribution of earthworms has been studied in four locations comprising of two
sandy loam and two clay loam soils of Madras, and correlated with the physical and chemical
parameters of the soil. L. mauritii and 0. serrata are the dominant earthworm species in the
sandy loams and clay loams respectively. Each location has limiting factors like temperature,
moisture and/or soil texture which strongly govern the density and diversity of earthworm
populations in that region.
Keywords. L. mauritii; 0. serrata; distribution; density.
1. Introduction
The ecology and the manner of life of the oligochaeta, especially the earthworms has
been extensively investigated since Darwin's contribution to the study of earthworms.
Earthworms prefer well aerated and moist habitats. They are frequently found in
medium textured upland soils where the moisture capacity is high, rather than in
droughty sands or poorly drained lowlands (Brady 1974). Distribution of earthworms
is usually irregular (Guild 1952; Satchell 1955; Svendsen 1957), the numbers varying in
relation to the type of soil (Evans and Guild 1947), with edaphic factors playing a vital
role in their distribution.
An attempt is made in the present study to investigate the distribution and
abundance of earthworms in the soils of Madras correlating their distribution to
physical and chemical parameters of the soils they inhabit. Atmospheric temperature,
soil temperature, moisture, pore space, maximum water holding capacity, organic
matter and pH have been investigated in the four locations studied for earthworm
distribution, including two sandy loams and two clay loams from May 1981 to May
1982.
2. Material and methods
The four locations studied in Madras (13°5'N, 80°18'E), have been categorically
designated as locations A, B, C and D. While locations A and B are located in Ameer
Mahal (a stretch of private land in Madras city), C and D are located in the Deer
Sanctuary which is a protected reserve forest at Guindy, on the outskirts of the city. Soil
is light brown sandy loam at A and B, brown clay loam at C and light brown clay loam
atD.
Location A is situated at the periphery of a heap of dung, which is cleared during
December-January. Field grass (Graminae) are the only floral associates of the
community.
558 Sultan A Ismail and V A Murthy
Location B lies adjacent to an open type domestic sewage about 0-5 km from A. Field
grass along with Coccinia indica W & A, Amaranthus spinosus Linn., and Euphorbia
hirta Linn, are the plant species.
Location C is richly inhabited by plants, the canopy of Cassia sp providing ample
shade and litter to this region.
Location D lies adjacent to the banks of the lake inside the Deer Sanctuary about
4 km from C. Organic matter to the location is contributed by leaf litter of Syzygium
jambolanum DC and droppings left behind by spotted deer and black buck which
frequent this lake.
Monthly samples were collected from three units in each location, each unit covering
a soil volume of 0-1 m3 (60 x 60 x 30 cm) from May 1981 to May 1982. Earthworms
were hand sorted and identified. Population density (no/0- 1 m3) was recorded for each
of the monthly samples analysed.
Atmospheric temperature (45 cm above soil level) and soil temperature (10 cm below
soil level) were recorded. Moisture content (Misra 1968), porosity and maximum water
holding capacity (Keen and Rackzkowski 1921), oxidizable organic matter (Walkey-
Black 1947) and pH (Hanna 1968) of the soil samples were assessed. Total monthly
precipitation was obtained from the Meteorological Department, Madras. Data were
subjected to appropriate statistical analyses for correlation and significance (Rao 1965).
3. Results
Soils of locations A and B are organically rich whereas at C and D only the surface
horizons can be distinguished as organically rich zones. The soil in location D is coarse
and gravel below 16 cm of the surface thus not being able to support earthworm fauna.
Mean annual values for atmospheric temperature, soil temperature, moisture, pore
space, water holding capacity, organic matter and pH are illustrated in figure 1.
Atmospheric temperature is negatively correlated to the distribution of earthworms
in all the four locations especially at A where it is significant (P < 0*05). Soil
temperature is also negatively correlated to earthworm distribution, being significantly
negative (P< 0-05) at the non-shaded locations A and B.
Moisture functions as a limiting factor at A and C, but not at B and D, which are close
to water sources. Correlation to earthworm distribution is positive in all locations,
being significant at A and C. Earthworm distribution in these areas is therefore co-
ordinated with the amount of rainfall. Significant amount of rainfall occurred between
16 July 1981 and 15 December 1981 precipitating 1042-5 mm of rain scattered during
the five months with a maximum of 305-5 mm during October-November 1981.
Pore space and the water holding capacity distinguish the sandy loams at A and B
from the clay loams at C and D, the latter soils having lesser pore space and greater
water holding capacity.
Organic matter of soils is generally positively correlated to earthworm distribution.
A piece of land of about 0-4 ha is reserved and protected for cultivation of fodder
within the Deer Sanctuary about 0-5 km from D. The soil is sandy here when compared
to C and D. This location, for convenience, called E supports earthworm fauna even
during summer due to its moisture content. Species of earthworms here include
Lampito mauritii Kinberg, Octochaetona pattoni (Michaelsen) and Octochaetona
thurstoni (Michaelsen).
Earthworm ecology
559
ATMOSPHERIC
TEMPERATURE
40
SOIL TEMPERATURE
(°C)
pH
*
A
ft
*
ft
7 .
n±\ T _
5 .
rh J-,
5 .
nn
B C D
ORGANIC MATTER (•/.)
/i a c D
Fl 1*1 ft
HMTT/? HOLDING
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i!
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P/4
C/]
T
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A . a p A
A B C D
MOISTURE (%)
ft
ft
A B C D
SOIL POROSITY (%)
50
Figure 1 . Annual mean with so for atmospheric temperature (°C), soil temperature (CC), pH,
organic matter ( %), moisture ( %), water holding capacity ( %) and soil porosity ( %) for
locations A, B, C and D (May 1981 to May 1982).
Lampito mauritii Kinberg is the dominant species at A and B distributed in
association with Drawida modesta Rao and Ramiella pachpaharensis Stephenson.
Octochaetona pattoni (Michaelsen) and Octochaetona thurstoni (Michaelsen)> have also
been observed in these locations, but not frequently.
Octochaetona serrata (Gates) occurs as the dominant and the only species at C while
L. mauritii and 0. thurstoni occasionally trespass the domain of 0. serrata at D,
probably from E.
Population density of earthworms at A, B, C and D are compared to the respective
mean monthly values for atmospheric temperature, soil temperature, pH, organic
matter and moisture in three dimensional models, the parameters being marked with
respect to the peak distribution of earthworms (figures 2 a-d).
560
Sultan A Ismail and V A Murthy
Earthworm ecology
561
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Sultan A Ismail and V A Murthy
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564 Sultan A Ismail and V A Murthy
4. Discussion
"Earthworms, though in appearance a small and despicable link in the chain of nature,
yet, if lost, would make a lamentable chasm . . . worms seem to be the great promoter
of vegetation which would proceed but lamely without them, ..." (Gilbert White of
Selbourne, 1 770; cf Satchell in Edwards and Lofty 1972). Much attention has been paid
in recent years to the importance of the earthworms in increasing farm yields and as a
source of protein for other organisms especially for fish, poultry, piggery, cattle and
man.
A number of ecological parameters play a vital role in the distribution and
abundance of earthworms, which may be due to the adaptability of that particular
species to the soil sample studied. While L. mauritii inhabits the sandy loams of Ameer
Mahal and neighbouring areas, 0. serrata occurs in the clay loams of Guindy. That L.
mauritii prefers sandy loams is evident by its presence at E, along with populations of 0.
serrata and 0. thurstoni. 0. thurstoni prefers the sandy loams of Ameer Mahal and
Guindy.
Close tree canopy at C and D prevents heating of the soil surface. Soil temperature on
an annual average is 3 1 -04°C at A, 3004°C at B and about 29°C at C and D. Location A
being exposed to sun absorbs heat significantly faster, while at B vegetational ground
cover as well as moisture from sewage check the effect of solar radiation. Soils at C and
D are not only protected by the close tree canopy but the layer of litter also insulates
the soil beneath it from getting heated up.
Temperature largely effects earthworm activity. Compared to temperate con-
ditions tropical species can withstand higher temperatures. L. mauritii is available at B
throughout the year where the annual temperature is 30 ± 2°C. At C population of 0.
serrata were available between 27 and 28°C. Occurrence of the tropical species of
earthworms at higher temperatures has been reported for Hyperiodrilus africanus
(Madge 1969).
Moisture is another limiting factor for earthworm distribution as water constitutes a
major portion of the body weight of an earthworm (Grant 1955). Soil moisture vs
population estimates are positively correlated at A, J3, C and D being significant
(P < 0-05) at A and C. Locations B and D being closer to water bodies (B near sewage
and D near lake) provide habitat to earthworms where water is not a limiting factor. A
and C depend on precipitation for their content of moisture which makes the soils of
these locations significantly correlated to moisture, for moisture controls earthworm
activity (Barley 1959; Dash and Senapati 1980). Earthworms avoid much adverse
conditions by either moving away to moist soils or by aestivating. Locations A and C
are exposed to such droughty conditions annually, when their population of
earthworms react suitably. L. mauritii and 0. serrata are both surface dwellers. L.
mauritii migrates either horizontally to moist soils or vertically to greater depths, while
0. serrata undergoes diapause by encircling itself. L. mauritii during dry seasons were
traced at A from depths of 80 to 105 cm, when their population within the quadrat was
zero. The most interesting recording is of O. serrata which had zero population
within the quadrat during July showed itself during August (ca. 77/0-1 m3) in a
quiescent state. September showed an activity of these worms which resulted in
their dispersal. However, the population of O. serrata at C decreased by January in
the field.
Earthworm ecology 565
Occurrence of active 0. serrata clitellate and non-clitellate at E during
January-February, when the worms at C get prepared for the summer diapause,
explains diapause to be facultative.
During August 1982 a large number of O. serrata in a quiescent stage were again
observed. Each cell or "house" that harboured quiescent earthworms contained only
one clitellate form, or two clitellates, or one clitellate and two non-clitellates, or just two
or more non-clitellates. This stage which is the commencing point for dispersal can be
termed as the quiescent stage and the stage during January-February when dry
conditions commence as the diapause. Diapause in 0. serrata commences when the
temperature is about 28°C and soil moisture is 12-14%, these values being almost
similar to those reported by Dash and Senapati (1980) for Octochaetona surensis.
Though the earthworm has no significant correlation with soil porosity and water
holding capacity of soils, they to a large extent alter these factors by their tunnelling
activity (Guild 1955). This is reflected at C where soil porosity is high compared to D,
where earthworm activity is negligible.
Organic matter in soils greatly influences the distribution of earthworms; soils with
low organic matter do not support earthworm populations." Organic matter is
comparatively high at A followed by B and C, and D having the least content of organic
matter (1-79 %). Location A gets its high percentage of organic matter from the closely
deposited dung heaps, while B gets it from the sewage. Location C draws organic matter
through the litter of Cassia sp. while D through the litter ofS.jambolanum. Location D
shows a mat of undisturbed organic matter on the soil surface which is also an
indication of a poor population of earthworms in this location. Such mats of
undisturbed organic matter occur in both woodlands (Richardson 1938) and grassland
(Raw 1962) where earthworms are scanty.
Location A which is enriched with organic manure, at an optimal condition of
temperature and moisture supports a high density and diversity of earthworm
populations (120/0-1 cum). Species recorded here include L. mauritii, D. modesta,
R. pachpaharensis, 0. pattoni and 0. thurstoni. Plots with organic manure are known to
support higher populations of earthworms (Satchell 1955) as food supply appears to be
the major factor for earthworm distribution (Abbott and Parker 1980).
Hydrogen ion concentration of soils is neutral at A and B and medium acidic at C and
D. L. mauritii occurs in large numbers at A and B, where the soil is a neutral sandy
loam. 0. serrata dominates the medium acidic clay loams. Of C and D, the pH at D tends
to be more towards the acidic range, which also probably limits the distribution of
earthworms in this location. Though species of earthworms may be acid tolerant (Guild
1951; Satchell 1955; Leger and Millette 1979) earthworms normally prefer a neutral pH
(Arrhenius 1921; Salisbury 1925; Bodenheimer 1935; Petrov 1946; Magdoom 1984).
Peak of earthworm distribution is during November at A (120/0-1 in3; L. mauritii in
November: 40/0-1 m3), April at B (221/0-1 m3; L. mauritii in February: 156/0-1 m3),
November at C (0. serrata — 86/0-1 m3), December at D (30/0-1 m3, 0. serrata in
November: 12/0-1 m3) (figure 2). Availability of earthworms at A, C and D coincides
with the peak rainfall of 305-5 mm during October-November 1981.
A review of the four locations A, B, C and D investigated show variations with
reference to physical and chemical parameters, each location having limiting factors
which strongly govern the density and diversity of earthworm populations in that
region; they being temperature at A and B, moisture at A and C, and soil texture at D.
566 Sultan A Ismail and V A Murthy
Acknowledgements
The authors thank the Prince of Arcot and the Wild Life Warden, Madras for
permission to conduct ecological studies at Ameer Mahal and the Deer Sanctuary
respectively. Thanks are due to Mr E G Easton, British Museum Natural History,
London, for identifying/confirming earthworm species.
References
Abbott I and Parker C A 1980 The occurrence of earthworms in the wheat belt of Western Australia in
relation to land use and rainfall; Aust. J. Soil Res. 18 343-352
Arrhenius O 1921 Influence of soil reaction on earthworms; Ecology 2 255-257
Barley K P 1959 The influence of earthworms on soil fertility. II. Consumption of soil and organic matter by
the earthworm Allolobophora caliginosa; Aust. J. Agric. Res. 10 149-158
Bodenheimer F S 1935 Soil conditions which limit earthworm distribution; Zoogeographica 2 572-578
Brady N C 1974 The nature and properties of soils (New York: Macmillan)
Dash M C and Senapati B K 1980 Cocoon morphology, hatching and emergence pattern in tropical
earthworms; Pedobiologia 20 316-324
Edwards C A and Lofty J R 1972 Biology of earthworms (London: Chapman and Hall)
Evans A C and Guild W J Me L 1947 Studies on the relationships between earthworms and soil fertility. I.
Biological studies in the field; Ann. Appl. Bioi 34 307-330
Grant W C 1955 Studies on moisture relationships in earthworms Ecology 36 400-407
Guild W J Me L 1951 The distribution and population density of earthworms (Lumbricidae) in Scottish
pasture fields; J. Anim. Ecol. 20 88-97
Guild W J Me L 1952 Variation in earthworm numbers within field populations; J. Anim. Ecol. 21 169-181
Guild W J Me L 1955 Earthworms and soil structure, in Soil zoology (ed.) D K Me E Kevan (London:
Butterworths)
Hanna W J 1968 Methods for chemical analysis of soils, in Chemistry of the soil (ed.) F E Bear (Calcutta,
Bombay, New Delhi: Oxford and IBM Publ. Co.).
Keen B A and Raczkowski H J 1921 Relationships between clay content and certain physical properties of
soils; J. Agric. SCL II 441-449
Leger R G and Millette C J F 1979 Distribution ecologique des vers de terre (Oligochaeta: Lumbricidae) dans
le foret du Morgan Arboretum; Nat. Can. (Que.) 106 369-376
Madge D S 1969 Field and laboratory studies on the activities of two species of tropical earthworms;
Pedobiologia 9 188-214
Magdoom K M M 1984 Studies on chemosensitivity of the megascolecid earthworm, Lampito mauritii. M.Phil.,
Dissertation, Madras University, India
Misra R 1968 Ecology workbook (New Delhi, Bombay, Calcutta: Oxford and IBH Publ. Co.)
Petrov B C f946 The active reaction of soil (pH) as a factor in the distribution of earthworms; Zool. J. 25
107-110
Rao C R 1965 Linear statistical theory and its applications (New Delhi: Wiley Eastern Private Limited)
Raw F 1962 Studies on earthworm populations in orchards. 1. Leaf burial in apple orchards; Ann. Appl. BioL
50 389-404
Richardson H C 1938 The nitrogen cycle in grassland soils with special reference to Rothamsted Park Grass
Experiments; J. Agric. Sci. Camb. 28 73-121
Salisbury E J 1925 The influence of earthworms on soil reaction and stratification of undisturbed soils; J.
Linn. Soc. (Rot). 46 416-425
Satchell J E 1955 Some aspects of earthworm ecology, in Soil zoology (ed.) D K Me E Kevan (London:
Butterworths)
Svendsen J A 1957 The distribution of Lumbricidae in an area of Pennine Moorland (Moor House, Nature
Reserve); J. Anim. Ecol. 26 411-421
Walkey-Black 1947 Oxidizable matter by chromic acid with sulphuric acid heat of dilution; Soil Sci. 63 251
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 5, October 1985, pp. 567-572.
© Printed in India.
Alkaline protease in the midgut of the silkworm Bombyx mori L:
changes during metamorphosis
S K SARANGI
R & D Programme in Sericulture, Bangalore University, Bangalore 560009, India
MS received 13 February 1985; revised 22 April 1985
Abstract. The protease activity in the midgut of bivoltine and multivoltine races of the
silkworm, Bombyx mori, was studied. The enzyme activity increased during fifth instar, reached
a peak, and decreased significantly through pre-pupal stage to the lowest level at the pupal
stage. During pharate adult period, the protease activity increased to reach another peak just
before emergence of the moth, and decreased thereafter. The enzyme activity in bivoltines was
about 2-3 times higher than multivoltines at the peak levels. Bivoltines showed a sex difference
in midgut protease activity while no significant difference was observed in multivoltines.
Larvae and pharate adults showed a difference in the pH optima for the enzyme activity. From
the results the possible role of midgut protease during the process of metamorphosis is
discussed.
Keywords. Bombyx mori; bivoltine; multivoltine; midgut protease; metamorphosis.
1. Introduction
The most common proteolytic enzymes in the alimentary canal of insects have long
been recognised as trypsin-like enzymes (Day and Waterhouse 1953; House 1974; Law
et al 1977; Jany et al 1978). Eguchi and Iwamoto (1982) have also established that
midgut protease, the activity of which has been earlier demonstrated in the alimentary
canal of the larva of the silkworm, Bombyx mori (Horie et al 1963; Eguchi and
Yoshitake 1967; Hamano and Mukaiyama 1970; Eguchi and Iwamoto 1976) is a
trypsin-like enzyme possessing certain properties (Eguchi and Arai 1983). However,
most of the studies on the midgut protease of silkworms are restricted to the larval stage
mainly because of its important role in protein digestion. Eventhough Eguchi et al
(1972) have studied the proteolytic enzymes in pharate adults, very little information is
available on the pattern of change in total protease activity during larval-pupal-adult
transformations in silkworms. It has already been reported that the trypsin-like enzyme
in mosquitoes changes significantly during metamorphosis (Chen 1978). The present
communication deals with the pattern of change in total protease activity of the midgut,
a comparison of the enzyme activity in different races and sexes and the pH optima for
midgut protease at different stages of metamorphosis.
2. Materials and methods
2.1 Animals
Bivoltine (NB18 and NB4D2) and multivoltine (Pure Mysore) silkworm races were
maintained under standard laboratory conditions at 25-28°C and a relative humidity
$67
570
S K Sarangi
40 -
'2 20
I
o>
BIVOLTINE
PURE MYSORE
o
a
0-4
0-2
PHARATE ADULT
• BiVOLTINE
-0 PURE MYSORE
10
PH
12
Figure 3. Effect of pH on the alkaline protease of the midgut.
bivoltines was about 2-5 times higher than that of multivoltine. During pharate adult
stage, the bivoltines interestingly showed two peaks of enzyme activity at pH 9 and pH
11. But in case of pure Mysore only one peak was observed at pH 9. The enzyme activity
in bivoltines was about 2*5 times higher than multivoltines during pharate adult stage.
4. Discussion
Midgut enzymes in insects are synthesized in the midgut epithelium and secreted into
the gut lumen (Engelmann 1969; Engelmann and Geraerts 1980; Eguchi and Arai 1983).
Thus changes in total protease activity of the midgut reflect changes in the overall
production of the enzyme during metamorphosis. A significantly high activity of
midgut protease during fifth instar larval development of silkworms shows a higher
rate of enzyme synthesis corresponding to enhanced food intake (Waldbauer 1968).
This might facilitate a greater utilization of proteins for larval growth and silk
Midgut protease in silkworms 571
production as well. Higher protease activity in bivoltines might result in better
conversion of exogenous proteins which ultimately lead to production of more silk
compared to multivoltines (Tanaka 1964). The sharp decrease in midgut protease
activity from larval to pupal stage shows the difference between active feeding and non-
feeding stages during metamorphosis. Thus it can be presumed that the rate of synthesis
of midgut protease is dependent upon the rate of food intake. Furthermore, traces of
protease activity during pupal stage might be the residual larval enzymes. In pharate
adults, protease activity increases and reaches another peak just before emergence. This
is in accordance with earlier reports (Eguchi et al 1972) and supports their view that
midgut protease in pharate adult might contribute either in part or full towards the
formation of cocoonase.
The present study shows that the optimum pH for total protease activity of the
midgut is around pH 1 1-0 in the larval stage. Eguchi and Iwamoto (1976) have shown
that the pH optima for protease from midgut tissue and digestive fluid are the same.
Interestingly a shift in pH optima for protease activity is observed during meta-
morphosis from pH 11-0 at larval stage to pH 9-0 at pharate adult stage. It cannot be
confirmed at present whether the larval protease is different from adult protease or
whether they are two isoenzymes of protease active at different stages of metamor-
phosis. The presence of two peaks, each at pH 9*0 and pH 11-0, in midgut protease
activity of bivoltine pharate adult, likewise, again provides a basis to speculate the
existence of two isoenzymes of protease having different pH optima characteristics.
However, the single peak at pH 9-0, in the enzyme activity of multivoltine pharate adult
requires further studies to draw any conclusion.
Acknowledgement
Grateful acknowledgements are due to the World Bank for financial assistance and to
the co-ordinator, R & D Programme in Sericulture, Bangalore University for facilities.
References
Chen P S 1978 Regulation of proteolytic enzymes In: Biochemistry of insects (ed,) M Rockstein (New York:
Academic Press) pp. 162-168
Day M F and Waterhouse D F 1953 The mechanism of digestion. In: Insect physiology (ed.) K D Roeder
(London: Chapmann & Hall) pp. 311-330
Eguchi M and Arai M 1983 Relationship between alkaline proteases from the midgut lumen and epithelia of
the silkworm; solubilization and activation of epithelial protease (6B3); Comp. Biochem. Physioi, B75
589-593
Eguchi M and Iwamoto A 1976 Alkaline proteases in the midgut tissue and digestive fluid of the silkworm,
Bombyx mori\ Insect Biochem, 6 491-496
Eguchi M and Iwamoto A 1982 Comparison of three alkaline proteases from digestive fluid of the silkworm,
Bombyx mori, L.; Comp. Biochem. Physioi, B71 663-668
Eguchi M and Yoshitake N 1967 Electrophoretic variation of Proteinase in the digestive juice of the
silkworm, Bombyx mori, L.; Nature (London) 214 843-844.
Eguchi M, Furukawa S and Iwamoto A 1972 Proteolytic enzyme in the midgut of the pharate adult of the
silkworm Bombyx mori', J. Insect. PhysioL, 18 2457-2467
Eguchi M, Iwamoto A and Yamauchi K 1982 Interrelation of proteases from the midgut lumen, epithelia and
peritrophic membrane of the silkworm, Bombyx rnori^ L.; Comp. Biochem. Physioi A72 359-363
572 S K Sarangi
Engelmann F 1969 Food-stimulated synthesis of intestinal proteolytic enzymes in the cockroach Leucophaea
maderae; J. Insect Physioi 15 217-235
Engelmann F and Geraerts W P M 1980 The proteases and the protease inhibitor in the midgut of
Leucophaea maderae; J. Insect Physioi 26 703-710
Hamano K and Mukaiyama F 1970 Some properties of digestive fluid proteinase in the silkworm Bombyx
mori with references to the relation between dissociation degree and nutritive value of some proteins;
J. Sericult. Sci. Jpn 39 371-376
Horie Y, Tenaka M and Ito T 1963 Proteolytic enzymes of digestive juice and midgut of the silkworm,
Bombyx mori; J. Sericult. Sci. Jpn 32 8-15
House H L 1974 Digestion In:7te physiology ofinsecta (ed.) M Rockstein (New York: Academic Press) Vol. 5,
pp. 63-117
Jany K D, Haug H and Ishay J 1978 Trypsin-like endopeptidases from the midguts of the larvae from the
hornets of Vespa orientalis and Vespa crabro; Insect Biochem. 8 221-230
Law J H, Dunn P F and Kramer K J 1977 Insect proteases and peptidases; Adv. EnzymoL 45 389-425
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with the Folin phenol
reagent; J. Biol. Chem. 193 265-275
Tanaka Y 1964 Silkworm Races. In Sericology (Bombay: Central Silk Board) pp. 99-104
Waldbauer G P 1968 The consumption and utilization of food by insects In: Advances in insect physiology
(eds) J W L Beament, J E Treherme and V B Wigglesworth (London: Academic Press) Vol. 5, pp. 229-288
Foreword
This issue brings together contributions of five speakers who made presentations at a
special symposium on "Animal communication" held on the occasion of the Golden
Jubilee celebrations of the Indian Academy of Sciences on 6-8 February 1985. We are
grateful to the speakers who readily agreed to have the papers based on their
presentations published together in a special issue of the Proceedings of the Indian
Academy of Sciences (Animal Sciences) and to the many organizations which so
generously supported this meeting.
Madhav Gadgil
Convener, Symposium on
Animal Communication
573
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 6, December 1985, pp. 575-586.
© Printed in India.
On the communication of well-being
MADHAV GADGIL, MALATI HEGDE, N V JOSHI and
SULOCHANA GADGIL
Centre for Theoretical Studies and Centre for Ecological Sciences, Indian Institute of Science,
Bangalore 560012, India
MS received 27 May 1985; revised 28 October 1985
Abstract. The form that any communicatory exchange takes would depend on the extent to
which the interests of the signaller and the recipient are at variance. Where such interests
coincide, i.e. in cases of mutualism, the signals may be conspicuous when an immediate
response is favoured, but rather subtle and variable otherwise. Over 80 % of the events of tactile
communication that we have noted in our study of the social behaviour of free ranging groups
of tame elephants appear to belong to this latter category. On Smith's standard classification,
they can only be classified as 'associative', related to remaining in the company of another
individual. However, such signals are commoner by a factor of 20-1 00 amongst elephant calves
and their mothers and allomothers when compared to exchanges between adult cows. We
suggest that the function of these signals is mutual monitoring of the state of well being
amongst related individuals. The considerable degree of altruistic behaviour displayed in social
groups, such as those of elephants is now believed to subserve the function of enhancing the
inclusive fitness of the individuals concerned. We explore a mathematical model of exchange of
social aid which suggests that animals in social groups may enhance their inclusive fitness
further by adjusting the amount of social aid exchanged in relation to the state of well being of
the donor as well as the recipient. Our model further suggests that optimal social aid depends
on the state of well being in a complex fashion making it difficult for the recipient to deceive the
donor so as to extract more aid. We therefore expect that by and large honest communication
of the state of well being would be characteristic of the higher social animals. Such
communication would be based on normal physiological changes consequent on a change in
well being. Thus animals with a superior degree of well being would take postures conducive to
greater activity, would be more receptive to sensory inputs and may also shift the balance of
production of various metabolites. This monitoring of the well being has greatly advanced in
the human species and may be at the base of the elaborate health care amongst human societies.
Keywords. Communication; well-being; kin selection; elephants.
1. Introduction
Animals, whether they be lowly soil amoebae or highly evolved elephants, are creatures
on the move. For them success depends on being alert to what is happening around
them. They have therefore evolved a variety of sense organs to receive signals of
relevance to their own welfare from the environment. Such signals may be picked up
opportunistically, regardless of the interests of the signaller. Thus many species of soil
amoebae possess on their body surface receptors for derivatives of folic acid. These
chemical compounds are involved in the biosynthesis of purines and pyrimidines, the
basic building blocks of nucleic acids that are essential for every living organism. It
turns out that they leak out of the bodies of bacteria grazed on by soil amoebae. Soil
amoebae then employ the folic acid derivatives as signals to locate their prey, moving up
the gradient of concentration. This is thus an example of interception of a signal against
575
576 Madhav Gadgil et al
the interest of the signaller. In another paper in this issue Stanley Rand will add
another, that of predatory bats homing on to male frogs calling to keep out the other
males and to attract the females.
2. Eavesdropping
In general, we can classify signals into 4 categories depending on whether the signaller
benefits or suffers from the recipient receiving it, and on whether the recipient benefits
or suffers from acting on it (Wiley 1983).
Signaller
R
Benefits
Suffers
e
c
Benefits
Mutualism
Eavesdropping
i
P
Suffers
Deceit
Spite
The genetic interests of the predatory soil amoebae and their bacterial prey are
obviously divergent, making the use of folk acid derivatives to locate them an example
of eavesdropping. But two of the species of soil amoebae Dictyolstelium lacteum and D.
minutum use folic acid derivatives as communicatory signals in quite another context —
to attract each other. These soil amoebae multiply by binary fission, feeding on bacteria
in their vegetative phase. When the food supply runs out, members of a clone come
together to form a many celled creature called the slug. Some cells of this slug forego
reproduction to form a stalk, while other cells turn into spores which disperse and may
enter again into the vegetative phase if they encounter a favourable environment.
In terms of our classification above, some of the soil amoebae that are thus attracted
by the signal suffer, since they forego their chances of reproduction to form the stalk,
while others benefit, since they form spores which retain the possibility of future
reproduction. In the process of aggregation of soil amoebae, all cells produce the signal
and all cells receive it. Is this communicatory episode then an example of deceit by
future spore cells signalling the future stalk cells, or eavesdropping on the signal of
future stalk cells by the future spore cells?
3. Altruism
A closer look suggests that in fact it is neither. The soil amoebae attracting each other
are all members of a clone, and therefore genetically identical. The assumption of the
role of stalk cells by some of the amoebae is an example of altruistic behaviour through
kin selection. As Hamilton (1964) has shown, natural selection will favour such
behaviour so long as:
Cost to the altruist < Benefit to the recipient x kinship coefficient
On the communication of well-being 577
where the kinship coefficient is the proportion of genes shared by virtue of common
ancestry. Members of a soil amoebae clone will have a kinship coefficient of 1. Suppose
now that a proportion x of them sacrifices a chance to reproduce by forming a stalk;
leaving (1 — jc) to reproduce. Then this behaviour would be favoured if raising on a stalk
improves the chance of survival of a spore by a factor greater than .
JL "~~" X
There are good reasons to believe that this is in fact so. In that case the genetic
interests of all the soil amoebae that are attracted to each other by signals employing
folic acid derivatives are served by producing and responding to the signal. This is then
an example of mutualism in our classification of communicatory systems. Vidyanand
Nanjundiah discusses these fascinating organisms in greater detail in another paper in
this issue.
4. Information and manipulation
We have thus far looked at two extremes — total divergence of genetic interests as with
bacteria and their predators, and total congruence of genetic interests as with members
of a clone of soil amoebae. There would however be many examples of only a partial
congruence of genetic interests, resulting in a rich structure of animal communication
(Dawkins and Krebs 1978; Krebs and Dawkins 1984).
Consider for a moment the call of male frogs. It is in the interest of female frogs to
locate a mate, preferably a mate that will contribute qualities that will help the off-
spring succeed in life. It is likely that the biggest available male of their own species will
be their best choice. It will therefore be in the interest of the females to be able to derive
information on the size of the calling male from its call. On the contrary, it will be in the
genetic interest of every male to misinform the females, conveying that the signaller is
much bigger than in fact it is. We could then consider the information being transmitted
during any communicatory exchange under 3 components:
Signaller
True information sought to True information sought to False information sought
be transmitted in mutual be suppressed to be transmitted
interest
True information sought to True information sought to False information sought
be received in mutual be received against the to be discounted in
interest interest of signaller interest of the recipient
Recipient
Any communicatory exchange could be characterized usefully by the relative values
of a, j? and y. In the case of bacteria and amoebae /? dominates, the bacteria would rather
produce no signal if it were metabolically easily possible; while a dominates in case of
members of a clone of soil amoebae attracting each other. With male frogs calling to
attract females, y must assume significance with males evolutionarily favoured to
convey an exaggerated impression of their size and females favoured to discount any
578 Madhav Gadgil et al
such bluff. As Wiley (1983) and Krebs and Dawkins (1984) have argued this should lead
to the evolution of rather stereotyped, repetitive and unbluffable signals. These are the
conspicuous displays that have so attracted students of animal behaviour.
5. Kinship discrimination
It appears reasonable to conclude that signals would tend to be conspicuous because of
their stereotype and repetition when y is significant, and rather inconspicuous when /?
dominates. In the context of mutualism, when a dominates, they may be conspicuous
when immediate response is favoured, as with soil amoebae attracting each other. In
other mutualistic contexts, however, the signals are expected to be rather subtle and
variable and therefore escape the attention of the ethologist (Krebs and Dawkins 1984).
One such context is that of discrimination of kinship level. This could be important in
social animals, for as J B S Haldane is supposed to have said — he would be
evolutionarily favoured to lay down his life to save two full brothers from drowning;
but if it were only first cousins he must save at least 8 of them for this to make genetic
sense. This is because while full brothers have a kinship coefficient of 1/2, first cousins
share only 1/8 of their genes by virtue of common ancestry.
Discrimination of levels of kinship should therefore be important in social animals.
But it may be based on very subtle and variable cues and it is only now that we are
finding out that in some social insects individuals can discriminate kinship levels based
on chemical signals, even in the absence of individual recognition. This is a topic that
will be reviewed in detail in another paper in this issue by Raghavendra Gadagkar.
Amongst higher animals such as mammals on the other hand, sociality is based on
individual recognition. Many mammals use chemical signals in the form of ratios of
several compounds for this purpose. This individual recognition coupled with ties
established from birth onwards between the mother and the young must permit a fine
tuning of social behaviour in higher mammals such as elephants.
6. Elephant societies
Along with Dr P Vijayakumaran Nair of Kerala Forest Research Institute, we have
investigated the structure of social behaviour in the Asiatic Elephant (Elephas
maximus). For this purpose we used tame elephants maintained in elephant camps in
South Indian forests. All the adults were caught from the wild in the same or nearby
forests and the tame elephants were left for grazing in their natural habitat. These tame
elephants often mingled with wild herds when thus left free. In fact all the calves bora in
captivity were sired by wild tuskers. We observed these elephants over a period of 23
months between February 1975 to March 1976 and January 1978 to August 1979. The
total amount of time spent in the field recording behavioural details spanned 645 hr.
Our parallel, but less detailed observations on the wild elephants confirmed that the
behaviour patterns within the artificially constituted social groups of tame elephants
closely resembled those of wild elephants. In particular, adult females reacted to calves
of other females as did wild females to calves of other females in their herd. Under
natural conditions, of course, the several adult females in a single herd are related to
each other as mother-daughter-half-sister-aunts-cousins (Douglas Hamilton 1972).
On the communication of well-being 579
The glue that holds the elephant society together is evidently the protection and
nurture of calves. These herbivores would only reduce their feeding efficiency by being
together; and the adults can resist any predation by themselves. The calves, however, are
still susceptible to predation, and in fact an elephant calf fell prey to a tiger in Bandipur
during our study period. Adult female elephants therefore stay together to help protect
each other's calves, which under natural conditions have kinship coefficients of
1/2-1/16 with the adult females. The adult males wander from herd to herd and can
have little clue as to which calves they have sired. As expected, they play no role in
helping the calves.
The social group of elephants therefore comprises related adult females with their
young sons, daughters, cousins, nephews and nieces. The calves station themselves in
between adult females who run to them on slightest alarm. The adult females stand
guard over the calves when the latter sleep, and also suckle them if they have no suckling
calves of their own. Elephants are thus model aunts or allomothers. The role of calves in
holding these females together was strikingly brought out when the only calf with 3
adult females of Bandipur Tiger Reserve was removed for weaning. As soon as the calf
was gone, the cows started grazing separately by themselves (Gadgil and Nair 1984).
7. Communication amongst elephants
Elephants have poor vision and do not seem to base much of their- social communi-
cation on visual signals. We have little information on the extent to which they depend
on chemical signals except that they seem to base individual recognition on it. They do
use vocal signals, especially to communicate alarm and aggression. Tactile communi-
cation however seems to be the dominant mode of communication, especially where the
calves are involved. These tactile contacts are largely initiated by the calf towards
mother or allomother . Thus in a group of 3 adult females and a calf of less than 6 months
of age in Bandipur Tiger Reserve the calf touched the mother and one of the
allomothers at a rate of about 4 times in 10 min while it touched a second allomother at
a lower rate of once in 10 min. The mother touched the calf at the rate of once in 7 min,
the first allomother did so at the rate of once in 20 min, while the second allomother
touched the calf at the rate of once in 50 min. The adults touched each other at low rates
ranging from once in 50 to once in 300 min.
A question of considerable significance is the function of the different acts of
communication. In some cases the function is evident as when a calf follows a contact
by suckling or rushes to the mother when the latter sounds an alarm call. A remarkable
result however is that in the vast majority of cases, of the order of 80 % , no such clear cut
immediate function can be attached to a communicatory exchange. The intriguing
question therefore is this: what are the adult elephant, cows and calves conveying to
each other most of the time?
I believe that our failure to assign any obvious function to the bulk of these
communicatory exchanges is due to the fact that it does not subserve any immediate
need. Consequently, in this mutualistic system the signals, as expected, are inconspicu-
ous and variable. On Smith's (1977) standard classification they can be only classified as
Associative', related to remaining in the company of another individual. But then
elephant females remain in company with each other often for their whole lives.
580 Madhav Gadgil et al
Nevertheless the adult females have a far lower frequency of tactile communication
amongst themselves -by a factor of 20-100 than females and calves.
8. Monitoring well-being
We therefore suggest that there is an additional function of communication in case of
higher social animals like the elephants that has not so far been clearly identified; this is
the monitoring of the state of well-being of the young by related adults.
To understand why such communication of well-being could be favoured during the
course of evolution, we have to go back to Hamilton's inequality:
An altruistic act will be favoured if:
Cost to altruistic donor <
Benefit to recipient x kinship coefficient
between donor and
recipient.
A superficial examination of this statement suggests the following paradox: the kinship
coefficient between a mother and her off-spring and between two full sisters is the same,
namely 1/2. Nevertheless, a female mammal displays much greater level of altruistic
behaviour towards her offsprings than towards her sisters, or towards her mother. The
resolution of this paradox lies in recognising that the costs and benefits of a given
altruistic act will differ substantially depending on the identity of the actors involved.
These benefits and costs must be measured as marginal changes in the reproductive
value of an individual as a result of a given social act. Since social aid will often make
greater difference to the chances of survival and future reproduction of a young
offspring than that of a grown sister or an old mother, a female mammal will be
generally favoured by natural selection to behave far more altruistically towards her
offspring rather than a sister or a mother. This need not always hold, of course, and one
needs a better defined model to explore this proposition further. Such a model cannot
of course reflect all the complexities of the real world; that would be too cumbersome to
handle. Rather, the model we develop should be rich enough to reflect the essentials but
simple enough to handle and be interpretable.
9. Modelling social interactions
In such a model the physiological status of the interacting individuals could be specified
by 3 parameters: the ability to convert resources into somatic or reproductive growth, a,
the cost of maintenance, f) and the extent to which physical growth has been completed
and independent abilities to gather resources achieved S. The extent of favourability of
environment is reflected in a parameter F. Let us assume that each individual i has at its
disposal some resources taken to be proportional to 5£F for maintenance and growth.
Of these it retains a fraction <^>£i for its own use and donates a fraction <^ to the ;th
individual. Then the total amount of resources available to it, 6h is given by
The marginal change in fitness or the reproductive value of the ith individual,
On the communication of well-being 581
depends on the change in Sf, i.e. ASf. We take:
AS,- = StMl-SJtt-
and Wi is given by:
Hamilton's (1964) extension of the genetical theory of natural selection tells us that
each individual will be so programmed as to maximise its inclusive fitness
where ytj is the kinship coefficient between i and 7.
We can therefore determine for any given af, ft, Si9 F values the 00- that will tend to
maximize the inclusive fitness of each individual involved. The problem is made
complex by the fact that the optimal allocation of resources for an individual i depends
on the allocation strategies adopted by other individuals. In such an interactive
population, evidently the allocation strategy </>{J of the ith individual will be such that
the inclusive fitness of this individual is maximum for the existing strategies <j>kj(k ^ i)
of other individuals. We know that such a point exists, and have a working algorithm
for determining this matrix for the case of two interacting individuals, say the mother(l)
and the offspring(2).
In this simple case of two individuals, it can be shown that 0H is always 1 for atleast
one of the two individuals; i.e., one of the individuals may be a donor and the other the
recipient keeping all of its own resources to itself, or both may keep all their resources to
themselves. We naturally identify the donor in our model with the mother(l), and the
recipient with the offspring(2). Our problem then is to determine the optimal level of
maternal investment, i.e., social aid <j>l2 from the mother to the offspring.
10. Optimal maternal investment
There are two immediately interesting results of our model. The first is that optimal
maternal investment is much more sensitive in variation to the offspring's cost of
maintenance /?2, extent of development completed S2, and the extent of favourability of
the environment F, than it is to efficiency of growth a2 (figure 1). This result is probably
related to the fact that the first 3 parameters appear in the exponential term of our
specific model. Secondly, we find that the optimal level of maternal investment need not
vary monotonically with the value of variables specifying offspring's state. That is to say
the mother will not necessarily provide more and more maternal care as offspring's
apparent needs increase. This is because the optimal level of maternal investment
depends on the extent to which an offspring can enhance its reproductive value by
receiving such help. An offspring not in much need can make little use of aid; at the same
time, an offspring too much in need may also be a bad investment. We are reminded of
Kafka's story 'Metamorphosis' in which the son of the family is turned into a
cockroach. Initially, while there is hope that he will turn back into man much help is
lavished on him. But as time goes on and he remains an insect, he is neglected and
ultimately allowed to die. There will thus be an intermediate level of need by offspring
582 Madhav Gadgil et al
1.3
°-2 0-4 0.6 — 08"
Variable parameter
1.0
" also
investment at the
offspring's state is availabl To te
make too much or too li
inclusive fitness. <^
offspring's well-being can i
on mother's
-lue of
' if inf°rmation on the
information, she will either
a decline in her
lon relating to the
,00,
1 1 • Mother-infant conflict
favoured
On the communication of well-being
1.0
0.8
0.6
"* 0.4
0.2
583
0 0.2 0.4 0.6 0.8 1.0
52
Figure 2. Optimal maternal investment, 012 as a function of S2 for different values of 02
The values of other parameters are at = 0-6, a2 « 0-6, £t = 0-4, St = 1, F = 0.1.
1.0
Figure 3. Contours of optimal maternal investment $12 as a function of S2 and 02. The
values of other parameters are a: = 0-6, a2 = 0-6, fl = 04, S{ = 1 and F = 0-1.
genetic interests will diverge, though within limits, from those of its mother. It would
then be expected to try to extract a higher level of maternal investment 0^, then the
mother would be selected to offer. Hence the weaning conflict in mammals with the
584
Madhav Gadgil et al
mother attempting to stop suckling and the offspring attempting to continue doing so
at a certain age. In context of the problem of our interest, the offspring can gain in its
inclusive fitness by miscommunicating to the mother the value of its well-being so as to
extract a higher level of $12. Our model has a very interesting result bearing on this
issue, namely that optimal <£12 that the mother would be programmed to adopt does
not change in a simple fashion with parameters specifying the offspring's condition
(figures 2 and 3). That is, a mother will not always increase her level of maternal
investment if the offspring's condition is better or worse. Under certain conditions she
will increase it, under others decrease it. There is therefore no simple strategy available
to the offspring of misinforming its mother of its own level of well-being so as to
prompt her to enhance the level of maternal investment. Hence natural selection would
tend to disfavour any manipulation of information relating to its own state from the
offspring to its mother (figure 4).
Our enquiry thus suggests that atleast in higher animals with a well developed
nervous system and high levels of maternal care such as elephants and human beings,
communication of well-being must be a significant component of social
communication.
12. Communication of well-being
We began by noting that soil amoebae use folic acid derivatives which are normal
products of bacterial metabolism as signals to locate their prey, and further that these
have been elaborated to serve as signals for aggregation in some species of soil amoebae.
Evolution has thus opportunistically siezed upon normal physiology of animals from
which to elaborate communicatory signals. Signals communicating well-being must
+ 24
+ 18
AH1
AK2
% ASSUMED VALUE OF
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
ACTUAL VALUE OF S2
Figure 4. Change in inclusive fitness of mother (A/^) or offspring (Aff2) when optimal
maternal investment is based assuming S2 to be 0-45, but is in fact different. The values of other
parameters are 04 = 0-6, «2 = 0-6, ^ = 0-4, St == 1, F = 0-1.
On the communication of well-being 585
similarly relate to normal physiological changes in an offspring brought about by a
change in its state of well being. An animal must adjust its metabolism, activity level
and behavioural patterns to its level of well being, though this problem does not appear
to have been specifically investigated with this view point. Coming back to the elephants,
Dr V Krishnamurthy of Tamilnadu Forest Department who has handled elephants for
30 years as a Veterinarian, tells us that a male elephant will come into musth only if he is
given light work and fed well and is in excellent physical condition. A male elephant's
physiology thus shifts into this mode only when its state of well-being is high. There
must be other chemical substances that an elephant calf, say, will start producing in
large quantities only when it is in good health; shifting to others as its health declines.
An elephant mother may continually monitor the state of well being of its offsprings
through monitoring such chemical signals.
The state of well being of an individual will also be reflected in its activity level.
Andrew (1972) talks of an exertion/immobility continuum along which an individual
mammal may be placed. We expect it to move towards greater exertion with an
improvement in its state of well being. This will be reflected in its posture; thus in horses
being more active leads to a high postural tonus with a raising of the tail. An active
mammal also tends to adopt postures which would lead to a loss of heat, an inactive one
curled up postures designed to conserve heat. We may also expect more active animals
to be much more receptive to sensory cues from their environment; this could be
reflected in their sense organs, for instance, cocked ears and dilated pupils.
13. The human species
We close with some less formal observations. In the human family the mother is
continually monitoring the state of well being of her offspring based on general activity
level, changes in sense organs such as 'sparkle* in the eyes, and what goes under the
broad title of 'moods'. Further clues are picked up if necessary by monitoring body
temperature, sensations of pain, excretions and so on. Beginning with this ancient
heritage, human societies with their complex social ties going beyond kinship level have
erected a whole system of monitoring of health status through increasingly specialized
professionals detecting increasingly subtle cues. Communication of well-being has
indeed been tremendously elaborated in human societies.
Acknowledgement
We have benefitted greatly from discussions with P Vijayakumaran Nair.
References
Andrew R J 1972 The information potentially available in mammal displays; in Non-verbal Communication
(ed.) R A Hinde (Cambridge: Cambridge University Press) pp. 179-203
Dawkins R and Krebs J R 1978 Animal signals: information or manipulation, in Behavioural Ecology: An
Evolutionary Approach (eds) J R Krebs and N B Davies (Oxford: Blackwell) pp. 282-309
Doublgas-Hamilton I 1972 On the Ecology and Behaviour of the African Elephant, D.Phil. Thesis, Oxford
University, Oxford
586 Madhav Gadgil et al
&
Gadgil M and Nair P V 1984 Observations on the social behaviour of free ranging groups of tame Asiatic
elephant (Elephas maximus Linn.); Proc. Indian Acad. Sci. (Anim. Sci.) 93 225-233
Hamilton W D 1964 The genetical evolution of social behaviour I and II. J. Theoret. Biol 7 1-52
Krebs J R and Dawkins R 1984 Animal signals: mind-reading and manipulation; in Behavioural Ecology. An
Evolutionary Approach (Second edition) (eds) J R Krebs and N B Davies (Oxford: Blackwell) pp. 380-402
Smith, John W 1977 The Behaviour of Communicating. (Cambridge: Harvard University Press) p. 545
Trivers R L 1974 Parent-offspring conflict; Am. Zool 14 24^-264
Wiley R H 1983 The evolution of communication: information and manipulation; in Animal Behaviour, 2.
Communication (eds) T R Halliday and P J B Slater (Oxford: Blackwell) pp. 156-189
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 6, December 1985, pp. 587-621.
© Printed in India.
Kin recognition in social insects and other animals — A review of
recent findings and a consideration of their relevance for the theory
of kin selection
RAGHAVENDRA GADAGKAR
Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560012, India
MS received 23 May 1985; revised 26 November 1985
Abstract. Kin selection is a widely invoked mechanism to explain the origin and evolution of
social behaviour in animals. Proponents of the theory of kin selection place great emphasis on
the correlation between asymmetries in genetic relatedness created by haplodiploidy and the
multiple origins of eusociality in the order Hymenoptera. The fact that a female is more closely
related genetically to her full sister than to her daughters makes it more profitable for a
Hymenopteran female, in terms of inclusive fitness, to raise full sisters rather than daughters or
full siblings with a female biased sex ratio rather than offspring. This is sometimes referred to
as the haplodiploidy hypothesis. In reality however, genetic relatedness between workers in
social insect colonies and the reproductive brood they rear is far below 0-75, the value expected
for full sisters, often below 0-5 the value expected between mother and daughter and, not
uncommonly, approaching zero. Such values are on account of queen turnover, multiple
mating by queens or polygyny. This situation raises doubts regarding the haplodiploidy
hypothesis unless workers can discriminate between full and half sisters and preferentially
direct their altruism towards their full sisters only. This would still mean an effective coefficient
of genetic relatedness of 0-75 between altruist and recipient. For this to be possible however,
workers should be able to recognise their full sisters inspite of growing up with and being
habituated to an assortment of full sisters, half sisters and perhaps other even less related
individuals. Even outside the Hymenoptera, social animals may find themselves growing up
together in the company of individuals of varying degrees of relatedness. An ability to tell apart
the more and less related individuals under such circumstances should favour kin selection.
Much effort is now going into assessing the abilities of animals to discriminate between kin
and non kin. In every case studied carefully so far, animals appear to be capable of recognising
their kin. Ants, wasps, sweat bees, honey bees, frogs, toads, mice, rats, voles, squirrels, monkeys
and even humans appear to be able to recognise their kin in one circumstance or another. An
ability to recognize true genetic relatedness requires genetically specified recognition labels
and these must therefore be present. Recent findings of the role of the histocompatibility
system provides some clues to the possible nature of recognition labels. An ability to recognise
full sisters for example, inspite of being habituated to full and half sisters requires not merely
genetically specified labels but also recognition templates which are based on the charac-
teristics of the individual animals making the recognition and not templates based on all
animals one grows up with. Some animals such as honey bees, tadpoles and ground squirrels
appear to have such templates but others such as sweat bees and some mice appear not to. It is
entirely possible that our inability to devise natural enough assays for recognition prevents us
from understanding the full potential of the kin recognition abilities of many animal species. In
any case, genetically specified labels and self based templates should greatly facilitate the
evolution of social behaviour by kin selection.
Keywords. Genetic relatedness; kin recognition; kin selection; hymenoptera; haplodiploidy;
evolution of social behaviour.
1. Introduction
There are two main grounds for expecting that animals must be capable of
distinguishing between close genetic relatives and non or distant relatives. The first has
587
588 Raghavendra Gadagkar
to do with inbreeding avoidance (or for achieving an optimum balance between
inbreeding and outbreeding; see Bateson 1980). On theoretical grounds it can be shown
that inbreeding leads to homozygosity of recessive lethal genes resulting in in viable
offspring. In conformity with this expectation inbreeding avoidance is widely observed
in most animal groups. The second has to do with models for the spread of 'altruistic'
alleles by natural selection. The basic idea of current models is that animals must behave
altruistically towards close genetic relatives and selfishly towards non relatives. Such
'nepotistic' behaviour has again been observed in a wide variety of animals. In spite of
such strong theoretical and empirical grounds, efforts to unravel animals' abilities to
recognise kin (other than parent-offspring recognition) began just over 5 years ago. In
this paper I will review experimental evidence of kin recognition from different animal
groups, both among insects and vertebrates. Given an ability to recognise kin, any
animal can potentially use it both for mate selection and for structuring altruistic and
selfish interactions. I will not specifically allude to the function of kin recognition in
each case.
Whether animals can discriminate between close and distant relatives inspite of being
habituated to both classes of relatives is of great theoretical interest. Such an ability is
essential for the tenability of a widely discussed form of kin selection theory (the
haplodiploidy hypothesis) that purports to explain the evolution of insect sociality.
Even outside the Hymenoptera, an ability to discriminate between close and not so
close relatives within a mixed cohort or family group will greatly facilitate the operation
of kin selection. This is because such an ability can raise the effective coefficients of
relatedness between donor and recipient in altruistic interactions. My intention here is
not so much to exhaustively review the literature on kin selection or kin recognition but
to examine the consequences of our present understanding of kin recognition and its
possible mechanisms to the theory of kin selection.
2. The theory of kin selection
The concept of 'inclusive fitness' first put forward by Hamilton (1964a, b) has promised
to provide a plausible mechanism for the evolution by natural selection of altruistic
behaviour in general and sterile castes and 'worker behaviour' in Hymenoptera in
particular. The basic idea is a very simple one and has now come to be known as the
theory of kin selection. Since organisms are ephemeral combinations of genes it is the
individual alleles that form the connecting link from one generation to the next. This
being the case one must be concerned with the changes in frequency of alleles per se in a
population and not merely with the numbers of offspring produced by the bearers of
the alleles in question. An allele can increase in frequency not only by programming its
bearers to produce more offspring (who are likely to carry the same alleles) but also by
programming them to aid genetic relatives (who too are likely to carry the same alleles)
and the latter could well be at the cost of offspring production. If an individual aids n,
relatives (other than offspring) who are related to it by r{ at the cost of producing n0
offspring who are related to it by r0 then, as long as
H,-rf>n0r0, (1)
even sterility could evolve by natural selection (this form of eq. is from Craig 1979).
(Notice that this argument rests on the assumption that the offspring given up and
Kin recognition 539
relatives reared are of equal reproductive value). Eq. (1) can be rewritten as
ni ^ ro
This inequality is possible either if n{ > n0 (i.e., the individual is able to rear more
relatives than it is capable of rearing offspring) or if r£ > r0 (i.e., if the individual is more
closely related to the relatives in question than to its own offspring). The possibility of
rearing more relatives in a social group compared to offspring in a solitary mode of
living is conceivable for any group of animals under certain severe ecological
conditions. One cannot say the same thing for the other alternative, namely, closer
genetic ties with relatives compared to offspring because, no other genetic relatives can
be more closely related to oneself than one's own offspring who bear a coefficient of
relatedness of 0-5 with their parents in any diplo-diploid system. The insect order
Hymenoptera is unusual in this regard because it is not diplo-diploid.
Two facts make Hamilton's arguments particularly attractive. First, haplodiploidy,
which is nearly universal in Hymenoptera but rare outside that order makes a female
more closely related (coefficient of genetic relatedness, r = 0-75) to her fullsister than to
her daughter (r = 0-5) (table 1) so that rt- can potentially be greater than r0. Second,
eusociality, a condition characterised by overlap of generations, cooperative brood care
and reproductive division of labour, has arisen at least eleven times independently in the
order Hymenoptera (Wilson 1971). On the other hand eusociality has only arisen twice
in the rest of the animal kingdom [namely termites and the naked mole-rat; see Jarvis
(198 1) for evidence of eusociality in the naked mole-rat]. In other words, haplodiploidy,
which makes possible for r, to be greater than r0 has been an important factor in the
multiple origins of eusociality in Hymenoptera. We shall henceforth refer to this as the
"haplodiploidy hypothesis". It is true of course that if Hymenopteran workers rear
sisters and brothers in equal numbers in place of sons and daughters, they gain nothing
as the low relatedness to brothers (r = 0-25) exactly cancels out the advantage due to
high relatedness to sisters. In other words average relatedness to fill-sibs ( r siblings
== 0-5 ) is the same as relatedness to offspring (r = 0-5). It has now been
2
pointed out however that if Hymenopteran workers (who are always females) skew their
investment in favour of sisters, then they would capitalise on the asymmetries in genetic
relatedness created by haplodiploidy. In fact at equilibrium workers would be expected
to invest in their full sisters and brothers in the ratio 3 : 1 (being the ratio of their genetic
relatedness to full sisters and brothers) (Trivers and Hare 1976; for a recent review see
Table 1. Co-efficients of relatedness under haplodiploidy assuming complete
outbreeding.
Daughter Son Sister Brother Mother Father
0-75 0-25
Female 0-5 0-5 ^ = ^ 0-5 0-5
i-o oo i-o o-o
^-- °'s °'5 —-
590 Raghavendra Gadagkar
Joshi and Gadagkar 1985). If workers who give up the production of a certain number
of offspring in fact invest in an equivalent number of siblings skewing investment in
favour of sisters, average r,- would be greater than r0, satisfying the condition for the
evolution of sterile or other altruistic behaviour by the haplodiploidy hypothesis. The
current status of theory and data on evolution of social behaviour has been extensively
reviewed (Hamilton 1972; West-Eberhard 1975; Wilson 1975; Starr 1979; Gadagkar
1985).
3. Lower than expected levels of relatedness — the evidence
The legitimacy of the haplodiploidy hypothesis outlined above is crucially linked to the
demonstration that values of rt greater than r0 in fact occur. Isozyme patterns revealed
by electrophoresis are now routinely used to determine genotypes of individual
organisms (see Lewontin 1974 for a comprehensive as well as historical introduction to
this subject). In recent years a number of methods with increasing levels of
sophistication have been developed to estimate levels of genetic relatedness within
subgroups of a population, using electrophoretic data (Metcalf and Whitt 1977a;
Lester and Selander 1981; Craig and Crozier 1979; Pamilo and Varvio-Aho 1979;
Pamilo and Crozier 1982; Pamilo 1984). Many social insect species have now been
subjected to such an analysis and a sample of the results (not an exhaustive list)
available in the literature are presented in table 2. With few exceptions most estimates
of genetic relatedness among workers or between workers and the female reproductive
brood they rear are very low; almost always less than 0-75, the value expected for full
sisters, often below 0-5, the value for mother and daughter and, not uncommonly the
values are not significantly different from zero. Most species listed in table 2, however,
are ants which are all highly eusocial. In the context of the evolution of social behaviour
by kin selection our focus should naturally be on the primitively eusocial species but
there have been surprisingly few attempts to estimate genetic relatedness in such
species. And the few attempts that have been made are not very encouraging (table 2).
Determining the frequencies of alleles in 5 polymorphic esterase loci Metcalf and
Whitt (1977a) showed that in the primitively eusocial wasp Polistes metricus:
(i) foundresses mate at least twice using sperm from the two males in the ratio 9:1;
(ii) a foundresses share reproduction with their subordinate /? foundresses, the former
contributing 78 percent of the females and 87 percent of the males;
(iii) workers lay male eggs if foundresses die and even here one worker produces 19
times as many eggs as another. Intra-nest genetic relatedness can vary drastically
depending on the fate of the foundresses.
On these criteria six different types of nests were defined and intra-nest relatedness
was calculated for each nest type: (a) solitary foundress alive, (b) solitary foundress
dead, (c) a and ft foundresses alive, (d) a foundress alive, /? dead, (e) a foundress dead, j?
alive and (f ) a and jS foundresses both dead. Using the data provided by Metcalf and
Whitt (1977a, b), Lester and Selander (1981) have calculated an average relatedness of
0-63 between a worker and her female reproductive siblings. Of the three studies
pertaining to primitively eusocial wasps listed in table 2, this is in fact the only case
where rf is greater than r0. In P. exclamans and P. apachus-bellicosus, respectively,
similar electrophoretic techniques revealed an average genetic relatedness of 0-390 and
0-429 between workers and their reproductive sisters. This value is not only far below
Kin recognition
Table 2. Genetic relatedness in colonies of social insects.
591
Species
Average genetic related-
ness between workers
and the reproductive
female brood they rear
or among workers
Reference
Wasps
Polistes metricus
Polistes exclamans
Polistes apachus-bellicosus
Bees
Apis mellifera
Ants
Aphaenog aster rudis
Myrmecia pilosula
Formica sanguinea
Formica sanguinea
Formica transkaucasica
Formica aquilonia (Espood)
Formica aquilonia (Vantaad)
Formica polyctena (Siuntiod)
Formica polyctena (Kauniainend)
Myrmica rubra Site A- 1975
Myrmica rubra Site A 1977
Myrmica rubra Site A 1978
Myrmica rubra Site B 1977
Formica exsecta (Espoo4)
Formica exsecta (Tuusulad)
Formica exsecta (Joskar4)
Formica exsecta (Kalvholm1*)
Formica pressilabris (Espood)
Formica pressilabris (Tuusulad)
Rhytidoponera mayri
0-63°
0-390°
0429"
Approaching
0-25*
0-75C
0-172 ±0-053*
0-378 ±0-1 73"
0-19*
0-33 + 0-07*
0-09 ±0-09*
-0-02±0-14*-e
0-19 ±0-34"
0-30 ±0-23"
0-1056*>e
0-0218*- e
0.0828b'e
0-5428*
0-04 ± 0-07*- e
0-09 ± 0-08*' e
0-62 + 0-13*
0-78 ±0-1 3*
0-29 ±0-1 3*
0-07 ± 0-08*- <
0- 1 58 ± 0-037*
Metcalf and Whitt 1977a,b;
Lester and Selander 1981
Lester and Selander 1981
Lester and Selander 1981
Page and Metcalf 1982
Crozier 1973
Craig and Crozier 1979
Pamilo and Varvio-Aho 1979
Pamilo 1981
Pamilo 1982
Pamilo 1982
Pamilo 1982
Pamilo 1982
Pamilo 1982
Pearson 1983
Pearson 1983
Pearson 1983
Pearson 1983
Pamilo and Rosengren 1984
Pamilo and Rosengren 1984
Pamilo and Rosengren 1984
Pamilo and Rosengren 1984
Pamilo and Rosengren 1984
Pamilo and Rosengren 1984
Crozier et al 1984
"Genetic relatedness between workers and the female reproductive brood they rear.
* Genetic relatedness among workers.
c Inferred because monogyny and monoandry were demonstrated.
''Localities from where the populations were sampled.
*Not significantly different from zero.
0-75, the value expected for full sisters in a haplodiploid system, but even lower than 0-5,
the value expected between a female and her offspring.
In the honey bee Apis mellifera, which of course is highly eusocial, Page and Metcalf
(1982) again used isozyme polymorphism and set out explicitly to study multiple
mating and patterns of sperm usage by queens. Their results showed that honey bee
queens used sperm from at least 3 males at any given time and mixing of sperm in the
spermatheca resulted in the average relatedness amongst her daughters approaching
0-25.
592 Raghavendra Gadagkar
4. The causes and consequences of low levels of relatedness
The main reasons attributed to such low levels of relatedness are polygyny, queen
turnover and multiple mating followed by sperm mixing. Queen turnover can reduce
the average relatedness between workers and the reproductives they rear in the kinds of
Polistes nests studied by Metcalf and Whitt (1977a,b) and Lester and Selander (1981).
If the a foundress lays worker eggs and dies paving the way for the /? foundress to lay the
reproductive eggs, then workers are not rearing their full sisters (r = 0-75) as future
reproductives, but their cousins (a and /? are assumed to have been full sisters) [r
= 0-1875; relatedness of workers to their mother a (0*5) x relatedness of a to jS (0-75)
x relatedness of /J to her daughters who are the future reproductives (0-5) = 0-1875]. In
tropical wasps the queen's daughters often replace the queens (see for eg.. Jeanne 1972).
Here the workers who are sisters of the new queens now raise nieces (r = 0-375) rather
than full sisters (r = 0-75). Notice that this value would be even lower if the original
foundresses a and jS were not full but half sisters in the temperate species and if the
workers in the tropical species were not full but half sisters of their new queens.
Polygyny or the simultaneous presence of more than one egg layer (a condition known
among many social insects) would also similarly lower the levels of relatedness between
workers and the reproductive brood they rear. For a discussion of the role of polygyny
see West-Eberhard (1978). Yet another factor contributing to low levels of relatedness
between workers and the reproductive brood would be usurpation of nests by
unrelated conspecifics (Gamboa 1978).
When the queen mates with more than one unrelated male, her daughters would not
all be full sisters of each other if she used sperm from more than one male at any given
time. Any two randomly picked daughters would be full sisters with a certain
probability p and half sister with a probability 1 - p. Suppose a female mated with n
males who are respectively responsible for proportions & , f2 , f3 , . . . ,fn of her female
n
progeny where, £ Jj = 1. The average coefficient of relatedness (r) bet ween daughters
i = 1
is then
and if all males contribute equally, we have
1/1 1
r = - - + - (Hamilton 1964b).
2V2 nJ
The larger the number of males she has mated with the closer the relatedness between
two average daughters approaches 0-25, the relatedness between two half sisters. This
perhaps partly accounts for the low relatedness in the wasp studies referred to above
and is in fact the reason behind the results in Apis mellifera (Page and Metcalf 1982).
Although Page and Metcalf (1982) first conclusively demonstrated multiple mating and
sperm mixing, these ideas have a long history. Indeed social insects have long been
known to be notoriously polyandrous (Wilson 1971; Page and Metcalf 1982; Cole
1983). In fact this was well known at the time Hamilton first proposed the ideas of kin
selection and the role of haplodiploidy. Hamilton's reaction to this was first (1964b) that
'multiple insemination will greatly weaken the tendency to evolve worker-like altruism
Kin recognition 593
and n > 2 . . . should prevent its incipience altogether' and later (1972) that 'the
occurrence of this special relatedness to sisters must not be over emphasized. Male
haploidy is certainly not the only prerequisite for evolving a sterile caste'.
Queen turnover might be of unpredictable accidental occurrence. But polygyny and
multiple mating by the queens, widespread as they are, are clearly evolved traits that
might have some adaptive significance. From the point of view of social evolution one
might wonder why polyandry has evolved at all in social hymenopterans. By mating
with only one male a queen ensures the highest possible relatedness amongst her
daughters and thereby might be expected to maximise the chances of their cooperating
and helping each other. It is possible that in ancestral hymenopterans natural selection
placed a higher premium on other factors correlated with multiple mating such as
greater brood viability (Page 1980; Page and Metcalf 1982; Woyke 1963) and larger
colony size (Cole 1983) rather than the evolution of sociality. In the few cases where it
has been studied, sex determination in Hymenoptera appears to be determined by one
or a few polymorphic loci. Diploid heterozygotes (heterozygous in at least one locus in
multi-locus systems) are females; haploids (hemizygotes) are males while diploid
homozygotes (homozygous at all loci in multi-locus systems) are inviable or sterile
males (see Wilson 1971 for a lucid treatment of sex determination in Hymenoptera). In
such a system (let us consider the single locus system for simplicity) a queen who mates
with a male carrying one of her own sex determining alleles is destined to produce 50 %
inviable offspring. It thus pays for the queen to mate with several males and thereby
reduce the proportion of inviable offspring ( Page 1980; Page and Metcalf 1982). On the
other hand Cole (1983) has shown that multiple mating is strongly correlated with large
colony sizes in ants, and argues that multiple mating ensures sufficient sperm in the
queen to make possible the maintenance of larger colonies (see Starr 1984 for a more
comprehensive account of the consequences of multiple mating). Similarly polygyny has
been considered as an adaptation against extinction in small or rare populations
(Wilson 1963).
5. Rendering the low levels of relatedness consistent with the haplodiploidy
hypothesis
There are several ways in which attempts have been made in the literature to explain
away the difficulties rendered to the haplodiploidy hypothesis by the low levels of
genetic relatedness especially when they result from multiple mating.
(i) If the two or more males that mate with a queen are very closely related to each
other then their sperm will be nearly identical thus negating the effects of multiple
mating (Wilson 1971).
(ii) If multiple insemination is restricted to the more highly advanced social groups and
absent in the primitive ones, it can be thought of as secondarily evolved after sterile
castes had already evolved by kin selection and had gone so far as to be now
irreversible. Such irreversibility could arise because workers may no longer have
any immediate reproductive options in response to low levels of relatedness to the
brood on their parental nests. Neither of the above ideas is however supported by
any strong empirical observations (Wilson 1971; Starr 1984).
(iii) Low levels of reiatedness in general and multiple insemination in particular can be
thought of as posing no special problems for the haplodiploidy hypothesis as long
594
Raghavendra Gadagkar
0.75
CO
in
LU
z
o
LU
< 0.50r
LU
cr
0.25-
10 15
NUMBER OF MATINGS (n)
20
Figure 1 . Mean relatedness among daughters as a function of the number of matings by the
mother in haplodiploid (Fw) and diploid (F^) genetic systems. This graph is drawn using the
following equations provided by Page and Metcalf (1982): Assuming that each mate
contributes equal amount of sperm 7H = 1/2(1/2+ l/n) and 7D — 1/4(1 H- 1/n).
as one does not find these phenomena more often in haplodiploid than in diploid
groups. At any level of multiple mating haplodiploid groups are always at least
slightly more predisposed towards sociality than their diploid counterparts (Page
and Metcalf 1982). Note from figure 1 that if the queen mates with two males, then
any two of her daughters on the average are related to each other by 1/2, which is
the same as that between mother and daughter. The asymmetry in genetic
relatedness caused by haplodiploidy is thus completely lost. This is probably why
Hamilton (1964b) believed that more than 2 matings should prevent altruism
altogether. It is true however that at any given number of matings two sisters in a
haplodiploid populations are more closely related than two sisters in a diploid
population. Ecological factors being identical haplodiploid populations are
therefore more likely to develop female altruism than diploid populations (Page
and Metcalf 1982).
(iv) A very popular way of getting out of the difficulty created by multiple mating has
been to assume that although queens mate with more than one male the sperms
from different males do not mix appreciably in the spermatheca. The queen is
therefore effectively monoandrous using sperm from only one male for long
stretches of time (Trivers and Hare 1976; Orlove 1975; Charnov 1978; Cole 1983).
Taber (1955) conducted perhaps the first detailed investigation of sperm usage
patterns using naturally and artificially inseminated queens. His results indicated a
non-random usage of sperm but clearly sperm from different males was at least
partially mixing in his experiments (Page and Metcalf 1982; Crozier and Bruckner
1981). Other studies on honey bees (Alber el al 1955; Kerr et al 1962) strongly
suggest sperm mixing. Comparable experiments have also been performed with
Solitary wasps (Wilkes 1966; Holmes 1974) and the conclusion here is that while
sperm from different mates is not used in a perfectly random fashion there is no
evidence of perfect sperm precedence either. Evidence for multiple mating and
Kin recognition 595
patterns of sperm usage had until recently to depend entirely upon dissections or
the use of genetic markers. In modern times however the use of isozyme markers
has begun to yield far more reliable data. Some 9 species of Hymenopterans have
been investigated using this technique (review in Page and Metcalf 1982; Starr
1984) of which 3 are polyandrous with a certain degree of sperm precedence in two
species. The varying degrees of sperm precedence or biased sperm usage
demonstrated in various species reduce the effective number of matings but the
resultant relatedness between daughters would nevertheless be less than 0-75, the
value expected for full sisters.
(v) Finally an ingeneous way of getting out of the difficulty caused by lower levels of
relatedness between the workers and the reproductive brood is to argue that
workers are capable of discriminating their full sisters apart from half sisters and
that workers selectively rear their full sisters. This would still make worker
behaviour advantageous by virtue of closer genetic ties between workers and their
full sisters compared to that with daughters (Getz et al 1982; Page and Metcalf
1982).
6. Kin recognition — the evidence
6.1 Sweat bees
Lasioglossum zephyrum is a primitively social sweat bee that lives in a system of burrows
under the soil. One of the bees usually assumes the role of a guard and, sitting at the
entrance to the burrow, prevents both parasites as well as non-nest mate conspecifics
from entering the burrow. Breeding these bees in artificial nests in the laboratory,
Greenberg (1979) presented guard bees with intruders who are of known genetic
relatedness to the guards (known to Greenberg!). Testing bees of 14 different
geneological relationships against one another in this fashion a highly significant
positive correlation between probability of acceptance into the nest and the genetic
relatedness between intruder and guard bee was demonstrated (figure 2). This clearly
implies a capacity to recognize different levels of genetic relatedness and there are
reasons to believe that such recognition is based on odours (Barrows et al 1975). Using
artificial laboratory colonies constituted by unrelated bees Buckle and Greenberg
(1981) concluded that the bees do not recognise genetic relatedness to themselves. The
guard bees appear to learn the odours of their nestmates and then, using these learnt
odours as a guide, they accept or reject intruders depending on the similarity of the
intruders' odour to those of the guard's nest mates (table 3). (see Getz 1982 for a
refutation of this conclusion). Previous work had suggested that both genetic
homogeneity as well as adult learning opportunities enhance nestmate recognition
abilities (Kukuk et al 1977). Since environmentally acquired difference in odours are
eliminated in these experiments it is presumed that similarity in odours reflect genetic
relatedness. In this species males are also capable of assessing the genetic relatedness
between successive female partners through a process of learning or becoming
habituated to female pheromones (Smith 1983). Notice that such a system of
recognition involving a template based on learning from individuals other than oneself
is unlikely to permit workers to distinguish between full and half sisters in the same
colony. This type of kin recognition will therefore not help get us out of the difficulties
596
Raghavendra Gadagkar
1.0-
~ 0.8
<
0.6
UL >- 0 A
r-> nn « • M
0.2
D NOT RELATED
A COUSINS
O AUNTS-NIECES
X SISTERS
_L
0 0.2 0.4 0.6 0.8 1.0
AVERAGE GENETIC RELATEDNES5 BETWEEN
GUARD AND INTRUDER
Figure 2. Individuals of the primitively eusocial bee Lasioglossum zephyrum were raised in
the laboratory. In artificially constituted laboratory colonies guard bees were presented with
intruder bees whom they had never encountered before. But these intruders were infact related
to the guard bees as sisters, aunts, nieces, cousins or were unrelated to them. The probability of
acceptance into the nest of the intruder bee by the guard bee was significantly positively
correlated with the average genetic relatedness between guard and intruder bees. After
Greenberg (1979). Copyright AAAS.
for the haplodiploidy hypothesis created by the low levels of genetic relatedness
between workers and their reproductive siblings.
6.2 Honey bees
While colony odours and the recognition of hive mates is well known in honey bees,
Breed (1981) first demonstrated a genetic basis in recognition 'of queens by workers.
Replacing existing queens by new queens and observing the response of workers, Breed
showed that the acceptance of new queens by the workers depends on genetic
relatedness of the replacement queens to former queens (table 4). Thirty five per cent of
the new queens were accepted if they were inbred sisters (r c~ 1) of the previous queen,
12 % if they were outbred sisters (r cs 0-75) and 0 % if unrelated. As in the case of sweat
bees recognition by the workers is a learned phenomenon. Carbon dioxide narcosis
abolished the memory of the recognition cue so that strange queens were now accepted.
With time, of course, the identity of the new queen was learned as shown by rejection of
subsequently introduced queens unrelated to that whose identity has been learned.
Similar results were obtained when workers were transferred from one hive to
another in the field or from a box containing one group of workers to one containing a
different group of workers (Breed 1983). Although environmentally acquirable cues
were held constant including the entire duration of larval development, genetically
unrelated workers were attacked more often than genetically closely related ones. There
Kin recognition
Table 3. Sweat bees don't smell themselves".
597
Accepted/
Colony
Guard
Intruder
rejected
3X 3Y"
X
Sister of X
Accepted
3X 3Y
X
Sister of Y
Accepted
3X 3Y
Y
Sister of X
Accepted
3X 3Y
Y
Sister of Y
Accepted
3X 1Y
X
Sister of X
Accepted
3X 1Y
X
Sister of Y
Accepted
3X 1Y
Y
Sister of X
Accepted
3X 1Y
Y
Sister of Y
Rejected
"Data from Buckle and Greenberg (1981).
*This means a colony consisting of 3 sisters from one inbred
genetic line X and 3 bees from another inbred genetic line Y.
Table 4. Acceptance of foreign queens by honey bee workers".
Percentage
Sample
transfers
Queen type transferred
size
accepted
Inbred sister of old queen
23
35
Outbred sister of old queen
26
10
Non sister of old queen from same genetic line
20
10
Non sister of old queen from unrelated genetic line
39
0
Disturbance control
10
90
Non sister of old queen from unrelated genetic line
transferred after CO2 narcosis of workers
20
90
a Modified from Breed (1981). Reprinted with permission.
s a strong suggestion of genetically determined odours and these appear to be already
present at 5 days after post emergence. Getz and Smith (1983) performed similar
experiments but with genetic relatedness more precisely defined. They set up
experimental hives using queens of known genotypes which were artificially in-
jerninated with sperms from males of known genotypes. In such hives full and half
dsters among workers were obvious to the experimenter because the genetic markers
ised led to different colour morphs. Groups of worker bees were then removed from
:heir parental hives little before their expected emergence and maintained as small
groups of full sisters for 5-6 days. Now, when bees were transferred from one group to
mother, they were found to be significantly more likely to bite half sisters than full
dsters. This result clearly indicates a genetic basis for the cue which is recognised
Because both full and half sisters were raised in the same hive and must therefore be
ilmost identical in any environmentally acquired odours. The ability to recognize cues
;ould however be based on learning the odours of one's nestmates because each test bee
598 Raghavendra Gadagkar
had been allowed to habituate to its full sister for 5-6 days prior to testing. It is therefore j
possible that the test bee had learned the odour of its full sisters during this period. j
In an earlier paper on the other hand, Getz et al (1982) suggest that distinction
between half and full sisters could be occurring even when both are present in the same .
hive and therefore habituated to each other. The basis for this conclusion is the result f
that in hives with two different genetic lines of workers, the patrilineal worker groups |
segregate non-randomly during swarming. One colony had 36,700 bees with 74 % of the \
cordovan mutant and 26 % wild type. After swarming the bees remaining in the hives j
were 64 % cordovan and 36 % wild type while those in the swarm were 79 % cordovan \
and 21 % wild type. Another colony had 30,000 bees with 54.5 % cordovan and 45-5 % \
wild type. The swarm contained 58 % cordovan and 42 % wild type while those staying
back in the parent hive were 43 % cordovan and 57 % wild type. The conclusion ( Getz et
al 1982) that, of the two patrilineal lines of workers, one line of full sisters leave while
another line stay home is obviously very weak because of the very slight differences in f
composition. The high statistical significance of the data appear to be due to the
inordinately large sample sizes ( > 30,000 bees). Besides, as the authors themselves
suggest, their data could simply 'reflect a propensity for cordovans to swarm more
readily than normal workers'. Significantly, the same author states in a subsequent
paper (Getz and Smith 1983) that 'at this stage there is no evidence that bees
discriminate between full and half sisters in the hive once they are habituated to both
sets of workers'. Unless this is shown we are still left with the difficulties posed by
multiple mating for the haplodiploidy hypothesis.
In more recent experiments (Breed et al 1985) honey bee workers were allowed to
mature (in cardboard boxes!) from day 1-5 after emergence either with other bees from
the same hive (i.e. with full or half sisters) or with unrelated bees. When such bees were
introduced into boxes holding other bees, an introduced bee is attacked depending only
on its genetic relationship to the recipient bees. There is no effect of mixed rearing ( from
day 1-5) so that no odours appear to be transferred from one bee to another. Bees,
however, appear to learn the odours of their nestmates (or boxmates!). If recipient bees
are housed together in mixtures of two different genetic lines, bees of both genetic lines
are equally likely to attack on introduced bee of either genetic line. This suggests that
discrimination of heterogeneity within a hive is not possible. On the other hand, there is
a tantalizing suggestion that bees learn the odour of their own genetic line as well as any
other genetic line in their association and store these two memories separately. When
feeding behaviour was studied, bees kept in mixed groups nevertheless interacted more
often with unfamiliar kin than with the other genotype, some individuals of which they
were also habituated to. In other words, as far as feeding behaviour goes, different
genetic lines appear to be distinguished. As a matter of fact, if kin recognition is to
counteract the effects of multiple mating and thereby rescue the haplodiploidy
hypothesis, differential feeding of different genetic lines is probably more important
than differential aggression. In any case, one cannot overemphasize the need for caution
in interpreting results based on assays of kin recognition which, almost always bear
unknown relationships to behaviour under natural conditions. Be that as it may, here is
the first indication of what we have been looking for — ability of workers in a social
insect colony to potentially discriminate between full and half sisters.
A conserted attack on the honey bee as a model system to unravel the possibility of
discrimination of different genetic lines within a hive appears to have been conspired.
Since the preparation of the first draft of this essay, I have received 4 unpublished
Kin recognition 599
manuscripts, each taking us a step closer to a decisive answer to this question. Briefly,
Getz W M, Bruckner D and Smith K B (unpublished results) asked if there is sufficient
variability in the odours of full and half sisters to permit their differential recognition in
the first place. The answer seems to be in the affirmative because they succeeded in
conditioning bees to extend their probosides to only one of the two choice odours
derived from their full and half sisters respectively. Getz W M and Smith K B
(unpublished results) have now eliminated a lacuna in their previous experiments by
showing that bees reared in complete isolation can still discriminate between their full
and half sisters. Their results also corraborate those of Breed et al (1985) that, while
odours from more than one genetic line can be learned, the respective templates used in
recognition are not confounded. There is however a suggestion of possible transfer of
labels from one bee to another unlike in the case of the experiments of Breed et al (1985).
Noonan K C (unpublished results) has now demonstrated that worker honey bees in
colonies of mixed patrilines show preferential care to queen and worker brood of their
own patrilines. It thus appears that at least in the honey bee effective genetic relatedness
between workers and the female reproductive brood they rear can be as high as 0-75.
One hopes that further experimentation will reveal similar phenomena in primitively
eusocial insects which is the critical focus for the haplodiploidy hypothesis.
6.3 Ants
Ants are a group where colony odours that help discrimination of nestmates from non-
nestmates have been suggested almost a 100 years ago. There has been much discussion
in the literature regarding the genetic versus environmental origins of such odour. It was
not until a series of simple experiments by Jutsum et al (1979) however that it became
clear that both exogenous (from diet) and endogenous (probably but not necessarily
genetic) components exist and act synergistically. These experiments also indicated that
even in the complete absence of any exogenous differences there exist sufficient
differences in the endogenous component to permit distinction between nestmates and
non-nestmates (table 5). The experiments just described used colonies of the leaf cutter
ant Acromyrmex octospinosus maintained in the laboratory and where the endogenous
and exogenous source of odour difference could be carefully controlled. Field
experiments on aggression between workers drawn from local versus widely separated
Table 5. Endogenous and exogenous components of colony odour in
ants".
Colony
(endogenous
factor)
Forage
(exogenous
factor)
Mean time spent
in investigating
(minutes)b
Sample
size
Different
Different
12-4
60
Same
Different
7-0
15
Different
Same
4-4
55
Same
Same
2-6
23
"All means are significantly different from each other (P < 0-05)
* Modified from Jutsum (1979). Copyright Bailliere Tindall.
600
Raghavendra Gadagkar
colonies confirmed these laboratory findings. More recently, experiments have been
performed with interspecific mixed laboratory colonies using 5 species of monogynous
carpenter ants belonging to the genus Camponotus (Carlin and Hdlldobler 1 983). When
worker larvae from an alien species were introduced into a queen-right colony of a
different species the larvae were accepted, groomed and fed to grow into adults. In
several such colonies studied no pattern of preference for kin or rejection of
heterospecific nestmates was observed. Moreover when interaction between an adoptee
from a mixed colony was tested with its non nestmate genetic sisters taken from a stock
laboratory colony, there was always intense aggression showing lack of recognition of
genetic relatedness (figure 3). In other words the alien adoptee workers had acquired as
Remove
t
Original
colonies
Queen of Workers of
type A ' type A
Experimental
colony
Queen of
type A
Workers of
type B
Experimental
set-up
Workers of Queen of
type B type B.
Workers
of type B
Workers
of type B
These two sets of
workers show intense
aggression towards
each other
Figure 3. In laboratory colonies of the carpenter ants belonging to the genus Camponotus
one colony (species A) was deprived of its workers and alien workers from a different
laboratory colony of another species (species B) were introduced. The experimental colony
thus consisted of a queen of one species (species A) while the workers were of a different genetic
line (species B). These workers of species B in association with the queen of species A showed
evidence of having learned as well as acquired the properties (odours) of their foster Queen.
IHis is inferred from the observation of intense aggression between these workers of species B
and other workers of species B drawn from the original species B colony. Data from Carlin and
Holldobler (1983). More recent experiments suggest that colony-specific odours and not
species-specific odours are involved in these phenomena (Carlin and Holldobler unpublished
Kin recognition 601
well as learned an odour from the queen in its new nest. The adoptee workers must have
acquired the queen's odour because they are discriminated against by their non
nestmates genetic sisters and they must have learned the queen's odour because they
now discriminate against their non nestmate genetic sisters. It is also clear that it is the
queen that is the source of the discriminator odour because the same results were
obtained with all-adoptee colonies where all the queen's brood had been removed.
These results suggest that the queen's odour is learned by all workers in a colony and
later used for discrimination of different individuals. Although interspecific colonies
were used in these experiments, more recent studies have confirmed that indeed,
colony-specific and not species-specific cues are being transferred from the queens to
the workers. It also appears that in the absence of the queen, worker-derived as well as
food-derived cues begin to exert their influence on recognition (Carlin N F and
Holldobler B, unpublished results). In any case, since the queen-derived cues are
dominant there is little scope for different genetic lines of workers in the colonies of
multiply mated queens to preferentially aid full sisters and discriminate against half
sisters. On the contrary, nestmate recognition in the Acacia-ant Pseudomyrmex
ferruginea appears to be of a rather different kind. The queen is not the source of
recognition pheromone in this system because groups of worker brood separated from
a stock colony and reared separately by different foster reproductives failed to show
antagonistic interactions; although workers from different colonies are normally
incompatible. Recognition must therefore be based on genetically specified cues
although, whether the ability to discriminate nestmates is learned or not is unclear
(Mintzer 1982). A follow-up study using colonies initiated by inbred lines suggests a
multiple locus model for the production of recognition pheromones (Mintzer and
Vinson 1985).
6.4 Social wasps
Comparable experiments have been performed using the primitively eusocial wasp
genus Polistes. These wasps have not been bred in the laboratory and it has therefore
not been possible to control genetic relatedness between experimental animals as
precisely as in the case of sweat bees or honey bees. The discussion of the wasp data will
therefore be in terms of nestmate discrimination. When nests are drawn from
reasonably well separated localities, nestmates (wasps emerging in the same nest) must
be significantly more closely related to each other than to non nest mates (animals
emerging in different nests). The exact degree of genetic relatedness may however vary
in an undefined fashion between different experiments. Wasps from the same nest will
certainly be related and the values of relatedness may even fall within narrow limits.
Wasps from nests drawn from distant localities will certainly bear low relatedness to
each other. But pairs of wasps drawn from different pairs of distant nests may have very
different values of relatedness. This can contribute to increase in variation in the results
from experiment to experiment. Nestmate discrimination has been studied in Polistes
in 4 situations: association of overwintered foundresses in spring, artificial associations
of nest mates and non nestmates under experimental conditions, recognition of brood
and mate preferences. For a review of the wasp work, see Gamboa et al (1985).
6.4.1 Foundress associations: Polistes wasps in the temperate regions terminate
602 Raghavendra Gadagkar
their nest cycle during the fall season and newly emerged females overwinter by
hibernating in places away from the nest sites. At the beginning of the following spring
season the overwintered females return preferentially to the natal nesting sites and
initiate new colonies, often by several females cooperating in what are termed multiple
foundress colonies. Because females preferentially return to their natal nesting sites it
has been possible to mark emerging females in the fall and observe association patterns
in the following spring. This has been done repeatedly in several species ofPolistes and
two species of Mischocyttarus (another genus belonging to the sub-family Polistinae).
The general result has been that cofoundresses are usually females emerging from the
same nest (see West-Eberhard 1969; Noonan 1981 and references therein). Klahn (1979)
working with Polistesfuscatus and Pratte (1982) working with P. gallicus have argued
that preferential association of nest mates as cofoundresses is not based on recognition
of nest mates per se but because ofphilopatry — the phenomenon of returning to the site
of emergence. Because nests can be located very close to each other, however,
philopatry is unlikely to be sufficient to ensure relatedness of cofoundresses. Besides,
other workers have since provided strong evidence of recognition of nest mates per se.
Ross and Gamboa (1981) collected nests from different localities and allowed the gynes
(females emerging in fall which are potential foundresses for the next spring) from each
nest to overwinter in the laboratory along with their nests and nest mates but separated
from other gynes and other nests. After thus overwintering in the laboratory for 6-5
months the wasps were exposed to spring conditions. At this stage nest mates and non
nest mates were introduced into enclosures where nest mates preferentially associated
with each other to initiate nests. Behavioural interactions among such overwintered
females who were isolated from all other conspecifics for 74-99 days showed that they
still retained the ability to recognise the nestmates with whom they had hibernated.
Similar results have been obtained with P.fuscatus where sample sizes were larger and
observations blind (Bornais et al 1983). Using P.fuscatus, Post and Jeanne (1982)
went a step further and showed that females overwintered in the laboratory
preferentially associated with former nest mates even if they had not hibernated along
with them and that they do not associate with non nest mates even if they had been
forced to hibernate with them. The characteristics of nest mates must thus have either
been learnt during the fall season soon after emergence and remembered or animals
must be able to recognise nest mates without the need to have to learn anything from
them. In other words, they must be selfsufficient in producing a template in their brain
with which to compare other animals and assess relatedness to themselves. That the
ability to distinguish nest mates from non nest mates does not depend on having to
arrive at the same nesting sites is further strengthened by the observation of clumping
patterns during overwintering in the laboratory by P. exclamans females. Artificial
hibernating boxes containing only nestmates show few and large clumps during
hibernation while those containing a mixture of nestmates and non nestmates contain
many small clumps throughout the period of hibernation (Allen et al 1982).
6.4.2 Associations of females emerging in the laboratory: Shellman and Gamboa
(1982) collected natural nests of P.fuscatus and kept them in the laboratory to allow
emergence of adults from the puparia. Upon emergence females were either (i) isolated
from the nest and nestmates within minutes of emergence, (ii) isolated from the nest but
kept along with other newly emerged nestmates or (iii) exposed both to their natal
nest and nestmates. After such treatment for 15-120 days each female was isolated into
Kin recognition 603
individual boxes for 14-20 days. Now two nest mates and one unrelated female were
introduced into a test box and the 3 females were observed for discrimination of nest
mates from non nest mates. Using time spent in close proximity (less than 5 cm apart) as
an assay of discrimination, Shellman and Gamboa (1982) showed that only females
exposed to their natal nests and nest mates are capable of discriminating between nest
mates and non nest mates. From more recent studies using newly emerged workers
exposed to unrelated nest fragments and to unrelated conspecifics (Pfennig et al
1983a, b) it is quite clear that nest mate discrimination depends on learning of chemical
cues from the natal nest or its brood by newly emerged adult wasps. Similar
experiments have recently been performed with the bald-faced Hornet, Dolichovespula
maculata (Hymenoptera: Vespidae) (Ryan et al 1985). These results are somewhat
difficult to interpret because there appear to be discrepancies between different
measures of recognition. Wasps isolated from their nests and nestmates also probably
are capable of nestmate discrimination (unlike Polistes, see above). However, the
authors conclude that the nest is somehow involved in the ontogeny of nestmate
recognition ability because there is much more variability in the responses of the
isolated wasps compared to those allowed to learn the characteristics of their nests and
nestmates.
6.4.3 Recognition of brood: Polistes fuscatus wasps destroy brood or desert a nest
significantly more often if their own nest is replaced by the nest of an unrelated female
than if their nest is replaced by those of their sisters (former nest mates). Sisters
normally nest in close proximity of each other and may therefore share common food,
nesting material and other environmental odours. But the involvement of such
environmentally originated odour is ruled out because brood destruction is based only
on genetic relatedness even when sisters nesting far apart and non sisters nesting in
close proximity were tested. A nest and its brood appear to be recognised as a unit and
there is no evidence of discrimination of differently related brood within the same nest
(Klahn and Gamboa 1983).
6.4.4 Mate preferences: In contrast to the female's demonstrable ability to dis-
criminate nestmates from non nestmates, males of P. fuscatus appear to lack the ability
of discriminating between nestmate and non nestmate females but, appear to recognize
nestmate males at least under certain experimental conditions. When paired with
nestmate and non nestmate females, males seem to choose their mates without regard to
relatedness. This has been shown both by visual observation of mating behaviour
(Larch and Gamboa 1981) as well as by actual assessment of insemination (Post and
Jeanne 1982). The males also do not discriminate between nestmate and non nestmate
males, as shown by Ryan et al (1984) in experiments where, spatial associations were
observed in artificial associations of males in the lab. The techniques used were similar
to those used for female-female recognition (e.g. Shellman and Gamboa 1982). Using
slightly modified procedures, however, Shellman-Reeve and Gamboa (1985) conclude
that mates can recognize their male nestmates. Here we must perhaps distinguish
between ability to discriminate and actual discrimination. As suggested by Post and
Jeanne (1982) these species are probably not selected to inbreed and therefore males,
while still being able to distinguish between sisters and non sisters perhaps indis-
criminately inseminate them. Similarly if the best strategy for males in natural
situations is to ignore all other males, they are not likely to pair more (or less) often with
nestmates in the laboratory.
604 Raghavendra Gadagkar
In summary, studies on social wasps have shown that discrimination of nestmates
from non nestmates depends on learning of recognition cues from their natal nests.
Discriminations of different levels of genetic relatedness among nestmates has not so
far been investigated. What we do know of kin recognition therefore does not help
overcome the problems of low levels of relatedness caused by multiple mating for the
haplodiploidy hypothesis.
6.5 Vertebrates
In recent years recognition of kin, other than offspring, in the absence of locational and
other indirect cues has been demonstrated in several species of vertebrates. These
studies also appear to have begun approximately around the year 1979 when the kin
recognition abilities of ants and bees were first demonstrated (Green berg 1979; Jutsum
et al 1979). In many species of vertebrates there is evidence that totally naive individuals
reared in isolation from all conspecifics also appear to recognize siblings. Although we
are no longer concerned with the haplodiploidy hypothesis, our interest in kin
recognition in vertebrates stems from a very similar logic. Given similar ecological
conditions, altruistic behaviour can evolve more easily by kin selection if there is a high
degree of genetic relatedness between the interacting individuals. Animals may however
grow up with atleast some individuals who are not their full siblings (due to multiple
mating for example). If kin recognition depends on learning the characteristics of all the
individuals one grows up with coupled with an inability to distinguish different levels of
genetic relatedness among them, the average coefficients of genetic relatedness between
participants in social interactions will be relatively low. On the other hand, if kin
recognition depends on matching encountered animals with oneself (either through an
innate or learned knowledge about oneself) different levels of genetic relatedness can be
recognised. Thus altruism can be so distributed that the effective coefficients of genetic
relatedness between donor and recipient is relatively high. We will therefore once again
be concerned with the possibility of discrimination between levels of genetic relatedness
within a family unit.
6.5.1 Toads and frogs: Tadpoles of the toad Bufo americanus associate preferentially
with siblings in the laboratory. Waldman and Adler (1979) released marked tadpoles of
two different genetic lines in an indoor test pool and measured the positions of all
tadpoles in repeated trials. The experiment was repeated 6 times with different sets of
tadpoles and in each experiment the mean nearest neighbour distance between siblings
was significantly less than that between non siblings, indicating a preferential
association of siblings. In 37 % of the trials the mean coordinates of the two groups
were different indicating that the sibling groups separated out into different regions of
• the pool But it was not as if the two groups of tadpoles preferred different regions of
the pool due to any possible environmental gradients. They simply preferred to stay
away from non siblings and closer to siblings. This is inferred because the position of
the tadpoles kept changing from time to time. These experiments were subsequently
repeated by releasing the tadpoles in outdoor ponds which are the natural habitats of
the tadpoles. Here the sibship composition of 64% of all schools sampled were
significantly biased in favour of one siblings or the other, once again demonstrating the
ability of tadpoles to preferentially associate with siblings. There was again no evidence
Kin recognition 605
of preferences for any specific habitats and the preference was clearly for siblings per se
(Waldman 1982). Some light has been thrown on the mechanism of sibling recognition
by rearing groups of tadpoles either in isolation or along with non siblings. Tadpoles
reared together with siblings and non siblings together throughout their development
failed to distinguish between familiar siblings and familiar non siblings in laboratory
tests (Waldman 1981) although they appeared to be capable of doing so in field trials
(Waldman 1982). It is now clear that the failure to distinguish siblings from non-
siblings upon being reared together is not because of any convergence in recognition
characteristics (labels) but because of learning of the characteristics of the non siblings
by the experimental animals (Waldman 1985). On the other hand, tadpoles reared only
with siblings for the first 18 days of their development and later exposed to non siblings
successfully discriminated between familiar siblings and familiar non siblings in
laboratory tests. Besides, tadpoles reared in total isolation from all conspecifics
beginning prior to neural plate formation distinguished between unfamiliar siblings
and unfamiliar non siblings. They also distinguished full siblings from paternal half
siblings but not from maternal half siblings (Waldman 1981). It can be concluded from
these experiments that tadpoles are capable of sibling recognition even without the aid
of post-embryonic experience with conspecifics. However learning of the cues from
conspecifics during early development not only reinforces recognition but also
probably over-rides any ability innately present, acquired environmentally but pre-
embryonically or acquired by learning from self (we must say 'probably' because here
there is a discrepancy between laboratory and field experiments).
A number of very similar experiments have been conducted with tadpoles of the frog
Rana cascadae. Here the testing procedure involved recording the time spent by test
tadpoles in two different halves of a tank each holding different kinds of stimulus
individuals. The basic result is similar to that with the toad study; tadpoles prefer to
associate with siblings over non siblings (O'Hara and Blaustein 1981). With many
controls and different kinds of rearing regimes it has been shown that tadpoles (a)
reared with siblings, (b) reared with siblings and non siblings, (c) reared in isolation with
their egg jelly mass, (d) reared in isolation without their egg jelly mass and (e) reared in
isolation with egg jelly mass of non siblings all prefer full sibling over maternal half
siblings, maternal half siblings over paternal half siblings and the latter are preferred
over non siblings (Blaustein and O'Hara 1981; 1982; O'Hara and Blaustein 1981). These
results reinforce our conclusion that there may be more than one mechanism of sibling
recognition but the Rana cascadae study argues very strongly in favour of either an
innate ability to recognise siblings or an ability dependent only on learning
characteristics of oneself. More recent studies have shown that even adult Rana
cascadae frogs prefer to associate with siblings over non siblings (Blaustein et al 1984).
6.5.2 Bank swallows: Bank swallows (Riparia riparia) breed in large dense colonies
where errors in recognition of one's own burrow by young birds is not uncommon.
Under such situations parent swallows recognise and evict their neighbour's chicks by
means of what has been termed a 'signature' call given by the chick (Beecher et al
1981a, b). Siblings also appear to recognize each other by means of a similar call.
Beecher and Beecher (1983) have recently shown that chicks recognise siblings by giving
more calls in response to the recorded calls of their own sibling groups than they did to
the calls of unrelated groups. Chicks hand reared in isolated from all their conspecifics
but who had heard calls of unrelated chicks responded however to the familiar calls of
606 Raghavendra Gadagkar
the unrelated groups rather than to the unfamiliar calls of their own siblings. Here again
is an example of a learned recognition system but the critical data do not exist which ,
might tell us if a residual innate recognition capacity persists in the absence of learning
stimuli.
6.5.3 Mammals: Among mammals most work on kin recognition has involved
rodents: mice, rats, squirrels and voles. Kin recognition can clearly take place in the
absence of prior contact in white-footed deermice (Peromyscus leucopus) (Grau 1982).
These mice were tested in pairs for frequency and duration of behavioural interactions. ;
The mice investigated related but completely unfamiliar individuals (non litter mate
siblings who were non cage mates) significantly more often than they did unrelated
strangers (non siblings, non cage mates) showing evidence of kin recognition without
prior contact with the very individuals tested with. All mice however had considerable
social experience with their siblings both before and after weaning which must have f
provided them sufficient opportunity to learn the odours of at least some of their \
siblings. It is possible that they could later have used this information to discriminate j
between related and unrelated individuals as in the case of sweat bees (Buckle and |
Greenberg 1981). Similar results have been presented by Hayashi and Kimura (1983). In \
contrast Hepper (1983) ensured that at least some of his rats had no post natal j
experience with any related individuals. Preferences of the pups were tested in a T maze. \
When presented with an unrelated cage mate and an unrelated non cage mate, the pups i
preferred to associate with the unrelated cage mate suggesting that the characteristics of
the cage mate had been learnt postnatally. On the other hand when pups were given a
choice between genetically related but unfamiliar individuals and unrelated unfamiliar
individuals, they now preferred genetically related strangers over unrelated strangers.
In this case the discrimination had obviously been made without an opportunity for
postnatal learning of sibling odours. Since the pups were not given a choice between
familiar but unrelated individuals and unfamiliar relatives it is not clear whether any
one mode of acquisition of information regarding the characteristics of relatives (self
based or non self based) can be dominant over the other mode.
Gavish et al (1984) investigated sibling recognition in Prairie voles in the context of
incest avoidance. They showed that individuals, whether related or not, but reared
together, did not mate with each other while those not reared together mated whether
or not they were genetically related. In nature this species has been shown to exhibit
incest avoidance and the capability to do so is obviously due to postnatal learning
abilities.
In two studies the importance of innate (or self based learning) and non self based
learning were specifically investigated. In laboratory mice full sibs, half sibs and non
sibs differed significantly from each other in aggressive interactions, but all such
differences disappeared completely when the tested partners were familiar to each other
(Kareem and Bernard 1982). The authors conclude that the mice use familiarity as a
'rule of thumb' during interactions. Porter et al (1983) applied artificial odours like
musk oil, oil of clove, lemon lime and cherry to spiny mice pups. Pups who had a
particular odour applied to themselves alone or to themselves and their littermates
housed with them later reacted preferentially to unfamiliar animals treated with the
identical artificial odour, indicating both the importance of postnatal learning in. the
recognition process as well as the possibility of using one's own odour as a standard of
comparison. These results, however, are in contrast to earlier results where untreated
Kin recognition 607
littermates isolated for a comparable period of time displayed no evidence of
recognition (Porter and Wyrick 1979). It is somewhat difficult however to compare these
results with the other mammalian studies because according to Porter and Wyrick
(1979) spiny mice appear to be incapable of recognising unfamiliar siblings, quite in
contrast to all the other studies described above.
Ground squirrels have been subjected to an impressive, array of laboratory and field
experimentation by Holmes and Sherman (1982). Each baby squirrel was marked for
identification within about 3 hr of birth and from then was raised by either its biological
mother or its foster mother along .with some siblings and some non siblings. When later
tested for aggressive interactions, animals reared together are much less aggressive than
those reared apart. Among animals reared apart, biological sisters were more tolerant
of each other than non kin. These results suggest that both genetic relatedness as well as
rearing conditions affect recognition. In field studies there was clear evidence that
animals were more tolerant to and cooperative with their full sisters than with their half
sisters. Here full and half-sisters were reared together and genetic relatedness was
assessed electrophoretically. As the authors have noted, there is an apparent
contradiction between lab and field studies in whether different levels of genetic
relatedness can be distinguished within a group being reared together, and this is the
crucial question we have been interested in throughout this survey of the literature.
Holmes and Sherman (1982) believe that their field tests are more sensitive and
therefore that full and half sisters can be distinguished inspite of being reared together.
The study most cited as an illustration of kin recognition, not only in the absence of
an opportunity to learn the characteristics of siblings or other relatives, but one which
persists even if the experimental animal grows up entirely with unrelated conspecifics is
that of Wu et al (1980). Sixteen infant pig tail monkeys (Macaca nemestrina) were
separated from their dams within 5 min of birth and reared separately while allowing
for social interaction with unrelated conspecifics for several hours a day. When tested
subsequently these monkeys preferred to associate with their paternal half siblings over
unrelated individuals. These results suggest that the monkeys were capable of
distinguishing between related and unrelated animals without any prior experience
with relatives and indeed inspite of prior experience with unrelated individuals.
Fredrickson and Sackett (1984), however, appear not to have been able to reproduce
these findings. Notice that the results of Wu et al (1980) in contrast to Buckle and
Greenbergs' (1981) sweat bees, Breed's (1981) honey bees, Polistes wasps (Gamboa and
colleagues, referenced above), Beecher and Beecher's (1983) bank swallows or Kareem
and Bernard's mice squirrels where subjects preferred relatives of those individuals
whose characteristics they had learned or those individuals emerging from nests whose
characteristics they had learnt.
MacKenzie et al (1985) have studied the effects of companionship, kinship and
rearing in social preferences of stump-tailed macaques (Macaca arctoides) using a
qualitatively different set of techniques. A whole range of social interactions were
observed as they occurred naturally in a heterogeneous group of 26 monkeys occupying
a large enclosure. The monkeys varied widely in age, kinship and rearing conditions.
Data on social interactions were subjected to partial correlational analysis. The results
suggest that familiarity was the most important variable affecting social preferences.
This was followed by correlation with kinship and very interestingly, kinship through
the father was important but not through the mother. These results are in broad
agreement with studies of other vertebrates suggesting an ability to recognise
608 Raghavendra Gadagkar
kinship per se, but a strong masking influence of familiarity. As the authors note
familiarity is probably sufficient to recognise matrilineal kinship (as the offspring of a
female will grow up together) but natural selection appears to have favoured a special
mechanism (not based on familiarity) to recognise patrilineal kinship. This is
reminiscent of the ground squirrel study (Holmes and Shermann 1982; Holmes 1984)
where, recognition abilities appear only at about the time that offspring are old enough
to emerge from their natal burrows, move about and get mixed up. This is true both
for the mother's ability to recognise her offspring as well as sibling recognition by the
offspring itself.
There is some evidence that humans too are capable of assessing degrees of genetic
relatedness amongst themselves. Dizygotic or fraternal twins are genetically no
different from any pair of siblings but are likely to have shared a very similar
environment during embryonic development. While the average genetic relatedness
between co- twins would be 0.5, any pair of same sex twins could share from 1 to 46
chromosomes in common. The exact degree of genetic relatedness between a given pair
of twins can be determined by the analysis of a large number of blood group factors. In
a couple of rather fascinating studies it was found that the degree of genetic similarity as
revealed by blood group analysis was significantly positively correlated with similarity
in physical appearance as rated by the twins themselves, their mothers or other
observers (Pakstis et al 1972; Carter-Saltzman and Scarr-Salapatek 1975). In retrospect
this result is not so surprising after all. Some human siblings appear so similar to the
casual observer that it is impossible not to guess their relationship. On the other hand,
we all have remarked at one time or another that it would have been impossible to guess
that certain pairs of individuals were siblings unless we were told of the fact. That same
sex siblings can vary in their genetic relatedness by as much as from 1 to 46 shared
chromosomes appears to be manifested in the widely varying degrees of similarity in
physical appearance apparent even to the casual observer. We do not however know if
this relationship between genetic relatedness and physical similarity is actually used by
humans in recognition of unfamiliar relatives. Besides, our logic would be of course be
in some trouble if all the genes controlling physical appearance (especially of facial
features) were clustered on a single or a very small number of chromosomes but there is
no evidence for or against this.
While sight may be more important than smell in the lives of humans we may be using
smell in subtle ways not obvious without carefully controlled experiments. Such an idea
is reinforced by a recent study showing that human mothers are capable of telling their
infants apart from other infants by means of smell alone if they have been allowed to
interact with their infants only for half an hour immediately after birth. Clearly here is
an imprinting like phenomenon. Interestingly enough, fathers were unable to show any
such capacity (Russell et al 1983).
7. The mechanism of recognition
There has been an explosion of studies on kin recognition in the last 5 years. Different
studies use different methods to assess animals' abilities to discriminate kin from non
kin and use a variety of different conditions for rearing experimental animals. Wilson
(1986) provides a glossary that helps to face up to a concomitant explosion of
terminology used by researchers in this field. There is also much discussion of
Kin recognition
609
RECOGNITION
Individual
or
group
recognition
Labels
environmentally
acquired and
templates
Self based or
Non self based
Labels
genetically
specified
and self produced
Template
non self based
True
recognition
of
genetic
relatedness
i
Labels genetically
specified but
have to be
acquired from
other animals
nest etc.,
Template
non self based
Figure 4. Facets of kin recognition.
In order for kin recognition to take place every animal must carry a label on its body and a
template in its brain. Animals must examine the labels of any individual encountered and
compare them with the template in their brains to determine the extent of relatedness between
themselves and the individual encountered. The labels may be genetically specified or
environmentally acquired. Genetically specified labels may be produced by each animal or
some animals may acquire them from other animals (eg. from queens in social insect colonies),
nests etc. Templates may be self based (either innate or dependent on learning from oneself) or
non self based (dependent on learning from individuals other than oneself).
genetically determined versus learned or acquired abilities to recognise kin. For a
discussion of quantitative genetic models for kin recognition see Crozier (1986). To
facilitate comparison between different studies I suggest that we view the phenomenon
of kin recognition in the following general frame work (figure 4).
7.1 Group or individual recognition versus recognition of genetic relatedness per se
This is an important distinction which is not always made. Group recognition by means
of colony specific odour has long been well known in social insects such as ants and bees
(Wilson 1971; Michener 1974). It is only in recent times however that the capabilities of
ants and bees to assess actual genetic relatedness have become evident (Greenberg
1979; Jutsum et al 1979; Breed 1981).
610 Raghavendra Gadagkar
7.2 Distinction between labels and templates
All that we know about kin recognition abilities of animals is consistent with the
following scheme. In order to make kin recognition possible every animal should carry
a label (or set of labels) on its body and a template in its brain. It should then compare
any animal encountered to the template in order to decide if the label is similar to the
template and therefore if the encountered animal is related to it. Although this scheme
and terminology are used by some (Lacy and Sherman 1983; Holmes and Sherman
1982; Sherman and Holmes 1985; Waldman 1985; Getz 1982; Breed and Bennett 1986)
the distinction between labels and templates has not always been made in discussing kin
recognition abilities of animals.
7.3 Labels
Since most experimental results suggest olfaction as the sensory modality involved in
recognition we shall refer to the labels as odours although a label could just as well be a
visual label as it probably is in the human twin studies (Pakstis et al 1972; Carter-
Saltzman and Scarr-Salapatek 1975) or an auditory label as in the case of bank swallows
(Beecher and Beecher 1983). Unless labels used in recognition are genetically specified
true recognition of genetic relatedness cannot occur. Entirely environmentally acquired
labels can only be used in group or individual recognition. It is conceivable however
that insects living in large colonies may use environmentally acquired labels for group
recognition and thereby maintain colony integrity without having the ability to assess
genetic relatedness. This is why much effort should go into discerning between group
recognition and recognition of genetic relatedness (eg. Kalmus and Ribbands 1952;
Boch and Morse 1979). Labels can be produced directly by the metabolic machinery of
an animal or it can be acquired either from other animals (who have produced it by their
metabolic machinery) or from the products of other animals (fecal matter, nests built by
other animals etc.). These two possibilities were explicitly contrasted by Crozier and
Dix (1979) who considered two kinds of models. In the 'individualistic' model each
colony member is expected to 'retain its pheromonal integrity with no significant
transfer of colony odour pheromones between colony members'. This clearly appears
to be the case in the acasia ant studied by Mintzer (1982), Mintzer and Vinson (1985) as
well as in Waldjnan's (1985) tadpoles. In contrast the 'gestalf model supposes transfer of
colony odour pheromones between different members of a colony by grooming and
trophallaxis so that each individual responds to a common gestalt odour. A case
intermediate between the gestalt and individualistic models is that where the queen is
the source of the label which the workers acquire. This certainly seems to be the case in
some ants (Carlin and Holldobler 1983; Carlin N F and Holldobler B, unpublished
results). In most studies one cannot really distinguish between each animal producing
its own label and labels being acquired from others because of the lack of distinction
between labels and templates. Taking the case of the Polistes wasps, for example, in the
experiments described so far, we do not know whether the wasps that were not exposed
to their natal nests lacked the labels or templates or both. If they lacked labels then we
may conclude that the labels must be acquired from the nest.
Getz (1981, 1982) has considered genetic models for the production of a sufficient
diversity in labels to provide for recognition of genetic relatedness. Applying these
Kin recognition 611
models to existing data on Lasioglossum zephyrum they suggest a genetic labelling
system of 4 or 5 loci with 2 to 3 alleles at each locus. Only if labels are individualistic
(self produced, figure 4) and not 'gestalf or acquired from a common source such as the
queen or nest (figure 4) will it be possible for animals to recognise different levels of
relatedness within a hive or family unit. From the point of view of the haplodiploidy
hypothesis or kin selection this distinction is thus essential.
7.4 Templates
Are templates the products of learning or are they somehow innately specified? This
seems to be a question of great interest (Holmes and Sherman 1982, 1983; Sherman and
Holmes 1985). The idea of innately specified templates by means of recognition alleles
was first suggested by Hamilton (1964b). Dubbed as the 'green beard' effect by Dawkins
(1976) in his inimitable style, the idea simply is that we need to postulate a gene that
makes its bearer not only have a 'green beard* but also program it to aid all individuals
in the population possessing 'green beards'. Such genes have been repeatedly
considered highly improbable (Hamilton 1964b; Alexander and Borgia 1978; Dawkins
1976; Holmes and Sherman 1982). In contrast Alexander and Borgia (1978) and Lacy
and Sherman (1983) have suggested 'phenotype comparison or matching' mechanisms
with a learned component. Lacy and Sherman (1983) have modelled situations where
an 'observer' assesses its genetic relationship to the 'observee' by means of 'templates'
determined by 'referants' where the referants could be known relatives such as a parent
or the observer itself. Given this definition of phenotype matching it cannot really be
disproved (Blaustein 1983). The results of even the most carefully controlled
experiments where learning of cues from relatives is ruled out (e.g. Blaustein and
O'Hara 1981, 1982) can be construed as phenotype matching where individuals use
themselves as 'referants' (see Holmes and Sherman 1982).
Holmes and Sherman (1982, 1983) and Sherman and Holmes (1985) distinguish 4
mechanisms of kin recognition namely, spatial distribution, association, phenotype
matching and recognition alleles. Kin recognition based on predictable spatial
distribution or predictable patterns of association are widespread and well-known
(the relevant literature is reviewed in the just mentioned three papers). It is only in
recent years that kin recognition in the absence of spatial and associational cues has
become apparent. All known cases of such recognition are lumped under phenotype
matching by these authors because recognition alleles have hitherto been defined in a
way that precludes their search and possibly their very existence! I will argue here that it
is useful to distinguish between two types of templates: (i) Self based templates where
the templates do not have to be learned or they can be learned from oneself without the
intervention of any other individual and (ii) non self based templates where the
templates are learned from individuals other than oneself or even from some structure
such as the nest as in some social insects. Once an individual produces a chemical label
by means of its own metabolic machinery then, whether it releases this substance to the
surface of its body, smells itself and then produces a template or whether a template is
produced without it having to smell the surface of its body postnatally is perhaps not a
terribly important distinction at the moment. Whether an animal acquires a template
by smelling other individuals in the population (non self b^sed) or whether the template
is produced without the intervention of any other individuals (self based) is perhaps the
612 Raghavendra Gadagkar
more important distinction. These two different mechanisms could drastically affect the
abilities of animals to recognise levels of genetic relatedness. Consider for example the
different lines of daughters in a honey bee hive with a multiply mated queen. The
workers could get habituated to both their full and half sisters and acquire a template
that prevents them from discriminating between full and half sisters as seems to happen
in the case of sweat bees (Buckle and Greenberg 1981). If on the other hand each worker
acquires a template by the action of its own alleles then even within a hive with a
multiply mated queen, workers can selectively aid their full sisters. The effective genetic
relatedness between a worker and the beneficiary of her altruism could thus be as high
as 0-75, thereby drastically altering the conditions for the evolution of sociality by kin
selection.
In this framework of the two kinds of templates there is evidence of self-based
templates in honey bees (Breed et al 1985; Getz and Smith unpublished results; Noonan
K C unpublished results) tadpoles of frogs and toads (Waldman 1981; Blaustein and
O'Hara 1981, 1982; O'Hara and Blaustein 1981), rats (Hepper 1983), pig tailed monkeys
(Wu et al 1980) as well as ground squirrels (Holmes and Sherman 1982). An example of
the absence of self based templates and the need for non self based templates is the
study of sweat bees by Buckle and Greenberg (1981). Several studies suggest a
combination of self based and non self based templates. Indeed, most studies cited
above as examples of self based templates are in fact instances of a combination of both
kinds of templates. Notice that familiar individuals are almost always treated as kin.
Given this fact what we need to be concerned about is how the self based and non self
based templates are weighted. If self based templates are dominant over non self based
ones then recognition of different genetic lines is possible within a mixed hive or family.
This is what appears to be happening in the cases of ground squirrels (Holmes and
Sherman 1982) and Raw cascadae tadpoles (Blaustein and O'Hara 1981, 1982; O'Hara
and Blaustein 1981), On the contrary non self based templates appear to override any
self based templates in sweat bees (Buckle and Greenberg 1981), Bufo americanus
tadpoles (Waldman 1981), and laboratory mice (Kareem and Bernard 1982). In
summary, what future experimental work should focus on is whether templates are self-
based or non self based and if a mixture of the two kinds, whether the two templates are
stored separately (as appears to be the case in honey bees, Breed 1985; Getz W M and
Smith K B, unpublished results; Noonan K C unpublished results) and also whether the
two templates can be differentially weighted during interaction. I suggest that we need
not concern ourselves with whether true recognition allels exist and whether true
genotypic comparison occurs simply because, these questions are not experimentally
tractable. It has been hard enough to understand whether animals come with fully
specified, hard wired knowledge of some features of the external world or they need
some experience for complete specification, without getting lost in an irresolvable
nature-nurture controversy. Understanding whether features of the animal itself can be
hard wired can only be much worse.
7.5 An experimental approach to distinguish between labels and templates
Let us now consider a specific example to illustrate an experimental approach to
discriminate between labels and templates. For the purposes of illustration let us use the
experiments of Shellman and Gamboa (1982) with Polistes as our paradigm [although
Kin recognition
Experienced Experienced
Recognition
613
I
©
Recognition
Naive
/-—^ No recognition
Hf A \ """v
Naive Either labels not
^-^ self produced or
( A J ^
( B j Template
— No recognition
non self based
or both
Experienced
©Recognition
^
Naive Labels genetically
©specified and self
No recognition
produced but
Template
non self based
Experienced
©No recognition
Naive Labels not self
©produced but
r
Recognition
Template
self based
Experienced .. ...
*-* No recognition
Naive Labels not self
^^ No recognition
Template not
self based
Naive
^_^ Recognition
UT /T^N x,
Naive Labels genetically
-^ specified and self
VI ( ) x
/ £j j oroduced and
^-^ Recognition
\v__X
Template
self based
Figure 5. An experimental approach ta distinguish between the roles of labels and templates
in kin recognition.
A and B are two animals (say, wasps) who may or may not recognise each other as close
genetic relatives depending on their rearing conditions. Based on this one can infer the
ontogeny of the labels and the templates. See text for details.
Gamboa et al (1985) have recently used a different approach to address these questions
and provided some additional information — see below]. Consider two wasp nestmates
A and B (figure 5). We shall refer to the animals exposed to their natal nests during early
life as 'experienced' and those animals isolated immediately after emergence from their
natal nests as 'naive'. Let both animals A and B be able to recognise each other when
both are experienced (figure 5, panel I). Let neither animal however be able to recognise
the other when both are naive (panel II). This suggests that either the label is acquired
after eclosion from the nest or the template is not self based or both. But notice that
only one of these may be true. Consider now an extension of the experiment where
animal A alone is experienced while animal B is naive. Now if animal A recognises B but
B does not recognise A (panel III) we can conclude that A must have a normal template
and B must have had a normal label. Since B did not recognise A this must mean that B
did not have a normal template because A, being an experienced animal, must
614 Raghavendra Gadagkar
necessarily have had a normal label. In other words it is the template that must be non
self based while the label must have been innately specified and produced by B itself.
This is because B is a naive animal. On the contrary consider a situation where A, the
experienced animal, fails to recognise B but B, the naive animal, recognises A (panel IV).
In this case we must conclude that A, being an experienced animal, must have had a normal
template and therefore B lacked a normal label. B however must have had a normal
template since it recognised A. In other words it is the label that must be acquired from
the nest while the template is self based. If neither A nor B can recognise each other
although one of them is experienced (panel V) then we must conclude that the label has
to be acquired from the nest and that the template is not self-based. Finally, it is possible
that even when both A and B are naive they may be able to recognise each other (panel
VI) in which case labels must be self produced and templates must be self based. It
should be possible to devise such experiments and understand the distinction between
the possible ontogenies of labels and templates. Lack of distinction between labels and
templates can potentially lead to erroneous conclusions. For instance Shellman and
Gamboa (1982) showed that wasps exposed to their nestmates alone did not acquire the
capacity to recognise nestmates. They therefore concluded that exposure to nestmates
is neither a sufficient nor a necessary condition for the development of kin recognition
abilities (Pfennig et al 1983a). But this conclusion will depend on the history of the
animals that were used for exposure. Consider a situation where labels have to be
acquired after emergence, which is certainly possible in the Polistes studied by them.
Now if the animals used for exposure were themselves naive ones then they would not
have acquired the required labels. On the other hand, if experienced animals were used
for the exposure, then exposure to such experienced animals may be sufficient for other
animals to acquire recognition abilities.
Recently Gamboa et al (1985) have used a different approach to address the same
questions. Assuming that the templates are non self based (which is suggested by their
earlier experiments) they set out to ask if the labels are self-produced or acquired from
the nest. Taking wasps from two unrelated nests they exposed the animals not to their
own nests but to each others nests. Now if labels are acquired from the nest (and
templates are any way assumed to be learnt from nests) the animals should have labels
and templates that match with each other (although corresponding to the unrelated
nests). Two animals coming from a nest when exposed to the same alien nest should
treat each other tolerantly. On the other hand, if labels are genetically specified and self
produced then each animal should have mismatched labels and templates and be
intolerant of each other. Since the results happen to be intermediate between these two
possibilities the authors assumed that both endogenous odours as well as odours
acquired from the nest are involved.
7.6 A possible genetic basis for recognition labels
True recognition of genetic relatedness must involve genetically specified labels that
vary in a quantitative fashion between animals of different levels of genetic relatedness.
This suggests a highly polymorphic multi locus system. A search for such a genetic
system no longer appears like looking for a needle in a haystack with the recent
demonstration of the role of the histocompatibility system in kin recognition. All
multicellular animals, especially vertebrates, have a well developed immunological
Kin recognition 6 1 5
system to prevent the invasion of their bodies by foreign cells. It is this histo-
compatibility system that frustrates transplantation of organs from one individual to
another. The body's immune system unfailingly distinguishes between self and non self
tissue by means of a set of antigenic molecules commonly referred to as transplantation
antigens which are present on the surface of all cells. As this implies, the exact nature of
the transplantation antigens present on the cells of any two individuals are different
from each other unless of course the individuals are identical twins. There is
considerable information on the genetic basis and Mendelian inheritance of the genes
coding for the transplantation antigens. A large number of transplantation antigens
coded for by an equally large number of genetic loci have been identified. Of the many
gene complexes, the one known as the major histocompatibility complex (MHC)
(designated as the H-2 in the mouse and the HLA in man) dominates the body's
reaction to a graft. The MHC is a rather complex and highly polymorphic set of loci (see
Roitt 1980 for an overview).
Recent work has unravelled another profound and rather surprising function for the
MHC. The H-2 locus in the mouse appears to produce genotypically variable odour
components on the basis of which mice can potentially assess their genotypic similarity
with other conspecifics (see Jones and Partridge 1983 for an interesting commentary
and Beauchamp et al 1985 for a non technical account). We know this from two kinds of
experiments. Firstly, by various tricks strains of mice have been bred which differ from
each other almost exclusively in the H-2 locus. Using such strains it has been
demonstrated that mice can be trained to distinguish specific H-2 types by scenting the
arms of a Y maze with the urine of an appropriate mouse (Yamazaki et al 1982). The
idea that the H-2 loci produce distinctive odours which enable mice to distinguish one
another is also supported by the observation that males of a certain H-2 type largely
prefer to mate with females of alternative H-2 types (Yamazaki et al 1976). The second
kind of experiment involves the use of the well known Bruce effect or 'pregnancy block'.
If pregnant mice are exposed to 'strange' (males different from the one they have mated
with) or even the urine or bedding of 'strange' males within the first 6 days of pregnancy,
a neuroendocrine imbalance results leading to abortion of the embryo. This
experimental situation has been utilized to show that the frequency of pregnancy block
is higher if the 'strange' male is of a different H-2 type compared to the stud male (the
original male with which the female was mated). Similar results were obtained with
'strange' females differing in H-2 type from the stud male although to a lesser extent
(Yamazaki et al 1983). This once again suggests a role for the MHC in chemosensory
recognition.
Another rather spectacular result that we should mention here is the recently
discovered role of the t locus in determining mating preference in mice. The t locus is a
highly polymorphic locus closely linked to the H-2. Most of the alleles at this locus are
recessive lethals in spite of which a considerable amount of polymorphism is
maintained in natural populations. It has been established that due to segregation
distortion heterozygous males produce about 95 % t-bearing sperm. There has been
considerable interest in the t locus because the frequency of t alleles in natural
populations is higher than would be expected on the basis of their lethality but less than
would be expected after taking segregation distortion into account. It is now known
that female mice given a choice between wild type males and males heterozygous for the
t locus preferred to mate with the wild type males. (Levine et al 1980; Lenington 1983).
The adaptive significance of this behaviour is easy to see because a wild type female
616 Raghavendra Gadagkar
mating with a heterozygous male would produce 50 % heterozygous offspring and
could therefore potentially have some inviable grandchildren. On this argument a
female who is herself heterozygous would be much worse off mating with a
heterozygous male because she would then have 50 % heterozygous offspring and 25 %
inviable offspring (homozygous for the lethal allele).
Females who are themselves heterozygous show a much stronger avoidance of
heterozygous males when given a choice of mating with wild type and heterozygous
males (Lenington 1983). Similarly males also prefer to mate with wild type rather than
heterozygous females (Lenington 1983). These results suggest that the mice are capable
of assessing their own as well as their potential mate's genotype at a single locus. There
is evidence that this assessment is also on the basis of chemosensory perception of
odours in the urine (Lenington 1983). It is unlikely that animals would be able to make
such assessment at every genetic locus and these results were obtained probably because
of the close linkage of the t locus to the H~2 locus. The H-2 is a highly polymorphic
locus which is well known to produce sufficient genetic diversity in cell surface
glycoproteins (the histocompatibility antigens) to permit recognition of self versus non
self at the cellular level. Perhaps the H-2 locus also produces a similar diversity of
odourous molecules that permits recognition of self versus non self at the behavioural
level.
8. The physical basis of recognition
Today we understand rather little regarding the physical basis of kin recognition. That
olfaction must be involved in most cases had however been suggested quite early. Most
recent studies have confirmed this (except possibly in the case of birds where
recognition could be acoustic and humans where recognition may be visual). Olfaction
also appears to be the most suitable sensory modality to combine metabolically
produced and environmentally derived cues in recognition, as many insects appear to
do. The most precise statements regarding the basis of kin recognition have been made
by Holldobler and Michener (1980) who have coined the terms 'discriminators' or
'recognition pheromones' for 'the odour Signals that differ among individuals in a
population' but 'not of extrinsic origin'. It has also been hypothesised as discussed by
Holldobler and Michener (1980) that the recognition pheromones consist of several
active components. What is believed to make a particular pheromone unique is not only
its qualitative composition but also the concentrations of its different constituents
(Barrows et al 1975). The resultant economy in producing and detecting pheromones
under such a scheme is obvious. The properties of several known pheromones are
clearly consistent with this idea (see for instance Cammaerts et al 1981).
There is one more aspect of the recognition system that we already know and that is
that an imprinting like phenomenon is involved. Notice that while this strongly
suggests a role for learning in kin recognition it does not rule out 'recognition alleles'.
Animals might have to smell themselves and get imprinted on their own odour before
acquiring the capacity to recognise kin. A recent neurophysiological study with Norway
rat pups reinforces the idea of odour imprinting. Experimental Norway rat pups were
exposed to peppermint odour during early postnatal development while control pups
were exposed to clean air. The experimental pups showed an enhanced olfactory bulb
response to peppermint odour as measured by radioactive glucose uptake, compared to1
Kin recognition 617
the control pups (Coopersmith and Leon 1984). It seems likely that a similar
phenomenon may be involved in kin recognition. The idea that a developing animal
forms olfactory representations in the nervous system which serve as templates to
which incoming odours are later matched has already been suggested in the context of
olfactory preferences in animals (Freeman 1981).
Apart from the involvement of olfaction and an imprinting like phenomenon we
know scarcely little else. It should however be possible to begin to identify the
recognition pheromones. Given that several complex pheromone systems have been
identified it is well within the present technological capabilities to chemically identify
recognition pheromones. One expects this to be a particularly exciting area of research
in the coming years. Our knowledge of the basis of kin recognition thus seems to be
poised for a quantum leap.
Acknowledgements
This paper has benefitted greatly from a very critical reading of an earlier draft by
Madhav Gadgil, N V Joshi, M S Shaila, Ramnath Sasisekaran, C D Michener, G J
Gamboa, M D Breed, W M Getz, C K Starr, R L Jeanne, R H Crozier, John Wenzel, Bill
Wcislo and two anonymous reviewers. I thank all of them for their time and patience. I
am also grateful to E O Wilson, W D Hamilton, R H Crozier, K C Noonan, M D Breed,
G J Gamboa, B Holldobler and W M Getz for their generosity in sending me their
unpublished manuscripts or those in press. V B Swarnalatha, Padmini Nair, C Vinutha,
Arun Venkataraman, K Muralidharan and K Chandrashekar provided a stimulating
atmosphere in which many of the ideas in this paper were discussed over and over again.
My wife Geetha helped substantially in the preparation of this paper. Supported in part
by a grant from the Department of Science and Technology, Government of India.
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© Printed in India.
Tradeoffs in the evolution of frog calls
A STANLEY RAND
Smithsonian Tropical Research Institute, Balboa, Panama, Central America
MS received 20 April 1985; revised 2 August 1985
Abstract. All biological characteristics are subject to conflicting selection pressures. This is
particularly true of those characteristics that are subject to sexual selection. The classic
example is the peacock's tail. Others are the calls used by male frogs and toads to attract their
mates. The forces which have acted in the evolution of these calls are varied and the calls that
we hear made by these animals are diverse.
Two kinds of factors can be recognized: constraints and forces. Constraints on the kind of a
call that a frog might evolve include its phylogeny, the energy required to produce different
kinds of calls, the risks incurred from attracting predators. Also important is the morphology
of the frog: both the structures used by the males to make the calls and the apparatus with
which the females hear the calls. For example, frog size has an important influence both on the
frequencies of the sounds that a frog produces and the acuity with which they are heard.
Both passive and active selective forces can be identified. Passive forces include the distances
that environments transmit sounds of different frequencies and the interference from other
sounds that calls encounter. Active forces include the reactions of conspecific males and
females to the calls. Males interact acoustically in a variety of ways to organize their choruses in
both space and time. They position themselves, and time their calls so that they minimize the
interference from other males while maximizing their chances of securing a mate. Female
choice has been studied in test arenas. Females choose louder calls, calls that are most easily
located, and the calls of their own rather than other species. In choosing among males of their
own species, females have been shown to pick the males controlling the best resources,
sometimes using calls to do so. They should also be expected to choose those males who can
contribute the best genes to their offspring. The extent to which they do this and the role of
calls in choosing is actively being argued.
Sexual selection, both interactions between males and female choice, have undoubtedly been
important in the evolution of frog calls but only within the constraints imposed by a variety of
other factors.
Keywords. Amphibian; anuran; frog; communication; vocalization; call; sexual selection;
evolution.
1. Introduction
All biological characters are subject to conflicting selection pressures and the states that
one sees represent points where opposing pressures balance. This is particularly evident
in those characters in which sexual selection is involved; the classic example is the
peacock's tail — frog calls are another.
Here, I will consider the various forces that shape the signals used in the long distance
vocal communication of frogs and toads. A frog call is as we hear it not because there is
no selection for it to change but because a change that might improve it in one way
would make it less effective overall.
Standard English makes a distinction between frogs and toads. In this paper I will use
frog as a general term for the Anura, including in this category the toads, the true frogs,
the treefrogs and all of their varied relatives.
623
624 A Stanley Rand
2. Vocalizations
Here I will be considering only advertisement or mating calls, one of the several kinds of
vocalizations that frogs use. These are the calls used to communicate at long distances.
They are given by males and serve to advertise their location, to attract receptive
females and commonly, to organize the chorus of males. Females are not known to give
advertisement calls.
There have been several review papers on frog communication, among them are:
Arak (1983a), Bogert (1960), Blair (1964, 1968), Littlejohn (1977), Straughan (1973) and
Wells (1977a, b).
Frog calls are diverse; no two species have exactly the same call, but they are not
nearly as diverse as are bird calls nor so complex as some of them are. A few examples
will illustrate the diversity of frog calls. Most calls are between 400 and 4000 Hz. Some
are high in frequency, others are low. All have a relatively simple frequency structure;
some are tonal, others are noisy. Some calls are frequency modulated usually with a
simple sweep up or down. Calls also differ in their timing. Some are long, others short.
Calls may be amplitude modulated with a simple change in loudness, modulated into
regular pulses, or more complexly patterned. Some calls have a single note, others
several identical notes, still others a long introductory note followed by one or more
shorter notes. Among the most complex frog calls are those that combine notes that
differ in several aspects as do the notes of the Physalaemus pustulosus advertisement call
(Rand 1983; Ryan 1985).
Frog calls seem to be genetically determined. Males of two closely related species of
Hyla, raised in isolation from tadpoles to adults, gave the appropriate species specific
call regardless of whether they were exposed to the calls of their own or of the other
species (Burger 1980, cited in Gerhardt 1982). Evidence that female call preference is
also inherited comes from experimentally produced hybrids between two species of
Hyla (Doherty and Gerhardt 1983). The hybrid males produced intermediate calls.
Female hybrids preferred the synthetic calls with the pulse-repetition rate of the
hybrids to synthetic calls with pulse-repetition rates of either parental species.
3. Constraints and selective forces
One can recognize two sets of factors that have been important in the evolution of frog
calls. One set includes the forces that select for calls that are more effective in their
social/sexual function, for example, the selection that would exist for low frequency
calls in a population in which females always preferred the lowest frequency call that
they heard. The other set are the constraints that seem to limit the effectiveness of the
first set, for example the fact that larger structures vibrate at lower frequencies. Call
evolution involves compromises and tradeoffs between and within both sets of factors.
3.1 Constraints
Constraints are imposed on frog calls by such things as: phylogenetic relationships,
energetic costs of calling, risks of predation, and morphology, particularly the size and
physical properties of the sound producing apparatus of the male, and the receptor
Tradeoffs in the evolution of frog calls
625
system of the female. These constraints do not specify what the call of a specific frog will
be, but they do specify that certain things are more difficult and unlikely to evolve than
are others.
3.2 Phylogenetic constraints
After reviewing the influence of phylogeny on frog calls, Schi0tz (1967, 1973) concluded
that between subspecies within a species there are sometimes differences; between most
Table 1. Groups within the genus Physalaemus.
Call groups*
Morphological groups**
A — long descending sweep
biligonigers
fuscomacu latus
riograndensi
albonotatus,
jordanensis
gracilis
B-— long rising sweep
fernandezae,
C — short descending sweep
santafecinus '
pustulosus
signiferus
aquirrei
cuvieri
D — noise
nattered
obtectus
maculiventris
centralis
henseli
-toadlike trill
cicada
1 — biligonigerus group
— biligonigers
— fuscomaculatus
santafecinus
— pustulosus group
freibergi
paraensis
pustulosus
pustulatus
schereri
stentor
3 — signiferus group
signiferus
olfersi
nanus
obtectus
maculiventris
. — cuvieri group
riograndensis
albonotatus
'jordanensis
gracilis
'fernandezae
aquirrei
cuvieri
enesefae
centralis
henseli
cicada
albifrons
barrioi
ephippifer
evangelistai
koeyeri
soaresi
ternetzi
*Barrio (1965, 1967)
** Lynch (1970)
626 A Stanley Rand
species within a genus there are clear differences, and in most cases there are similarities
between the species in one genus that separate them from species of other genera; on the
family level it is impossible to find characters common to the genera in one family, that
are not shared by genera in other families.
One way to evaluate the phylogenetic constraints on the evolution of calls is to
compare the calls of frogs within the same lineage with those from differing lineages.
As an example, table 1 shows the five types of calls that Barrio (1965, 1967) defined
within the genus Physalaemus and the four groups into which (Lynch 1970), in his
revision of the genus, arranged the species, on morphological grounds. The lack of
correspondence between the two groupings is striking. Phylogeny does not seem to
constrain this system in a simple way. However, the same call types do recur in related
species. There do seem to be substantial phylogenetic constraints and major changes in
call do not occur often.
3.3 Energetic constraints
A single frog call takes only seconds, at most, and a small amount of energy. Some frogs
like the Panamanian forest species, Eleutherodactylus fitzingeri, may give only one or
two calls in a night, but others calling nearby, like Physalaemus pustulosus may give
5,000 calls on the same night.
Physalaemus pustulosus males can be persuaded to call inside a respirometer (Bucher
et al 1982). The energetic cost of a single call is small; an average of 0-024 joules. They
will also nest in a respirometer and using these data energetic costs for a whole season
can be estimated. Though a male calls many times in a night for a number of nights, he
does not use as much energy in reproduction in a season as does a female (Ryan et al
1983). But he may be using all the energy available to him. Calling not only uses energy
but it takes time that a male would have to spend feeding. In two species of Australian
leptodactylids in the genus Ranidella, males have been shown to loose about 35% of
their dry body mass in the course of a breeding season; between seasons, males gather
enough energy to make up this loss but not enough to grow much (MacNally 1981).
Thus, in at least some frogs, there is a tradeoff between expending energy in calling now,
or using it for growth. Bigger males, as we shall see, are generally better at securing
mates but a male that postpones calling in favor of growth risks dying without trying to
breed at all.
3.4 Risk from predators
A frog, when it gives an advertisement call, takes a risk of attracting a predator.
During the 7 nights that Ryan et al (1981) watched a Physalaemus pustulosus chorus
they saw predation by crabs, opossums, bigger frogs and bats. These bats, Trachops
cirrhosus, Tuttle and Ryan (1981) showed, were using the frog's calls to locate them.
Ryan (1985) lists several ways that a male frog might increase its probability of
mating: call more intensely; more frequently; use call types that are more attractive to
females; call longer; and remain at the calling site during disturbances. All of these
probably also increase the risk of predation, so that bat predation is a selective force
opposing the action of sexual selection. This tradeoff between probability of success
Tradeoffs in the evolution of frog calls 627
and risk of predation is an important one and we are just beginning to recognize the
kinds of behavior that frogs seem to have evolved as antipredator tactics. One such is
seen in the neotropical hylid, Smilisca si/a, that does most of its calling on moonlight
nights when the frog-eating bats are least active (Tuttle and Ryan 1982).
3.5 Morphological constraints
The morphology of the apparatus that a male uses to produce his advertisement call,
and the auditory apparatus with which conspecifics hear it, both may influence the
nature of the call.
3.6 Emitter
3.6.1 Size and frequency: A call is produced when air from the lungs is forced
through the larynx into the vocal sac, causing the vocal cords to vibrate. The frequency
of the vibrations is determined by the size of the vocal cords, their structure, and their
tension (Martin 1972). The vibrations of the vocal cords seem to excite the air filled
vocal sac to resonate so that some frequencies are emphasized. The frequencies at which
a system resonates would be expected to decrease as its size increased.
Many groups of frogs, such as the neotropical hylids (Duellman and Pyles 1983),
show an inverse correlation between size and call frequency. Slopes differ in different
groups and there is substantial scatter around the line. The correlation shows that size
influences call frequency but the scatter suggest that other factors are able to modify
this.
Size also may have another effect. Acoustics textbooks say that, for a surface of a
given area, frequencies above a threshold are radiated more or less equally, while
frequencies below it are radiated less intensely. For a Physalaemus pustulosus male,
Ryan (1985) calculated that threshold frequency should be about 3,500 Hz. Since most
of the sound energy in a P. pustulosus call is below this, the call apparently is being
radiated less effectively than it would be if the frequencies were higher. Presumably the
advantages to having a call that would radiate better are outweighed by the advantages
of having a lower frequency call. We will return later to what some of these advantages
may be.
As we saw among species, within a species individuals in a population frequently
show an inverse relationship between body size and the dominant frequency of their
call. But this correlation is not nearly as ubiquitous nor always as tight as one might
expect. In eastern North America, Zweifel (1968) found a significant relationship in
Bufo americanus, but not in Bufo woodhouseifowleri. In a widespread North American
hylid (Nevo 1969, 1973) size differences between populations have been interpreted as
adaptations to differing environmental aridity and differences in dominant frequency
as a direct consequence of these differences in size. There are also situations where
closely related species, or isolated populations differ in frequency in ways that can not
be explained by size alone. For example, there are populations of small toads that give
calls of lower frequencies than those of their larger relatives (Porter 1966, 1968). Again,
within, as among species, body size influences call frequency but does not determine it.
628 A Stanley Rand
3.6.2 Size and loudness: Frog calls are surprisingly loud. Most species measured
have calls with sound pressure levels between 90 and 120 dB at 50 cm. The loudest calls
are almost painful when heard at close range.
Big frogs do not always make more intense noises than small ones. There have been
three studies comparing the sound energy in calls of different species: one in Australia
(Loftus-Hills and Littlejohn 1971), one in North America (Gerhardt 1975), and one in
Africa (Passmore 198 1). In Australia bigger frogs call more loudly than do smaller ones.
Loftus-Hills and Littlejohn (1971) suggest that the size of the vocal sac may be
correlated with the efficiency of coupling between the frog's acoustic generator and the
atmosphere, giving an advantage to larger frogs. Likewise, the larger animals with
larger larynxes and more powerful muscles should be able to produce more energy.
Despite this very plausible argument, the 21 North American species (Gerhardt 1975)
and the African species (Passmore 1981) show no consistent relationship between size
and loudness.
Intraspecifically, Gerhardt (1975) reported a correlation between body size of male
Bufo americanus and the loudness of their calls. However, Passmore (1981) did not find
any such relationship within the African species that he studied. This seems anomolous.
If small frogs can make such loud calls, why don't big frogs make even louder ones. One
possible explanation lies in the very peculiar hearing of frogs.
3.6.3 Receiver: Frogs have very specialized, highly peculiar ears (Wilczynski and
Capranica 1984). They have two areas sensitive to sound, one is horjaologus to that in
other vertebrates; the other is unique to amphibians. Frogs have peaks of sensitivity
at two, or three, different frequency bands (Capranica 1976). Advertisement calls
commonly have two bands of emphasized frequencies and these coincide with peaks in
hearing sensitivity. In at least some species conspecifics respond best to artificial calls
that contain sound energy in both frequency bands (e.g. Rana catesbeiana, Capranica
1965, and Hyla cinerea, Gerhardt 1974, 1976, 1981).
The advantages that a male might gain from giving a call of a different frequency
might be more than offset if the female's hearing was less sensitive at the new frequency.
In all species studied, the lower frequency peak in an individual is more sensitive than
are the peaks at higher frequencies. When different species are compared the
frequencies at which maximum sensitivity occurs goes up as size decreases. Further,
Loftus-Hills (1973) showed that large species are more sensitive at their peak sensitivity
than are smaller species. Thus size influences hearing so that big frogs hear better at
lower frequencies than do small frogs and when compared at their most sensitive
frequencies bigger frogs are able to hear fainter sounds. We saw that big frogs didn't
generally call more loudly than small frogs. Is this possibly because they can hear
better?
3.6.4 What influences size: Body size is, of course, strongly influenced by a variety of
ecological factors. Among these are food, predators, competitors, mortality and
fecundity schedules, and physical factors like climate. In addition, success in social
interactions, particularly competition both for mates and for resources, may also
influence size.
A frog's ecology influences its size and in turn its size influences sounds that it makes
and hears. The reverse is at least conceivable, that selection for making or hearing
certain sounds may influence the evolution of a frog's size and thus in turn its ecology.
Tradeoffs in the evolution of frog calls 629
I. Selective forces, passive and active
rwo sorts of factors increase the effectiveness of an advertisement call. The first, Parker
1983) called passive attraction and the second, active discrimination. Factors that
ncrease passive attraction are those that maximize transmission through the physical
environment and minimize interference from ambient noises, including calls of
;onspecifics. Active discrimination involves differential responses to different calls even
:hough they are equally audible.
U Passive forces
Fhe first of the passive forces to consider is transmission.
U.I Transmission: In general, lower frequencies transmit through air better than
lo higher frequencies and so are audible from greater distances. Environments may,
Because of their structure, absorb, reflect and transmit different frequencies in more
complex ways. An environment may transmit an intermediate band of frequencies
setter than those either higher or lower (Morton 1975).
In their review of bird vocalizations Wiley and Richards (1982) conclude that; "For
naximum efficiency, long-range acoustic communication in any habitat should employ
:he lowest frequencies possible" p. 148.
Calls of monkeys (Waser and Waser 1977) and birds (Brenowitz 1982; Gish and
Morton 1981) may be adapted to the transmission characteristics of their environ-
ments. But, when Zimmerman (1983) looked for similar adaptations in frogs in the
\mazon, by comparing the calls of species from different habitats, she concluded that
my differences among them that could be attributed to habitat were small and
overshadowed by differences associated with differences in size and phylogenetic
relationship.
Transmission experiments with P. pustulosus calls in the environments where it
occurs showed that the call component which has more energy at lower frequencies,
ioes in fact transmit further than does the component with more energy at higher
?requencies (Ryan 1985).
Thus, in an otherwise silent environment, for maximum transmission a call should
:oncentrate its energy in frequencies that are low, but not too low.
1.1.2 Acoustic interference: Frog calls aren't given in isolation. They compete with
the sounds of a variety of other animals and with noises produced by wind and by water.
Ihe problem a frog confronts is akin to trying to shout instructions across a busy street,
[n Panama there are two bands of potential interference (Ryan and Brenowitz 1985).
One from about 100-200 Hz, is produced by wind noise and a second, from about
1-7 kHz by insects, particularly cicadas and orthoptera. In the window between these
two bands there may be interference from other kinds of frogs and, in some ways the
most difficult to deal with, interference from the calls of conspecifics.
That a frog's ear is tuned so that it is more sensitive to the frequencies in its
idvertisemept call reduces the interference from noise at other frequencies but the
:uning of the ear is broad enough that noise interference can still be important (Narins
1982). There is direct evidence that this is true. Female Hyla cinerea will respond to
630 A Stanley Rand
conspecific calls if the noise level is more than about 22 dB below that of the call, but not
if the noise is relatively louder than this (Ehret and Gerhardt 1980). This is equivalent to
the levels found for the few other animals that have been tested.
Wiley and Richards (1982) assert that in communities of birds there are no clear
allocations of the frequencies and that, most passerine birds use approximately the
same frequency band for their long-range acoustic signals. In contrast, frogs that call
together, tend to use different frequency bands (Littlejohn 1977; Drewry and Rand
1983). Often there is at least some overlap between the frequency bands used, and
species calling together usually also differ greatly in temporal characteristics. Usually
there are differences among these species in addition to those in the calls. The species
may call at different times, from different habitats, or like these same Australian frogs,
from different kinds of perches. Species calling together may interact so that they
partition signalling time and so reduce acoustic interference as has been reported for
birds (Cody and Brown 1969; Moynihan 1963) and primates (MacKinnon 1974).
Alternation between calling bouts is illustrated by the two neotropical hylids Hyla
phleboides and Hyla ebraccata (Schwartz and Wells 1985).
Noise is usually a problem for a calling frog but perhaps not always. Tuttle and Ryan
(1982) have argued that the neotropical hylid Smilisca sila uses the sounds of waterfalls
to hide its calls from predators.
4.2 Active discrimination — social /sexual selection
4.2.1 Responses of males: Breeding male frogs may react to the advertisement calls
of other conspecific males in a variety of ways.
(i) In some species, males moving to breeding sites are attracted by the calls of other
males (e.g. Ptychadena taenioscelis in South Africa, Passmore 1976), in other species,
breeding aggregations form with little or no calling (e.g. Rana temporaria in England,
Savage 1962).
(ii) Males tend to space themselves out at calling stations in one small North
American treefrog, Hyla crucifer, Wilczynski et al (1984) report that males position
themselves so that each male can just hear the calls of its neighbors.
(iii) In some species at least, non-calling males may sit near calling males and attempt
to intercept females attracted by the calls (e.g. Hyla cinerea Perril et al 1978).
(iv) Males are often stimulated to call by hearing the calls of other males, or even
rather crude imitations or the calls of other species (e.g. Hyla cinerea Gerhardt 1974a).
(v) In many species, such as Hyla microcephala (Wells and Schwartz 1984), males
react to calls of nearby conspecific males by giving aggressive calls that are responded to
by males but ignored by females. Eleutherodactylus coqui has a two note call, the first
note is responded to primarily by males as an aggressive call, the second, higher
frequency note is responded to primarily by females as a mating call. There are
corresponding differences in maximum sensitivity of hearing in the two sexes (Narins
and Capranica 1976).
(vi) Males might by synchronizing their calls produce a louder sound than would be
heard by females from further away. Certainly some frogs do synchronize their calls
(e.g. Bufo marinus) but it is not clear that the resultant chorus is enough louder than an
individual call to provide a selective advantage.
(vii) Males may time their calls so that they minimize overlap and presumably reduce
Tradeoffs in the evolution of frog calls 631
interference. A Hyla microcephala male responds to the call of a conspecific so that the
notes of his call fall in the periods of silence between the notes of the other frog
(Schwartz and Wells 1985). That the alternation of calls is due to active adjustment of
timing, not the coincidental result of similar call rates as has been shown by measuring
the responses to synthetic stimuli (e.g. Eleutherodactylus cogui Zelik and Narins 1982,
Hyla regilla Awbrey 1978).
(viii) The calls may be structured to reduce interference. The whines of Physlaemus
pustulosus and similar frequency modulated calls of other leptodactylids allows the
calls to overlap in time and still be recognizable.
Thus males interact acoustically in ways that are more complex than the classic
model of a shouting match for the attention of females.
4.2.2 Preferences of females: Still, attracting female attention is what the male is
trying to do, and one of the advantages of studying frogs is that, in at least some species,
female preferences for different mating calls can be tested directly. One captures a
mating pair, separates them and introduces the female into an arena where she hears
calls or other acoustic signals played through one or more loudspeakers. Many females
will make a clear choice, approaching a speaker with a series of slow and deliberate
hops.
There are several possible reasons why a female might select one call instead of
another if both were equally audible. These include selecting a call that could be more
easily located; a call of her own species; or a call that signalled a male that controlled
more desirable resources, or had a better genotype.
4.3 Locatability
For a call to attract a mate, she must be able to find the sound source, as they clearly do.
Konishi (1970) listed three ways in which animals could localize the direction of a sound
source: sound shadows, time differences, and phase relationships. The mechanism by
which frogs locate calls is not completely understood. At the relatively low frequencies
that frogs use, their heads are too small to produce strong sound shadows, and the
difference in times when a sound reaches the two ears is very short. A comparison of
phase relationships between the two ears seems to require lower frequencies or bigger
frogs than we observe. Gerhardt and Rheinlander (1980) suggested that anurans use
some form of sound pressure gradient system, where directional sensitivity is caused by
the interaction of two sound waves on the tympanic membrane, one acting on the
external surface and the other travelling via an indirect route to the internal surface.
Other neurophysiologists suggest that the situation is more complicated (Wilczynski
and Capranica 1984). Speculation about how selection should act to maximize
locatability will be on firmer ground when this issue is resolved.
Still, a female should select the mate who is easiest to find because by so doing, she
minimizes the time and the energy she must invest. Evidence suggests that P. pustulosus
females do this (Rand and Ryan 1981). Complex calls are preferred by females.
Complex calls contain a wider range of frequencies and more transients and therefore
should be more easily located. The parallel between the two components in the
P. pustulosus complex call and with bird alarm calls evolved to be easy or hard to
localize (Marler 1955) is striking. However, that female P. pustulosus really can locate
632 A Stanley Rand
complex calls more easily than simple calls has not yet been demonstrated.
Increasing locatability, like increasing sound intensity, may bring gains in terms of
females attracted but it also may bring increased attention from predators. The
complex P. pustulosus calls that females find most attractive are also most attractive to
the bats that hunt frogs by call (Ryan et al 1982).
4.4 Species isolating mechanisms
Since advertisement calls have as a primary function the attraction of a mate, one would
think it important to both sexes that females be attracted to the call of their own species.
That species calling together always differ in call, even when they are virtually identical
morphologically (e.g. Crinia Littlejohn 1959), supports this idea. Cases of character
displacement in advertisement calls have been interpreted as the results of selection for
species isolating mechanisms in the sympatric populations. There are at least two well
documented cases of character displacement in frog calls: between Litoria ewingi and
L. verreauxi in Australia (Littlejohn 1965; Littlejohn and Loftus-Hills 1968) and in the
Pseudacris nigrita complex in the southeastern United States (Fouquette 1975).
However, such well documented cases are quite few. In most cases studied, the species
are as distinctive in allopatry as in sympatry (Blair 1974). Paterson (1978, 1981) argues
that mate recognition systems diverge only in allopatry. As Passmore (1981) restates
this: "Sympatric species of frogs do not signal differently because they coexist, they are
capable of coexisting because they signal differently," p. 95.
On the other hand, Gerhardt (1982) argues strongly that interactions between species
are important. One of the cases he discusses is of two very similar hylids in the eastern
United States. Their calls differ most distinctively in pulse repetition rate. This rate is
positively correlated with the temperature of the calling male. In choice tests, females of
both species prefer calls with rates appropriate to their species at the test temperature
and discriminate more strongly against calls that resemble those of the other species
than against those that differ by the same amount but in the other direction. This
asymmetry in discrimination seems strong evidence of interaction between the two
species. Selection has favored stronger discrimination where confusion with the other
species is possible.
It appears that in frogs, species recognition may be an important force in the
evolution of the advertisement calls, but it is clearly not the only one.
4.5 Better resources
Territoriality is much less common in frogs than in many other vertebrates, but it does
occur and, in some species, larger males control better resources than do smaller
ones. For example, Howard (1978a, b) has shown that territories of larger Rana
catesbyiana have better oviposition sites, eggs laid in them have lower predation from
leeches; and that these larger males are more successful at securing mates.
Unfortunately, the territorial species are not easy ones with which to do female choice
experiments, so we don't know if females are choosing males or choosing territories.
In a very real sense, effective fertilization is a resource that males provide for females.
In at least two cases, Bufo bufo, (Davies and Halliday 1977) and P. pustulosus (Ryan
Tradeoffs in the evolution of frog calls 633
1983), certain combinations of sizes of male and female are optimal for maximum
fertilization, and size-biased mating is observed. In the case of the Bufo9 physical
combat between males for females seems the important factor. In Physalaemus female
preference is involved. Ryan (1980, 1983) tested a number of females and was able to
show that they preferentially approached calls in which the chucks had a lower
fundamental frequency. Further, Ryan (1983) showed that such a preference resulted in
females mating with males whose size was such that the maximum number of eggs were
fertilized.
Licht (1976) reported assortative mating by size in one Bufo americanus population
and suggested that it was due to an advantage gained by the female from having a male
big enough to achieve a secure axillary grip, but small enough to ensure cloacal
alignment. He suggested that female choice based on dominant call frequency was
involved, but he had no experimental evidence.
4.6 Better genes
Wilber et al (1978) reported that in several populations of Bufo guercicus, larger males
mated relatively more frequently than did smaller ones. They advance the hypothesis
"that females choose the largest males available because there is a selective advantage in
large body size per se. . . . Large males may be more attractive to females because they
are either older than the average male in the chorus or have had faster growth rates.
Both are a sign of vigor and, perhaps, of a good genotype." p. 267. They suggest, as Licht
did, that the females may be selecting mates on the basis of aspects of their calls that
correlate with male size. However, Arak (1983a) reviewed the cases where size
dependent and size assortative mating have been reported. He argues that "Non-
random mating is not sufficient evidence for female choice since it may come about
through incidental effects, such as size-related patterns of arrival at breeding
ponds ... or more likely, through male-male competition" p. 202.
The evidence of female preference for calls with lower frequencies is convincing for
P. pustulosus (Ryan 1980); but it does not seem to occur in all species of frogs. Gerhardt
(1982) argues that a species may be constrained by interactions with others and shows
that in Hyla cinerea female choice of low frequency calls is constrained by the
coexistence of another species With a similar but lower call. Bufo calamita in Britain
does not breed in choruses with any other species of toad that uses an advertisement
call. Females do not show any frequency preferences but males do (Arak 1983b).
Shine (1979) provided indirect evidence on the mating advantage of larger males. He
showed that there is a correlation between those species in which males are larger than
females and those in which fighting between males has been observed and/or in which
the males have morphological structures that appear to have evolved for fighting.
All this argues that in many species, male-male competition is probably iftore
important than female choice in giving large males mating advantages.
Frequency is not the only characteristic on which a female might select among calling
conspecifics. There is evidence that females select the males that call more often, e.g.
Bufo woodhousei australis (Sullivan 1982). The argument that by selecting the most
vigorous males females are selecting for the best genes could plausibly be suggested
here, but that it is true remains to be demonstrated.
In summary, the argument that females should be selecting males whose calls in some
634 A Stanley Rand
way signal their greater fitness (in terms of natural selection) is persuasive; the evidence
that they actually are doing so is still scant and some of it open to other interpretations.
4.6.1 Runaway sexual selection: Fisher (1930) suggested that selection would favor
females who selected males that were attractive to other females, because in so doing
they were increasing the probability that their sons would be attractive. This in turn
would increase selection for the traits that made the males attractive. The positive
feedback loop would continue with increasingly strong female preference and
increasingly exaggerated male characteristics until strong counter-selection prevented
further change.
The ways in which frog calls are subject to both active and passive selection have been
discussed at length. Despite the constraints of phylogeny, morphology and the physics
of sound, there is little question of the importance of female choice. There are instances
of geographical variation in frog call characteristics, such as pulse rate, that can not
easily be explained by differences in the sonic environment or frog size. The European
treefrogs Hyla arbor ea show this kind of variation (Schneider 1977) and Gerhardt and
Schneider (1980) showed that in the isolated population on the Canary Islands
(H. meridionalis) most, but not all, females preferred calls of local males to those of
males recorded in Germany. This kind of geographical variation is strong evidence
for the importance of female preference in the evolution of frog calls as
West-Eberhardt (1983) has argued for other organisms.
Curiously, since the evidence seems to implicate female choice as important, runaway
sexual selection has not often been invoked to explain any particular frog call (an
exception is Ryan 1985 for P. pustulosus). Indeed, do frog calls, diverse as we have seen
them to be, include any cases of extreme elaboration? Is there a frog call equivalent of
the peacock's tail? I would argue that selection for such extremes exists; that it is
generated by competition among males, directly for mates and indirectly for resources,
and by female preferences for calls that are maximally audible, minimally interfered
with by other noises, locatable, and, where appropriate indicative of males in control of
critical resources and perhaps with a good genotype. However, this selection is limited
in its expression by the wide variety of constraints and counterselections; including the
morphology of both the vocal apparatus and the hearing mechanism, the physics of
sound production and environmental transmission, the presence of other sounds, and
the activities of predators hunting by sound. However, the elaborate vocal apparatus
and the loud and persistent calling of many frogs is itself an extreme elaboration that
can only be interpreted as the result of sexual/social selection.
Acknowledgements
Over the past couple of years I have had opportunities to discuss frog vocalizations
with a number of colleagues. These conversations with Mike Ryan, Kent Wells, Josh
Schwartz, George Drewry and Carl Gerhardt have contributed both ideas and
information. Pat Rand, Josh Schwartz and Kathy Troyer read and improved the
manuscript. I must thank Madhav Gadgil and the Indian Academy of Sciences for the
opportunity to participate in this symposium.
Tradeoffs in the evolution of frog calls 635
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© Printed in India.
The evolution of communication and social behaviour in Dictyostelium
discoideum
VIDYANAND NANJUNDIAH
Molecular Biology Unit, Tata Institute of Fundamental Research, Homi Bhabha Road,
Bombay 400005, India
MS received 6 May 1985; revised 28 October 1985
Abstract. Exceptionally for a developing system, the pathways of intercellular communi-
cation are fairly well characterised in the cellular slime molds. This paper attempts to provide
adaptive explanations for the origin of the following features and consequences of communi-
cation between cellular slime mold cells: the tendency to congregate, chemotaxis to a released
signal, signal relay from cell to cell, oscillatory signal release and an invariant ratio of the
terminally differentiated cell types. For the sake of specificity attention is directed at the species
Dictyostelium discoideum. Central to the entire analysis is the assumption that contiguous
groups of feeding cells are, and in the past were, genetically identical. It is suggested that, in
respect of most of the features listed above, the critical event which started things off must have
been the acquisition by the cell membrane of permeability for a substance normally produced
intracellularly as a response to the stress of starvation. An argument is presented for treating
social behaviour in these organisms, and in particular the suicide by cells which differentiate
into stalk, as an example of group selection.
Keywords. Dictyostelium; slime mold; evolution; group selection.
1. Introduction
The aim of this paper is to advance adaptive explanations for some manifestations of
organised behaviour in a primitive microorganism. A property will be said to be
"adaptive" in the sense of being more useful for the survival and reproduction of the
organism than the absence of the property and, sometimes, than other possible
alternatives.
Almost by definition, communication or mutual signalling is a prerequisite for social
behaviour (Wilson 1975). Excepting cases wherein sociality is only apparent and due to
spatial proximity caused by other factors, the observation of social or cooperative
behaviour indicates the existence of an underlying system of communication.
Specifically, some form or forms of communication must be responsible for the
integrated multicellular behaviour displayed by a developing organism. This leads one
to the question of why any particular mode of communication, either identifiable
directly or inferred from its effect on social behaviour, exists in development In general
there are two sorts of answer possible to such a question. One can think of a
'developmental' explanation, meaning basically an explanation in terms of whatever is
known of the embryological process at a more basic level, ideally of its physics and
chemistry. On the other hand one can aim for an 'evolutionary* explanation; that is, one
can try to invoke natural selection. Seen in this light, the interesting questions which
need to be answered are, in what manner is a particular form of communication
adaptive? And how could it have evolved? Hardly anything is known about the details of
639
640 Vidyanand Nanjundiah
intercellular communication in most developing systems for these questions to be
considered seriously. The cellular slime molds, especially their best studied member,
Dictyostelium discoideum, provide an exception to this general rule (Bonner 1967;
Loomis 1975).
In D. discoideum multicellularity arises on account of aggregation of single amoebae
because of the emission of, and attraction to, a known chemical signal (Bonner 1967).
During aggregation the signal is released periodically and also relayed from cell to cell
(Gerisch and Wick 1975: Shaffer 1975; Roos et al 1975). Strong circumstantial evidence
indicates that the cells continue to communicate — albeit by other, not yet understood
means — in the multicellular aggregate (Raper 1940; Lokeshwar and Nanjundiah 1983).
Eventually, after a series of morphogenetic movements, essentially all the cells in the
aggregate differentiate into one of two types whose relative numbers are constant
(Bonner 1967).
The present work is the result of an effort to look at the following 4 features of
communication from an evolutionary point of view: aggregation by chemo taxis,
periodicity of signal release and signal relay, patterning within the multicellular
aggregate and cell number proportioning in the terminally differentiated structure. In
order to fix our frame of reference we restrict our attention to D. discoideum. A word
about methodology: the approach — not necessarily in the same order — will be to (i)
describe an existing property; (ii) suggest an adaptive value for it; (iii) conjecture an
ancestral state (by which is meant, somewhat loosely, a state in which the property is
missing); and (iv) point out that starting with the ancestral condition, the presently
existing property could be acquired via plausible mutational steps.
In what follows I first sketch the development of D. discoideum in just sufficient detail
for our purpose and then go on to suggest specific evolutionary hypotheses for the 4
features mentioned above. General issues related to the hypotheses are discussed at the
end. Comparative development in the cellular slime molds has been recently discussed
in a paper by Bonner (1982) which has strongly influenced this one.
2. The life cycle of Dictyostelium discoideum
As this description is highly condensed, the literature (Bonner 1967; Loomis 1982 and
references therein) should be consulted for a fuller picture. D. discoideum is a free living
soil amoeba. Controlled experiments performed in the laboratory enable one to build
the following plausible account of its life in nature (figure 1). In their vegetative phase,
individual cells feed on bacteria, grow and divide by mitosis. Under extreme
environmental conditions cellular slime mold cells are capable of following one of two
protective strategies, that of forming microcysts or macrocysts. Both are resistant
structures. Microcysts are not known as yet in D. discoideum, and macrocysts are the
first step in the sexual pathway. Since they are not formed under standard experimental
conditions, we will not refer to them any further (Bonner 1982 has considered their
possible significance). In the laboratory, amoebae starved of food go through a
seemingly quiescent phase for a few hours prior to aggregating. Aggregation is by means
of chemotaxis, usually accompanied by relay of the chemoattractant. The attractant is a
chemical, cyclic AMP (cAMP), which is produced and periodically released by the cells.
A cell which senses external cAMP moves towards its source and in turn itself releases a
burst of cAMP. Consequently local variations in cell density get amplified. Ultimately
Communication and social behaviour in D. discoideum
641
Q
Figure \ . Schematic representation of the life cycle of D. discoideum (not to scale). (1 ), Free-
living amoebae; (2), aggregation; (3), migrating slug, anterior to the right; (4), early culmination;
(5), advanced culmination; (6), fruiting body with spore mass held aloft stalk; (7), spores.
all the cells in a neighbourhood end up piled on top of each other in a mound which is
held together by specific adhesion molecules. The aggregate — now called the slug —
often falls down and adopts a cylindrical form with a nipple-like protrusion, the tip, at
its anterior end. After moving for a length of time which depends strongly on external
conditions as well as the genotype, the slug stops, reverts to a mound-like shape, and the
cells in it move in the manner of water in a reverse fountain and the entire mass rises
upwards (the words 'up' and 'down' mean away from the two-dimensional substratum
on which aggregation has occurred, or towards it). Irreversible differentiation sets in
concurrently, most of the cells either turning into spores or, alternatively, dying and
turning into the units of a cellulose-sheathed stalk. The final structure, called the fruiting
body, has a disc-shaped base (also made of dead cells), an erect stalk and, above the stalk,
642 Vidyanand Nanjundiah
a spheroidal mass of spores. Under suitable conditions each spore can germinate and
give rise to an amoeba, setting off the life cycle anew.
3. Aggregation
If not the earliest, aggregation is certainly the most striking example of social behaviour
in the cellular slime molds [mutual repulsion, observed in the vegetative phase of some
slime molds, is conceivably a device for maximising the efficiency of feeding (Keating
and Bonner 1977)]. Since starvation is the primary trigger, it seems intuitively
reasonable to suppose that aggregation, like the coming together of individuals in more
evolved social groups, must be a behavioural trait which has evolved to improve the
chances of survival of the individual itself or of its genetic relatives. Therefore one must
make it plausible that because of aggregation there is an increase, either in the
probability that an amoeba turns into a viable spore or in the probability that a spore
disperses to a favourable environment. A large mass of spores probably stands a better
chance of dispersal than many isolated spores (Bonner 1982). In a fruiting body, just the
fact that the spores are on an elevation should also help in dispersal, and we will take this
up later. That apart, the assumption that aggregation might enhance sporulation seems
justified, since cell-cell contact is normally a requirement for development, and so for
sporulation (Gregg 1971; Newell et al 1971; Takeuchi and Sakai 1971). Precisely how
contact aids development is not known; one possibility is that the binding or recognition
of sets of cell-surface molecules is necessary. Alternatively, from observations on
mutants which can sporulate in the absence of contact if simulated by exogenous
cAMP (Kay and Trevan 1981), we might conjecture that 'contact' really means a degree
of proximity sufficient for some substance emanating from one cell to reach a level at the
site of a second cell high enough to induce the latter to turn into a spore (extracellular
cAMP by itself is a non-specific differentiation signal in present-day D. discoideum; in
conjunction with other substances it can influence either stalk or spore formation
(Morrissey 1982) ). The suggestion is, then, that in the ancestor of D. discoideum a single
cell could sporulate in isolation but that the process could be helped along by a stimulus
emanating from another cell in the same condition. What sort of substance would be
appropriate to provide such a stimulus? A natural candidate would seem to be a
chemical which was already being synthesised in the (ancestral) isolated cell during
sporulation. Such a substance would reinforce a cell's response to starvation if, in
addition to being produced intracellularly, it moved from one cell to another through a
leaky' membrane. The leak itself would be selected for when it first arose by mutation.
This would be so since sufficiently small clusters of post-feeding amoebae very likely
consist — and consisted — of clones; the mutation would be simultaneously expressed in
an entire group of cells. The possibility that the leak could be disadvantageous to a cell
on account of its losing some of the signal substance must have been compensated by
the benefit it conferred when it entered and stimulated other, genetically identical, cells.
To sum up, a group of amoebae which stayed together after feeding was over would
hold a selective advantage over one whose members continued to move randomly and
so drifted apart. Thus the mechanism of 'aggregation', when it first arose, may have been
nothing more than a decrease in cell motility once the density of food became too low to
be reliably sensed; the effect may have been further strengthened by the acquisition of
the ability by cells to adhere to one another.
Communication and social behaviour in D. discoideum 643
4. Chemotactic attraction
Continuing with the line of argument we have used so far, it is not difficult to see how
mutual attraction, by decreasing the spatial spread of post- vegetative cells, would also
have been selected for. The agent of attraction could either be the same one that
enhanced sporulation or, perhaps, a substance which worked inside the cell and guided
cell movement by acting on contractile filaments, thereby causing local psuedopodial
extrusion (the observed rapid accumulation of cytoskeletal actin in D. discoideum
following an external cAMP stimulus (McRobbie and Newell 1984) could be a pointer
to the original situation). The same substance entering from outside would have the
same effect, but this time in a definite direction. A mutation which made the cell
membrane porous for this substance would polarise cell movement centripetally
towards wherever the cell density was slightly higher than in the neighbourhood, or
wherever — if development were not perfectly synchronised in all cells — the first cell to
leak out the substance was situated.
Until now I have tried to suggest how non-directed aggregation could be adaptive in
itself, and how — by improving the efficiency of aggregation — a further beneficial
adaptation could be conferred by either the simultaneous and mutual attraction of all
cells in a neighbourhood or the attraction of most of the cells towards a center.
5. Relay
Consider now the possibility that a primary chemotactic stimulus might excite a
sensitive cell to act as a secondary source of the stimulus. The advantage of having an
attractant relayed outward from a centre, rather than having it released at about the
same time by all cells, would seem to be the prospect of increasing the size of an
aggregate. Since the signal should have the same range whether it is released by a cell on
its own or whether a cell releases it in response to an external stimulus, it appears that
the ability to relay would not have been favoured if all cells had — in the course of their
development — started to signal simultaneously. In the absence of a means for
synchronising development simultaneous signalling does seem unlikely; the special care
needed to achieve developmental synchrony in the laboratory suggests this. Therefore,
in order to associate a selective advantage with the ability to relay, it needs to be assumed
that under natural conditions some cells in a group are developmen tally more advanced
than others and, specifically, are capable — purely on statistical grounds — of releasing
the chemoattractant earlier than most others. Given that relaying the chemotactic signal
causes aggregates to increase in size, how might this be beneficial? It could be that large
aggregates, with large volume-to-surface ratios, imply a reduction in the amount of the
hypothetical substance which leaks to the outside world and is lost. For the same
energetic investment, the yield in terms of effective signal strength would be higher.
Another possibility, alluded to earlier (Bonner 1982), is that a large aggregate might
improve the prospects for spore dispersal. Significantly, large slugs of D. discoideum
move faster than small ones (Bonner et al 1953). Consequently if the time available for
dispersal (before sporulation begins) is limited, the distance of dispersal would be
greater for cells contained in the larger of two slugs. If this is to be used as a justification
for increased aggregate size via relay, one will have to make the further supposition that
644 Vidyanand Nanjundiah
aggregate motility existed earlier, probably having being selected as an aid to dispersal
by itself.
There is another way of looking at the phenomenon of relay (Nanjundiah 1978). So
far we have assumed that chemotaxis-mediated aggregation came first and that relay
was a later refinement. Suppose things had been the other way round: consider a stage in
evolution at which a clone of cells, lying close to one another, were faced with starvation.
Just as we made a case for the original aggregation signal being a pre-existing
component of the cell's internal response, we can argue that the cell-to-cell transfer of a
substance, until then produced inside the cell in response to starvation and remaining
there, would have enabled an entire group of cells to cope with a deteriorating
environment faster than they would have in the absence of relay. Both clustering and
chemotactic attraction could have arisen, in this way of looking at things, as later
adaptations. Note that the first hypothesis (relay as a device for increasing the range of
the chemotactic signal) makes it essential that the relay substance and the chemoattract-
ant be the same (as is indeed the case in D. discoideum); on the second hypothesis, though
it would probably be of advantage to have them the same, they could well be different.
An interesting point regarding the origin of relay is that on the second hypothesis the
relay substance must have been able to activate its own synthesis. In other words, the
reaction pathway responsible for forming the substance must have had in it an
autocatalytic (positive feedback) step. The critical mutational event would then have
been, as already suggested, a specific membrane leak'. If, on the other hand, chemotactic
aggregation had arisen as an earlier adaptation (the first hypothesis), the important
mutation would have been the one which made chemoattractant synthesis autocatalytic:
that is, an early step in the synthetic pathway would need to be activated by a later step.
6. Oscillations
Given relay, and therefore an autocatalytic step; given that the living cell is
thermodynamically an open system; and given that most biochemical reactions in vivo
operate far from equilibrium, oscillatory reaction fluxes are almost a "natural"
consequence (Higgins 1967), meaning that no special adaptive explanations are called
for. Goldbeter and Segel (1977) have in fact shown that the observed cAMP oscillations
in D. discoideum can be successfully modelled by assuming that the condition for relay
exists, that is that an extracellular cAMP stimulus activates the intracellular synthesis of
cAMP. Oscillations are on the other hand ubiquitous in biological systems (Winfree
1980); in particular, morphogenetic oscillations are found in myxobacteria (Kaiser et al
1979) as well as in other cellular slime molds, possibly with the involvement of cAMP
(Schaap and Wang 1984), Even temporal patterning in chemical communication,
though rare, is not unknown (Conner et al 1980). So it is worth considering whether the
relatively rapid (period ca. 8 min) oscillations of cAMP in D. discoideum might be
adaptive after all.
Under certain conditions, the course of glycolysis in yeast is oscillatory, also with a
periodicity of some minutes (Hess and Boiteaux 1971). Since these oscillations involve
the adenine nucleotide pool and therefore the adenylate charge (Atkinson 1968) of the
system, Goldbeter (1974) has conjectured that the cell benefits by partitioning each
oscillatory cycle into distinct energy-utilising and energy-yielding phases, something
which is supposed to make for an improved metabolic efficiency. How far this argument
can be carried over to cAMP oscillations in D. discoideum, where ATP levels do not vary
Communication and social behaviour in D. discoideum 645
significantly within a period (Gerisch et al 1977), is not certain. Richter and Ross (1981)
have offered yet another adaptive explanation for the glycolytic oscillation. Using a
quantitative model they have calculated that energy dissipation in the latter part of
glycolysis (the pyruvate kinase step) is minimal precisely when an earlier step (the
phosphofructokinase reaction) is periodic with a frequency within a defined range.
Again, one does not know how good this explanation, depending as it does on the
"tuning" of one reaction step by another, would be for the slime mold oscillations. The
oscillations in this case occur — as far as is known, unlike glycolysis in yeast — both
extracellularly and inside the cell, and it is easier to speculate on a possible adaptive role
for extracellular oscillations. It has been shown (Nanjundiah 1973) that when the
relevant parameters are assumed to have reasonable values, a pulsatile source of
cAMP has a spatial range which is about an order of magnitude more than the range of
an equivalent steady source which releases cAMP at the same average rate. The
reasoning is based on the facts that (a) (in three dimensions) the concentration of a signal
diffusing from a steady source falls off inversely with the distance, whereas the peak
concentration due to an impulsive source decreases as the cube of the distance, and (b)
the lower the frequency of successive pulses, the weaker the effect of the equivalent
steady source. Then, given a plausible threshold for the sensitivity of a cell, either for the
concentration of cAMP or for its spatial gradient, the threshold is reached at a farther
distance from the source when the signal is released in a series of brief pulses than when
it is released at a uniform rate. The conclusion remains valid even when the released
signal profile is no longer a sharp pulse but is somewhat sinusoidal However, in the
latter case the range of the signal is enhanced by roughly a factor of two rather than by
one order of magnitude. A periodic signal has two other advantages. One is that the
receiver would not get adapted to it as readily as it might to a steady source. It is to be
expected that a train of stimuli reiterated at an appropriately intermediate frequency
would be much better at eliciting a response than the same train of stimuli applied either
at a very low frequency (in which case each stimulus would — so to speak — be like the
first stimulus, there being no reinforcement) or at a very high frequency (in which case
there would effectively be one constant stimulus, to which the system could adapt). Just
this has been observed in the case of D. discoideum cells with folic acid, a
chemoattractant for feeding cells (Wurster and Schubiger 1977). Also, periodic pulses
of cAMP are efficient at eliciting cell differentiation whereas steady levels are not
(Gerisch et al 1975; Darmon et al 1975). Unfortunately, none of these experiments
tested the effect of irregular pulses or different waveforms; one is still unsure as to
precisely which stimuli constitute signals and which ones do not. The other advantage
of a periodic signal is that at close distances there would be a lesser degree of ambiguity
in locating a periodic source than a steady source (the amplitude of oscillations will, in
any case, get damped with increasing distance from the source, the higher frequencies
dropping out first).
7, Pattern formation and tissue proportioning
7.1 Pattern in the slug
The slug is a facultative migratory structure formed by an aggregate of D. discoideum
before it differentiates into a fruiting body. It is in some ways a preparatory phase; the
646 Vidyanand Nanjundiah
future stalk and spore cells are identifiable in its front and rear portions (Raper 1940).
Depending on the type of experiment carried out, this separation into pre-stalk and pre-
spore can be seen either as an expression of autonomous cellular predispositions
(Takeuchi et al 1977) or as a consequence of the relative positioning of cells along the
long axis of the slug (Raper 1940). Probably both factors are important and reinforce
each other. Whatever be the cause, a spatial separation of presumptive cell types occurs
within a few hours after aggregation is completed. It is difficult to avoid the conclusion
that this spatial segregation must have co-evolved either with, or following, the
evolution of the process of fruiting body formation and the geometry of the fruiting
body. The chain of events could have been first, the transition of an aggregate into a
fruiting body of a certain structure (a spore mass held aloft on erect stalk) and later, the
appearance of an intermediate migratory phase in which the form of the fruiting body
was anticipated. Migration would most likely be on account of an extension in time of
the mechanism of amoeboid movement. However, morphogenesis in two other cellular
slime molds displays features which indicate that this cannot be the whole story. In
Dictyostelium mucoroides, stalk cells are formed and continuously released from a
common pool consisting of all the cells in the migrating slug; in Acytostelium the stalk,
which is acellular, is made up of material extruded from cells. Bonner (1982) treats this in
some detail and goes on to speculate that patterning might be related to some
developmental constraint imposed by the use of cAMP in chemoattraction.
It must be mentioned that the reasons for believing that cells in the slug communicate
with one another go beyond the facts of integration (in a general sense) and pre-stalk —
pre-spore patterning (in particular). The oldest and most convincing reason is that slugs
belong to the class of regulative embryos; a fragmented slug gives rise to a diminutive
but normally proportioned fruiting body (Raper 1940). Regulation was probably an
adaptation in response to the selective pressure provided by physical fragmentation
(Bonner 1982). Quantitative studies (Lokeshwar and Nanjundiah 1983) directed at a
very early event in regulation — the regeneration of a new tip at the anterior margin of
tipless fragments — indicate the involvement of long-range communication within the
slug. How communication actually occurs is not known; the evidence from experiments
on the rate of tip regeneration in genetically mosaic slugs is in agreement with, but does
not prove, cell-to-cell relay (Lokeshwar and Nanjundiah 1985). If this result is
confirmed, and if the same signal is responsible for both the rate of tip regeneration and
long-range communication in the slug, one might conjecture that cell-to-cell relay
during aggregation was a preadaptation which facilitated patterning in the slug.
7.2 Tissue proportioning
Under constant environmental conditions the number of spore cells in a fruiting body is
in a more or less constant ratio to the number of stalk cells. The data of Stenhouse and
Williams (1977), as analysed by Lokeshwar (1983), shows that 87-6 % ± 4-5 % of the cells
in small fruiting bodies (630-3901 cells) form spores; the corresponding figure for large
fruiting bodies (8404-18301 cells) is 87-1 % ±4-8 %. Lokeshwar also makes a case for
neglecting, in comparison with stalk and spore, the number of cells which form the basal
disc (less than 1-5 % of the total); his own studies reveal no undifferentiated amoebae in
the fruiting body. Two questions follow: (i) Why is the ratio of spores to stalk constant?
(ii) Why do a certain fraction of cells — those that form the stalk — die and so sacrifice
Communication and social behaviour in D. discoideum 647
their genetic potential? Wilson (1975) and Bonner (1980) cite stalk formation in
Dictyostelium as an example of altruistic behaviour and invoke kin selection in order to
explain it. The following simple argument (V Nanjundiah, unpublished results) makes
this explicit. However, I suggest (see later) that 'group selection' is a more appropriate
term than 'kin selection' to describe the phenomenon.
The critical assumption will be that, at least under the conditions obtaining when the
present form of the fruiting body first evolved, an aggregate consisted of genetically
identical cells. The problem facing this aggregate would have been to transfer all or part
of itself elsewhere because food was finished at the site of aggregation. Migration of the
entire aggregate towards food (not known to occur in D. discoideum slugs), might have
been a possibility. The alternative, given that some of the cells had committed
themselves to form spores, would have been to ensure that as many spores as possible
dispersed and helped to give rise to viable amoebae. With an erect stalk and a spore mass
on top, the distance of dispersal would depend on the elevation of the fruiting body and
so oh'the number of stalk cells, but too high a stalk would mean that there would not be
many spores left to propagate. An optimal balance would be reached at some
intermediate value for the proportion of cells turning into spores. The actual ratio of
spore to stalk at this optimal balance will depend on the mode of spore propagation —
which could be by means of water, insects or other small animals, or wind. Purely as an
illustrative exercise let us imagine that the spore mass is detached by a gust of wind and
free-falls on account of its own weight (turbulence being neglected). The distance of
dispersal will be the horizontal distance covered by the mass in the time it takes to fall to
the ground. For a fruiting body consisting of AT cells of which JVX are spores and N2 =
N—N! make up the stalk, the height of the structure will be proportional to AT2. Then the
time of fall, and so the distance covered, will be proportional to •N/A^~ or ^/N — N^.
Now, suppose that the availability of food, and so the probability of a spore
germinating, is proportional to the distance of dispersion. A single spore, N of whose
clonal descendants formed a fruiting body, would give rise to N!. ^/N — Nt (times some
constant) spores. It is easily seen that the product Nx. +jN-Ni is a maximum when Nj
= ^ N, that is, at a fixed ratio ofNi to N. Therefore according to this highly simplified
model, 3 of the cells in an aggregate should differentiate into spores and the rest into
stalk if reproductive fitness is to be maximised. This conclusion still leaves us with the
difficult problem of speculating on the possible mutational steps leading to proportion-
ing. An intriguing but attractive possibility would be for proportioning to have evolved
in parallel with patterning and spatial segregation of presumptive cell types, either in the
late aggregate or in the early fruiting body.
8. Discussion
I have tried to provide adaptive explanations for some observed and inferred examples
of communication between cells of D. discoideum. The sequence of evolutionary stages is
suggested to have been as follows (table 1): sporulation of single spores -* non-specific
aggregation and sporulation in a group -» aggregation by chemotaxis -» relay of
chemoattractant and oscillations -> formation of the fruiting body with division of
labour into stalk and spore ~> motility and patterning in the aggregate. Relay could have
preceded chemotaxis instead of following it. The types of mutational events which could
648
Vidyanand Nanjundiah
Table I. A hypothetical- sequence in which the later mutational events enhance, but do not otherwise
interfere with, the effects of the earlier ones. The initial situation is supposed to be one in which cells sporulate
individually when starved; 'X' is a substance synthesised intracellularly in the course of sporulation. For
simplicity X is assumed to be the same in each step; this would correspond to the roles played by cAMP in D.
discoideum.
Effect of mutation
Consequence
Possible reason(s)
for increase in
fitness
Starvation-induced leak
of substance (*X*) through
cell membrane
Tendency of cells to
stick to each other
X can enter a cell
from outside
Cells form clumps
(a) Improved ability
to sporulate;
(b) Cell responds to
deteriorating environment
more rapidly than it would have
Improved prospects
for (post-feeding)
dispersal, but probably
also a decrease in
feeding efficiency
Stickiness inducible
byX
Cells clump only
after feeding is over
As above, but no
decrease in feeding efficiency
Chemotaxis to X
Cells actively
congregate
Eventually, better
dispersal
X relayed from cell to
ceil
Aggregate size
increases
As above
have led to each new stage have been conjectured. In this discussion I confine myself to
making a few general points regarding these explanations.
Two questions which occur at the outset are, how reasonable are adaptive
explanations in this context? And how reasonable are the arguments that have been
advanced suggesting a specific form of selective force for the appearance of a particular
phenotypic trait? The answers to both of these questions will necessarily be indirect and
partial, and so not conclusive.
Cellular slime molds differ quite a bit in their life cycles, but aggregation and the
formation of fruiting bodies consisting of spore masses held aloft stalks are common to
all species. The reason why the details of development differ from one species to another
could have to do either with differing adaptations to different selection pressures or to
the same selective pressures leading to more than one adaptive peak. The possibility that
the more "primitive" species are in the process of evolving towards the D. discoideum
state cannot be ruled out, but appears to be unlikely. More relevant, the adaptive
explanation itself might be incorrect or, at any rate, no less likely than a non-adaptive
one (Gould and Lewontin 1979). It has already been mentioned that the phenomenon of
oscillations is a possible candidate for a selectively neutral trait, even though a plausible
adaptive explanation exists. The extracellular release of a cyclic AMP phosphodies-
terase might be a second such candidate; arguments for and against the viewpoint that
this is a nonadaptive (or maladaptive) trait will be found in the literature (Nanjundiah
and Malchow 1976; Gerisch 1976; Dannon et al 1978). So also the widespread
occurrence of regulation — the constancy in relative proportions of adult cell types —
Communication and social behaviour in D. discoideum 649
might well have a purely developmental, as opposed to evolutionary, explanation.
All the same, in the absence of a specific rival hypothesis — based on selective
neutrality, developmental correlations, even maladaptation — presenting itself, ex-
perience indicates that it is a useful exercise to see whether an adaptive explanation can
be constructed at all and if it can, whether the construction is natural or forced.
A necessary (though not sufficient) condition for a trait to have evolved by selection
amongst differentially adapted phenotypes is that it be subject to genetic control; and a
"natural" construction of an adaptive explanation is one that makes use of plausible
mutational steps. In the case of intercellular communication in D. discoideum both these
requirements are satisfied. Consider the following properties of known mutations in D.
discoideum: (i) an ability to differentiate without cell-to-cell contact (Kay iand Trevan
1981) or normal aggregation (Ishida 1980); (ii) a failure to produce cAMP (Bonner el al
1969); (iii) a failure to respond to cAMP by chemotaxis (Bonner et al 1969); (iv) an
inability to amplify and so relay an external cAMP signal, with the consequent absence
of oscillations (Wurster and Bumann 1981); (v) a failure of normal stalk-to-spore
proportioning (Morrissey et al 1981); and (vi) the absence of a correlation between the
spatial patterning of the presumptive cell types in the slug, which can be normal, and
proportioning in the fruiting body, which can be highly aberrant (Morrissey et al 1981;
Amagai et al 1983). The intention is not to suggest that these mutations must represent a
reversion to an evolutionarily primitive situation, but rather to indicate the plausibility
of the origin and evolution of (present-day) wild-type genes with the properties
demanded. It must be admitted that in one respect this could be a misleading argument:
because selection acts on the genotype and not on single genes, or in other words
because the relation between genes and phenotypic traits is normally not one-to-one,
the life cycle must have evolved as a whole — and not, as in a sense we have imagined, in
bits and pieces. It might appear that caution is all the more warranted when discussing
D. discoideum, given the manifold effects that extracellular cAMP has on its
development (Loomis 1982). Nevertheless, if the effect of a mutation is such as to
influence one aspect of the phenotype more than any other, and if the mutation arises
early enough in evolution for phenotypic effects to be significant, one can assume, as a
good first approximation, that the components of (in this case) the communication
pathway have been independently selected (table 1).
Even if we accept that adaptive explanations for the various features of intercellular
communication we have considered might be valid, could the same end result have been
achieved by other selective forces? The forces we have taken into account have been (a)
selection for an improved efficiency of sporulation and (b) selection for dispersal. The
assumption which runs through our entire analysis is that spatially contiguous,
genetically identical amoebae form the units on which selection can act. Bonner (1967)
starts with the opposite assumption — that in nature cells of diverse genotypes
commonly come together in an aggregate — and comes to quite different conclusions
regarding the function of aggregation. One is that aggregation serves as a partial
substitute for sexuality, because the nuclei of the many genotypes gathered together in
one spore mass have the potential to be redistributed as new combinations. He also
suggests that genetically diverse cells living in close proximity might have developed
mutual deficiencies which could have been overcome by aggregation. There are hints
from the literature that two other selective agencies, predation and the occurrence of
symbiotic or commensal organisms, might favour aggregation and fruiting. Waddell
(1982) has reported the isolation, from bat guano, of a species of predatory cellular slime
650 Vidyanand Nanjundiah
mold. When tested in combination with a number of other species, the predator is able
to delay the morphogenesis and (very probably) consume the amoebae of the prey
species. Ellison and Buss (1983) have found a case of synergism between field specimens
of D. mucoroides (a species similar in many respects to D. discoideum) and the fungus
Mucor hiemalis: the presence of the fungus induces stalk formation in D. mucoroides and
fungal extracts advance rate of fruiting in this and other species (though not in a
laboratory strain of D. discoideum, which conceivably had already been selected for very
rapid development). The stalkless variant of D. mucoroides, which forms in the absence
of the fungus, aggregates normally but neither forms a slug nor migrates. The
aggregating mass rounds up and differentiates straightaway into a mixture of spores
and dead amoebae. While it is not possible at present to seriously consider which
features of communication in D. discoideum might have been selected as adaptations to
predation (to discuss commensal living is even harder), we can imagine that anything
which lowered the risk of predation must have been strongly favoured. So, for instance,
both the tendency to form fruiting bodies as well as an increase in the overall rate of
development would have helped (rate, because in a communication network the same
set of events can have different effects on fitness if the temporal relationships of the
events differ (Bonner 1982)). Both environmental conditions (Schindler and Sussman
1977) and the genotype (Kessin 1977) can influence the life cycle of D. discoideum by
affecting the speed of development. Similarly, using a substance which was harmful to a
predator as an agent of communication -or using a misleading signal (imagine that
cAMP is an alarm pheromone for some predator) would clearly improve the prospects
of survival.
It is important to note that the speculations in this paper refer only to the first steps in
the evolution of pathways of communication in D. discoideum; what one finds today
must be the result of many further steps of refinement. The most important refinement
was probably the origin of receptors (Newell 1977). Receptors would enable signals to
work their effect without having to enter a cell, allowing for increased efficiency and
specificity. Also, the same substance used as a signal could then serve different ends,
depending on which subset of receptors it happened to interact with at any given time.
Other refinements could result in the signal becoming ritualised as has happened in
higher animals (particularly in the case of courtship signals; see Wilson 1975). The
signalling pathway would pick up successively arbitrary modifications and develop into
a link between source and receiver which is highly reliable and specific but displays few
clues as to how it could have evolved. Whatever be the extent to which the primitive
signal has become ritualised, if the view adopted here is correct it must follow that all the
conjectured "signal substances" must be components of the cell's internal response to a
deteriorating environment. Haldane (1955) has asserted as a general principle that a
signal is always a sign of a physiological or psychological condition within the sender;
what I am suggesting is that in the present context the signal is so to speak the condition
itself. To take a specific example, the choice of cAMP as a chemoattractant would not be
totally arbitrary (in the sense that human language is said to be), but would be related to
a role played by cAMP in the internal metabolism of a starved cell, even if the role were
that of a waste product to be discarded. This is a testable statement.
Cellular slime molds are social organisms, and the origin and continued evolution of
sociality all the way to the extreme altruism displayed by stalk cells must surely be a
consequence of, in part, the high degree of genetic relatedness, or even genetic identity,
of the amoebae in an aggregate (Wilson 1975; Bonner 1982). Nevertheless, I believe the
Communication and social behaviour in D. discoideum 651
term 'group selection' is better suited to describe the situation than 'kin selection*. Kin
selection is usually invoked when an individual sacrifices part of its potential
reproductive fitness for the benefit of another individual related to it by common
descent (Hamilton 1964). In the slime mold case, leaving aside the suicide by
presumptive stalk cells for the moment, each of the adaptations we have considered has
the characteristic that it is adopted by every member of the group; therefore its cost or
benefit is the same for every member. It is the group which must succeed or fail, in
comparison with other groups, with these adaptations. Even if this is not true at any one
time because an aggregate consists of more than one genotype, it must be a factor which
comes into play once the less fit genotypes have been eliminated (we take it of course
that the genotypes differ in respect of the adaptations referred to in this paper). An
implicit assumption is that all pre-fruiting cells are functionally identical: if this were
not so, 'kin selection' might well be an appropriate description. An important example
of functional non-identity would be if, for example, only the future stalk cells were to
release the chemoattractant.
What if a cell belonging to a cheater genotype fortuitously gets into an aggregate, a
genotype which responds to external signals with the rest of the group but does not
produce the signal itself? Armstrong (1984), in analysing the survival of "cheaters"
which never form stalk cells, suggests that division of labour would not be an
evolutionary stable strategy under conditions which favour such cheaters. I feel this
side-steps the basic problem, which is to explain why division of labour is observed even
though a "cheater" (of whichever sort) is always at an advantage whenever it arises. The
correct answer, according to me, is that the frequency of the cheater genotype will
increase from one generation to the next until an aggregate consists of so many cheaters
that the strategy does not pay any more. If the cheater only responds to signals released
by the wildtype but does not produce them itself, at some stage the signal strength will
be so weak essentially everywhere except at the locations of the signalling cells that
viable aggregation will simply not occur. If the cheater always differentiates into spores
the final state in evolution will be an aggregate consisting only of mutant spores; in line
with the assumption under which we have operated the spores will be unable to
disperse and therefore to survive. The cheater genotype increases in fitness but
eventually drives the group to extinction. Therefore the normal division of labour is best
described as a case of group selection.
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 6, December 1985, pp. 655-665.
© Printed in India.
Communication asid synchronization of circadian rhythms in
insectivorous bats
G MARIMUTHU and M K CHANDRASHEKARAN
Department of Animal Behaviour, School of Biological Sciences, Madurai Kamaraj
University, Madurai 625021, India
MS received 11 April 1985; revised 12 June 1985
Abstract. There is communication and social synchronization of the circadian rhythm in the
flight activity of the microchiropteran cave-dwelling bat Hipposideros speoris. Thus captive
bats surrounded by free-flying conspecifics synchronize their activity to the colony activity.
The circadian rhythm of a solitary bat in a solitary cave freeruns. Even the rhythm of an 'alien'
bat (Taphozous nudiventris kachhensis) held captive in the hipposiderid bat cave freeruns. But
the rhythms of a closely-related species, Hipposideros fulvus partially entrain to social cues
from Hipposideros speoris. Social synchronization of circadian rhythms in bats may be species-
specific. This synchronization is abolished when continuous light of 10-20 lux is shone inside
the natural cave.
Keywords. Circadian rhythms; bats; ultrasonics; communication; synchronization.
1. Introduction
Circadian rhythms, in nature, entrain to the light-dark cycles (LD cycles) generated by
sunrise/sunset. If the organisms displaying such entrained 24 hr rhythms are brought
into constant conditions of continuous light (LL) or constant darkness (DD) and
invariant temperature of laboratories they 'freerun' (Moore-Ede et al 1982).
Freerunning circadian rhythms have also occasionally been observed to be induced by
the LL of the arctic summer and DD of the arctic winter (Swade and Pittendrigh 1967;
Mueller 1968; Erkinaro 1969). LD cycles are clearly the most universal and dominant of
all zeitgebers (synchronizers). It is now becoming clear, however, that there may be
zeitgebers other than LD cycles. Cyclic variations in temperature (Hoffmann 1968),
cyclic availability of food (Aschoff 1981), noise-silence cycles (Sulzman et al 1977), the
state of the tides in the oceans and even the phases of the moon may act as zeitgebers
(Neumann 1981). Apart from these abiotic fluctuating factors there are also biotic
factors such as bird songs (G winner 1966; Menaker and Eskin 1966), mother-pup
interaction (Davis 1981; Viswanathan and Chandrashekaran 1984) and such other
social stimuli that can synchronize circadian rhythms. In humans social zeitgebers are
more effective than physical zeitgebers (Conroy and Mills 1970; Aschoff 1981; Minors
and Waterhouse 1981). We have been investigating (Marimuthu et al 1978) how the
members of a colony of microchiropteran bats inhabiting a true cave under DD and
constant temperature and constant humidity conditions still time their activity. Such
conditions normally as already explained release circadian rhythms into freeruns.
Abbreviations used: LD, Light-dark cycles; LL, Continuous light; DD, Constant darkness; T, Period length
(time between onset of activity from one day to the next); CF, constant frequency; FM, frequency modulation.
655
656 G Marimuthu and M K Chandrashekaran
Experiments with trapped bats and flight activity monitoring inside a few such caves
indicate that there is clear-cut evidence for social synchronization of the circadian
rhythm in the bat Hipposideros speoris (Marimuthu et al 1978, 1981), Bats held captive
some 40 m inside a cave in DD still began their nightly activity to coincide with the
onset of the foraging activity of the colony.
We now have information that the circadian rhythm of Hipposideros speoris is indeed
being synchronized by social communication and that such social communication is
inexplicably operant in DD but not in LL.
2. Materials and methods
2.1 Study site
The site where the observations and experiments were carried out is a 'true cave' (cave
1), i.e. a cave with just one opening (Twente 1955), situated in a rock complex close to
the Madurai Kamaraj University campus (lat. 9°58' N, long. 78° 10' E). The cave has
several labyrinthine ramifications 1 5-50 m from the cave mouth, which opens on the
northern flank of the rock complex. The bats use several of these pockets as their day-
time roosting place. We chose a site, ca. 40m inside the cave, that showed great
constancy of temperature (27°C±0-5°) and relative humidity of 95% (Lambrecht-
Goettingen thermohygrographs) and was absolutely dark (no light measurable over
periods of 1,000s even on the energy scala log scale of a United Detector Technology
Optometer). A second cave (cave 2) was situated in the same rock complex but with the
cave mouth on the southern side. The conditions obtained in cave 2 were very similar to
the conditions in cave 1; temperature was constant at 30°C, relative humidity was 85 %
and darkness complete. Since the depth of cave 2 was only approximately 5 m an
artificial mud wall had to be constructed and a black cloth curtain erected to ensure
absolute darkness.
2.2* Recording techniques
Our behavioural observations and feeding of captive experimental bats were made
using a noctovision apparatus with a far-red source of light projection and a viewing
scope-screen. The bats did not respond to the switching on of the noctovision by either
turning their heads towards the light source or flying away. We also used battery-
powered torch lights with a combination of filters transmitting red light of > 610 nm,
which seemed not to disturb the animals as much as white light.
Bats were captured on their return flight to the cave in the early hours of the morning
before sunrise and placed in light aluminium-framed activity cages wrapped on all sides
with synthetic gauze material The dimension of the cubical cages (30 x 30 x 30 cm)
permitted flapping flight. Bouts of flight jiggled the cages, which were suspended from
the arms of a metal column held by retort stands. The movements of the cages were
transferred directly to mechanically wound thermohygrograph drums with the aid of
bamboo strip stylets fitted with felt writing tips. Observations and experiments were
made with a minimum of disturbance to the bat population within this rather restrictive
cave, in which the ceiling in its roomiest region was 1-1-5 m from the floor. The captive
Orcadian rhythms in bats 657
bats were hand-fed at irregular hours of day and night with minced cockroaches.
LL conditions were created by using incandescent bulbs and an automobile lead-acid
battery. The incident light at the cage level varied between 1 and 20 lux. The battery was
changed after 36-60 hr at random intervals for recharging, by replacing a second
freshly charged battery. The caged bats could obviously hear orientation and/or
communicative sounds from their unrestrained conspecifics.
3. Results
The hipposiderid bats awaken well before sunset. They then stretch, preen themselves
and undertake short flights within the dark recesses. The bats then fly to a chamber
proximal to the cave mouth. The bats fly around in this chamber 'sampling light* as
other bats have been reported to do (Twente 1955; Voute et al 1974). When it gets
sufficiently dark outside after sunset they begin flying out to forage. Figure 1 A, setting
out the results of a 50-day experiment inside cave 1, indicates that 3 captive bats placed
and experimented upon 40m deep into the cave and surrounded by free-flying
conspecifics still 'knew time'. They timed their flight within their confining cages, to the
flight pattern of the colony. In further confirmation of the need for conspecific-
communication, the results of another 50-day experiment with a solitary bat (in cave 2)
with no conspecifics (figure IB) reveal an impressive 'freerun'. No free-flying
conspecifics, no social communication and no synchronization.
Interestingly even the rhythm of an 'alien' bat (Taphozous nudiventris kachhensis, an
emballonurid species) held captive in the hipposiderid bat cave (cave 1) freeran. Figure 2
shows that even though the social cues emitted by the members of the colony of H.
speoris, during their outflight and return before sunrise, were available to the captive
emballonurid bat T. n. kachhensis the locomotor (flight) activity of the latter exhibited a
spectacular freerunning rhythm with a T shorter than 24 hr. But the rhythms of a closely
related species H.fulvus partially entrain to social cues of H . speoris. Figure 3 illustrates
how the flight activity rhythm of only one of three members of captive H. fulvus
showed, synchronization to the colony activity of its sympatric species H. speoris.
To find out the effect of constant illumination on the social synchronization of the
circadian rhythms we have recorded the flight activity of H. speoris inside cave 1 in LL.
The circadian rhythms of H. speoris freeruns in LL in the laboratory. LL would tend in
itself to induce a freerun and the social input would tend to entrain it. Figure 4 shows
that under LL conditions of 1 lux two of the three captive bats still entrained to the
social cues of their free-flying conspecifics, but the rhythm of the third bat freeran with a
T more than 24 hr. Since we suspected that the LL intensities reaching the bats might
have been sub-threshold, the experiment was repeated with an increased light intensity
of 10-20 lux. Figure 5 sets forth data for one bat. During the initial period of DD its
daily activity rhythm coincided precisely with that of the free-flying conspecifics thus
undergoing social entrainment. However, the same bat (and 3 other bats whose activity
data are presented in figure 6) freeran in LL of 10-20 lux in spite of the social cues still
available to them. The i is longer than 24 hr in LL. LL seems to have abolished
the social synchronization of the circadian rhythm in this bat. Bats in figures 6A, B
exhibited in addition to freerunning rhythms, brief activity bouts for 28 days and 13
days, respectively, corresponding to onset of activity of the conspecifics. The bats flew
inside activity cages for about 1 1-100 min just as the conspecifics flew out of the cave to
658
G Marimuthu and M K Chandrashekaran
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Circadian rhythms in bats
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Figure 2. A typical example of the freenmning of the flight activity pattern of an
emballonurid bat confined in a hipposiderid cave for 60 days. The original felt pen tracings are
double-plotted. The hatched area at the bottom of the figure indicates the time over which the
members of the hipposiderid colony would be active, leaving the cave in a mass exodus during
early night and returning individually for the rest of the night. Other details are as in figure 1
(after Marimuthu and Chandrashekaran 1983b).
Figure 1. A. The flight activity patterns of 3 captive bats of Hipposideros speoris for 40 days
in one case and 50 days in the other two cases recorded 40 m inside a narrow 4true cave* in
Madurai. The bats could fly within the flight activity cages and the movements of the cages
were directly recorded. Activity bouts are indicated by vertical patches and the horizontal lines
indicate rest. The activity/rest data are schematized from original data and presented one
below the other for successive days (after Marimuthu et al 1981). B. A double plot of
activity /rest pattern of a solitary male H. speoris recorded in a cave without any conspecifics
over a period of 50 days. The activity data for day 1 leading horizontally to data for day 2, data
for day 2 to data for day 3 etc to facilitate visual evaluation. Triangles indicate feeding time.
The details are schematized from original felt pen tracings. Other details are as in "A" (after
Marimuthu et al 1981).
660
G Marimuthu and M K Chandrashekaran
Tim« of doy <h)
.2°°
Figure 3. The flight activity patterns of three captive bats of Hipposiderosfulvus confined in
H. speoris cave for 50 days. Activity bouts indicate the original felt pen tracings. Other details
are as in figure 1.
Time of Day (h)
Figure 4. The flight activity patterns of 3 captive bats of H. speoris in LL of 1 lux.
The activity /rest data are schematized from original felt pen tracings. Other details are as in
figure 1.
forage. Such flights of these bats coinciding with the onset of the colony activity account
for an exogenous component which expresses itself regardless of the phase of the
freerunning rhythms. These exogenous components merge into the activity bouts when
the rhythm crosses them during the freerun. The onset of the freerunning oscillatory
component for bat in figure 6A crosses the exogenous component around days 37 and
38 without any sign of even a temporary synchronization, i.e. there is no 'relative
coordination' (Hoist 1939) during the whole run. The freerunning rhythm re-entrained
to the social cues as seen in figure 5 when DD was restored. The light was turned off at
phase which was 1 80° off course relative to colony activity. Re-en trainment set in after a
few 'transient cycles' with the onset component of the re-entrained rhythm coinciding
Orcadian rhythms in bats
661
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+*fc. f— '-. ,,1t Ui*.. .*. . . *• •
^« - - -f - - . *» «UZ^- «*•»-!•» ff»« HP *.V . *.
* — r* W* ffBB *••« H» «V " *** *• * * *• * j
. '*** » 4> « «. 4 * j — -'- <«•*** «W»* *H- ' _f-
« <M- -W * <MM «V ' _»-1- - - • «•»"" «P - _ _•
DD
W 4 *^W >•€ *>" ' «**•* » *•
tt»li»- » «• . L- - BM- 4T* *-»*-»
fe«;<v
C. * . t • *
Figure 5. The flight activity patterns of a captive bat for 62 days. Days 1-7, DD; days 8-41,
LL of 10-20 lux; days 42-62, DD. The activity/rest data containing the original felt pen
tracings are double-plotted and other details are as in figure 1. (after Marimuthu and
Chandrashekaran 1983a).
with the onset of colony activity. Another possibility that cannot be ruled out, however,
is that the freerunning rhythm persists even after turning LL off and what is seen in
figure 5 is some form of 'masking' (Aschoff 1965).
4. Discussion
Literature on communication and synchronization of circadian rhythms is altogether
sparse. Even those of the reports tha| impute social synchronization are only rarely
based on rigorous experimentation. One of the earliest reports to impute social
synchronization among conspecifics was that of Johnson (1926) for the mice of the
genus Peromyscus. Subsequent reports described similar effects for blinded mice Mus
musculus (Halberg et al 1954), male chevrotain antelopes (Dubost 1975), wolf-coyote
hybrids (Roper and Ryan 1977), beaver colonies of Castor canadensis (Potvin and
Bo vet 1975), deer mice (Crowley and Bo vet 1980), macaque monkeys (Rohles and
662
G Marimuthu and M K Chandrashekaran
I
&«.
&
• T~
o
00
O
(SI
h*! i
!!
«••'•* " K '-it;'!i:; •* i
1-4 --'f-.^.-f^ill.!,;,. *? !*
''"'
o
in
h *o
S ^ 'S
- -s
Circadian rhythms in bats
on. Several other reports seeding 10 utusoiijr ^ ^g ^^ ^ present
are anecdotal (Rusak 1981; ChandrasneKa - ication for the cave-
synchronization of the circadian rhvthm by social co ^ _
communication. The communication ^^^^rt^ftoMm**
pheromones and (iii) acoustics. ^%**£S£to* which of these factors plays a
Lve environment, laboratory ***** £™™™™ors have to be simultaneously
dominant role. It is also possible that ^all the se synchronization.
available; Another feature (figure ^) »^^£ocal bimodality in their activity
*' colony and the second p
synchronize. We postu late thaU he frej "^^?ultrasonics of 135 kHz and
between the hipposidend bat (H.^ speons) wru i consisting of a family of
the emballonurid alien (T. n. kachhensis) whid i emits pu^ ^ ^ ^ n
harmonics up to 80 kHz. **«*»# ^f^/^hich is the species specific
fcac^nsis cannot indeed 'hear ^^ 1 1984). Threshold of hearing may not
specfficcues did not synchro^ he acuity my
of Lohmann and Enright (W^jJ^,;!! other non-specific noise.
™
o oma ,! oter non- .
human circadian rhythms ^r™£S^d synchronization of the circadian
K is tempting to assume ^ commu^caUon * ^ dosely related species,
7%^^^^
Figures « represent ^ responses o the b^ca^ ^ ^ ^^
conflicting zeitgeber sitoation. W°^v^_ ta fa ite of the social cues available
rhvthmsofthecaptivebatsfreeruninLLo ^ synchronization of the Orcadian
thus aarently ed *e sync ^ fi)
rv syncron
to them. LL thus apparently ^f ed *e ^ sync ^ ^ fi)
rhythm. The exogenous ««*M^«™2v not merit osciUator status. This may
represent a stimulus-response situation and I may no meffl , (Aschoff 1965, 1981;
Jthephenomenonofr^^^^^^^
Daan dnd Aschoff 1975). ^ igure ^ « .vnchVonized and the third did not.
•- 'SS. — a«a, a few ba* « »—
664 6 Marimuthu and M K Chandrashekaran
otherwise in and around the area inside the cave where the recording was carried out.
To that extent the 'social cues' might indeed have attenuated in the immediate vicinity.
However, within a week or so the bats returned to their original roosting sites, closer to
the captive bats. The exogenous component seen so clearly in bats in figures 6A, B is
further evidence that the onset of the activity of the bat colony had made itself felt, even
though it could not synchronize the rhythm. The freeruns indicate that whatever social
cues that prevail are not 'reaching the clock'. In LL the circadian rhythm apparently
uncouples from social 'zeitgebers'. The reentrainment of the rhythm by the social cues
after the light was turned off (figure 5) further confirmed that it is LL that uncoupled
the circadian rhythm from the social cues. Sulzman et al (1977) reported that the
locomotory activity of squirrel monkeys in noise/silence cycles of 24 hr periods did not
synchronize the rhythm in LL of 600 lux, but the food cycles did synchronize it. It would
be of great interest in the context of our findings to know whether squirrel monkeys
would have entrained to the noise cycles in DD.
Acknowledgements
We are grateful to the Department of Science and Technology, and the University
Grants Commission, New Delhi for financial support and Council of Scientific and
Industrial Research, New Delhi for a fellowship to GM. Part of the work was carried
out in connection with the Indo-German Project on Animal Behaviour. We thank
Professors J Aschoff, E Biinning, W Engelmann, H Markl and G Neuweiler for
critically reading several earlier versions.
References
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Aschoff J 1981 in Handbook of behavioral neurobiology (ed.) J Aschoff (London/New York: Plenum Press)
Vol.4
Chandrashekaran M K 1982 Social cues and circadian rhythms; Curr. Sci. 51 158
Conroy R T and Mills J N 1970 Human circadian rhythms (London: J and A Churchill)
Crowley M and Bovet J 1980 Social synchronization of circadian rhythms in deer mice (Peromyscus
maniculatus); Behav. Ecol Sociobioi 7 99-105
Daan A and Aschoff J 1975 Circadian rhythms of locomotor activity in captive birds and mammals: their
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Davis F C 1981 in Handbook of behavioral neurobiology (ed.) J. Aschoff (London/New York: Plenum Press)
Vol. 4, pp. 255-274
Dubost G 1975 Le Comportement du chevrotain african, Hyemoschus aquaticus Ogilby (Antidactyla,
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Erkinaro E 1969 Der Verlauf desynchronizierter, Circadianer Periodik einer Waldmaus (Apodemus
flavicollis) in Nordfinnland; Z. Vgl. Physiol 64 407-410.
Gwinner E 1 966 Periodicity of a circadian rhythm in birds by species-specific song cycles (Aves, Fringillidae:
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Halberg F, Visscher M B and Bittner J J 1954 Relations of visual factors to eosinophil rhythm in mice; Am. J.
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Hoffmann K 1968 Synchronization der circadianer Aktivitaetsperiodik von Eidechsen durch Tem-
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Hoist E v 1939 Die relative Koordination als Phaenomen und als Methode Zentralnervoeser
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Lohmann M and Enright J T 1967 The influence of mechanical noise on the activity rhythm of finches; Comp.
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Marimuthu G and Chandrashekaran M K 1983a Continuous light inside a cave abolishes the social
synchronization of the circadian rhythm in a bat; Behav. Ecol. Sociobiol 12 321-323
Marimuthu G and Chandrashekaran M K 1983b Social cues of a hipposiderid bat inside a cave fail to entrain
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Marimuthu G, Subbaraj R and Chandrashekaran M K 1978 Social synchronization of the activity rhythm in
a cave-dwelling insectivorous bat; Naturwissenschaften 65 600
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flight activity of the microchiropteran bat Hipposideros speoris; Behav. Ecol. Sociobiol. 8 147-150
Menaker M and Eskin A 1966 Entrainment of circadian rhythms by sound in Passer domesticus; Science 154
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Proceedings (Animal Sciences)
Volume 94, 1985
SUBJECT INDEX
Transpiration rates and acclimation to water
-JST7— r-Jtt^S
laevis Latreille
id phosphatase activity
f a hill stream teleost
(McClelland)
^So-chemical factors in the acridid tad*
behaviour (Orthoptera: Acndidae)
Activity-time budget ._
Activity-time budget in blackbuck 5'
is of feeding and breeding
Orthopteran insects
roscu in the termite genus
Amitermes (Tennitidae, Amitermnmae) 517
feoffs in the evolution of frog calls 623
^nfadvances in animal behaviour 173
A p?and dissolution of crystalline style in some
brvatve molluscs of Porto Novo coastal watery
MAn Analysis of the superparasitic behaviour
arTd hS discrimination of chalcid wasp,
(Hymenoptera: Chalcidoidea)
prawn Macrobrachium lamerru
«»•»»• »
5 on the adrenal gland of rats
j.iia.v*A**».«v** ny
oline dihydrochlonde
*S«ss«=s
ascorbic acid
623
gradus lydekkerianus (Cabra)
feoffs in the evolution of frog calls
brinjal crop
Aphrodisiacs . 17<i
Recent advances in animal behaviour 173
review
(McClelland)
feeder, Purtu «*&*" W
^tionofinsectsociality-Areviewofsorne
'
in animal behaviour 173
^fprtUcesofrodentsintheirnaturalhabitat
-elates of agonistic behaviour in
bengalensis: Hepatic cholesterol and
ascorbic acid
of Scchthonous feeder, ^ra
(Ham.) and of the autochthonous
feeder, Puntius amphibius (Val.) «
Bait
in the termite genus
(fermitidae" Amitermitinae) 517
Baitpreferences of rodents in their natural habitat
667
668
maex
Bandicota bengalensis
Biochemical correlates of agonistic behaviour in
Bandicota bengalensis: Hepatic cholesterol and
ascorbic acid 123
Bats
Communication and synchronization of cir-
cadian rhythms in insectivorous bats 655
Behavioural analysis
Behavioural analysis of feeding and breeding in
Lamellicorn beetles ' 303
Behavioural energetics
Behavioural energetics of some insects 219
Behavioural strategies
Behavioural strategies of emergence, swarming,
mating and oviposition in mayflies 351
Behaviour
Behavioural analysis of feeding and reproduction
in haematophagous insects 225
Functional morphology of air-breathing fishes: A
review 359
Bellamya bengalensis
Effects of starvation on respiration and major
nutrient stores of the prosobranch snail Bellamya
bengalensis (Lamarck) 99
Biochemical changes
Some biochemical changes in the reproductive
cycle of a hill stream teleost Ptwtius chilinoides
(McClelland) 67
Biochemical constituents
Changes in some biochemical constituents of
the fiddler crabs Uca annulipes Latreille and U.
triangularis (Milne Edwards) in response to eye-
stalk removal 475
Bivalve molluscs
pH and dissolution of crystalline style in some
bivalve molluscs of Porto Novo coastal waters
383
Bivoltine
Studies on the silk gland of Bombyx mori: A
comparative analysis during fifth instar de-
velopment 413
Alkaline protease in the midgut of the silkworm
Bombyx mori L: changes during metamorphosis
567
Blackbuck
Activity-time budget in blackbuck 57
Blinding
Extraretinal photoreception involved in photo-
periodic effects on gonadal activity in the Indian
murrel, Channa (Ophiocephalus) punctatus
(Bloch) 153
Bombyx mori
Studies on the silk gland of Bombyx mori: A
comparative analysis during fifth instar de-
velopment 413
Alkaline protease in the midgut of the silkworm
Bombyx mori L: changes during metamorphosis
567
Brain extract
Seasonal variations and the role of neurose-
cretory hormones on the androgenic gland of the
prawn Macrobrachium lamerrii 503
Brinjal
Behavioural response (feeding preference and
dispersal posture) of Aphis gossypii Glover on
brinjal crop 295
Brodifacoum
Laboratory evaluation of anticoagulant treated
baits against Indian field mouse, Mtts booduga
Gray 93
Bromadiolone
Laboratory evaluation of anticoagulant treated
baits against Indian field mouse, Mus booduga
Gray 93
Bursicon
Hormonal rhythm and behavioural trends in
insects 207
Call
623
Tradeoffs in the evolution of frog calls
Calling hormone
Hormonal rhythm and behavioural trends in
insects 207
Canarium euphyllum Kurz
Community and succession of the round-head
borers (Coleoptera: Cerambycidae) infesting the
felled logs of White Dhup, Canarium euphyllum
Kurz 435
Castration
Effect of castration and androgen treatment on
the androgen dependent parameters in the ac-
cessory glands of the slender loris, Loris tardi-
gradus lydekkerianus (Cabra) 427
Cellular slime mold
The evolution of communication and social be-
haviour in Dictyostelium discoideum 639
Cerambycidae
Community and succession of the round-head
borers (Coleoptera: Cerambycidae) infesting the
felled logs of White Dhup, Canarium euphyllum
Kurz 435
Cestoda
Observations on the histology and histochem-
istry of Penetrocephalus plerocercoid (Pseudo-
phyllidea: Cestoda) 1 1
Chalcids
Analysis of the superparasitic behaviour and host
discrimination of chalcid wasps (Hymenoptera:
Chalcidoidea) 325
Channa punctatus
Effect of carbaryl on esterases in the air-breathing
fish Channa punctatus (Bloch) 73
Extraretinal photoreception involved in photo-
periodic effects on gonadal activity in the Indian
murrel, Channa (Ophiocephalus) punctatus
(Bloch) 153
Index
669
Characterization
Effect of carbaryl on esterases in the air-breathing
fish Channa punctatus (Bloch) 73
Chemical composition
Influence of food plants on the food utiliz-
ation and chemical composition of Henosepi-
lachna septima (Coleoptera: coccinellidae) 161
Chemical signals
Recent advances in animal behaviour 1 73
Cholesterol
Some biochemical changes in the reproductive
cycle of hill stream teleost Puntius chilinoides
(McClelland) 67
Biochemical correlates of agonistic behaviour in
Bandicota bengalensis: Hepatic cholesterol and
ascorbic acid 123
Chorion
Ultrastructure of the eggs of Reduviidae: I. Eggs
of Piratinae (Insecta-Heteroptera) 533
Circadian rhythm
The Drosophila circadian clock 187
Circadian rhythms
Communication and synchronization of cir-
cadian rhythms in insectivorous bats 655
Cirrus
Male reproductive system of some digenetic
trematodes 145
Cirrus sac
Male reproductive system of some digenetic
trematodes 145
Cockroach
Electrical stimulation— -Effects on the protein in
the ventral nerve cord of cockroach, Periplaneta
americana 407
Communication
The evolution of communication and social be-
haviour in Dictyostelrjm discoideum 639
Tradeoffs in the evolution of frog calls 623
Communication and synchronization of cir-
cadian rhythms in insectivorous bats 655
Community and ecological succession
Community and succession of the round-head
borers (Coleoptera: Cerambycidae) infesting the
felled logs of White Dhup, Canarium euphyllum
Kurz 435
Communication signals
Recent advances in animal behaviour 173
Corcyra cephalonica
A behavioural assessment of the impact of
some environmental and physiological factors
on the reproductive potential of Corcyra cepha-
lonica (Stainton) (Lepidoptera: Pyralidae) 249
Corpora allata
Development, endocrine organs and moulting
in the embryos of Dysdercus cingulatus Fabr
(Heteroptera: Pyrrhocoridae) 489
Corpora cardiaca
Development, endocrine organs and moulting in
the embryos of Dysdercus cingulatus Fabr
(Heteroptera: Pyrrhocoridae) 489
Crassostrea
pH and dissolution of crystalline style in some
bivalve molluscs of Porto Novo coastal waters
383
Crop fields
Bait preferences of rodents in their natural habitat
117
Crystalline style
pH and dissolution of crystalline style in some
bivalve molluscs of Porto Novo coastal waters
383
Cyprinids
Bionomics of hill-stream cyprinids. I. Food, para-
sites and length-weight relationship of Labeo
dyocheilus (McClell)* 377
Degree of preference
Behavioural responses in terms of feeding and
reproduction in some grasshoppers (Orthoptera:
Insecta) 443
Density
Distribution of earthworms in Madras 557
Deterrent
Physico-chemical factors in the acridid feeding
behaviour (Orthoptera: Acrididae) 283
Diapause
Hormonal rhythm and behavioural trends in
insects 207
Diaphanosoma Senegal
Laboratory culture of Diaphanosoma Senegal
Gauthier, (Crustacea, Cladocera) from south
India * 87
Digenea
Topography of nervous system in two pouched
paramphistomes 129
Distillery effluent
Influence of distillery effluent on growth and
metamorphosis of Rana malabarica (Bibron)
111
Distribution
Distribution of earthworms in Madras 557
DNA
Studies on the silk gland of Bombyx mori: A
comparative analysis during fifth instar de-
velopment 413
Donax
pH and dissolution of crystalline style in some
bivalve molluscs of Porto Novo coastal waters
383
Drosophila
The Drosophila circadian clock 187
Dysdercus cingulatus
Endocrine influence on protein synthesis in the*
fatbodies of female red cotton bug, Dysdercus
cingulatus Fabr 1
Development, endocrine organs and moulting
in the embryos of Dysdercus cingulatus Fabr
(Heteroptera: Pyrrhocoridae) 489
Eavesdropping
On the communication of well-being 575
Eclosion
Hormonal rhythm and behavioural trends in
insects 207
Egg
Ultrastructure of the eggs of Reduviidae: I. Eggs
of Piratinae (Insecta-Heteroptera) 533
Egg development
Studies on mating, spawning and development of
egg in Macrobrachium nobilii (Henderson and
Mathai) 421
Egg viability
A behavioural assessment of the impact of some
environmental and physiological factors on the
reproductive potential of Corcyra cephalonica
(Stainton) (Lepidoptera: Pyralidae) 249
Electrical stimulation
Electrical stimulation — Effects on the protein in
the ventral nerve cord of cockro? ~h, Periplaneta
americana 407
Embryonic moulting
Development, endocrine organs and moulting
in the embryos of Dysdercus cingulatus Fabr
(Heteroptera: Pyrrhocoridae) 489
Emergence
Behavioural strategies of emergence, swarming,
mating and oviposition in mayflies 351
Endocrines
Hormonal rhythm and behavioural trends in
insects 207
Environmental factors
A behavioural assessment of the impact of some
environmental and physiological factors on the
reproductive potential of Corcyra cephalonica
(Stainton) (Lepidoptera: Pyrralidae) 249
Esterases
Effect of carbaryl on esterases in the air-breathing
fish Channa punctatus (Bloch) 73
Ethogram
Behavioural analysis of feeding and breeding in
Orthopteran insects 265
Ethological analysis of drug action
Recent advances in animal behaviour 173
Ethology
Recent advances in animal behaviour 173
Behavioural analysis of feeding and breeding in
Orthopteran insects 265
Evolution
The evolution of communication and social be-
haviour in Dictyostelium discoideum 639
Tradeoffs in the evolution of frog calls 623
Evolution of social behaviour
Kin recognition in animals — a review of
recent findings and a consideration of their
relevance for the theory of kin selection 587
Extrapineal
Extraretinal photoreception involved in photo-
periodic effects on gonadal activity in the
Indian murrel, Channa (Ophiocephalus) punctatus
(Bloch) 153
Extraretinal
Extraretinal photoreception involved in photo-
periodic effects on gonadal activity in the
Indian murrel, Channa (Ophiocephalus) punctatus
(Bloch) 153
Eyestalk ablation
Changes in some biochemical constituents of the
fiddler crabs Uca annulipes Latreille and U.
triangularis (Milne Edwards) in response to eye-
stalk removal 475
Seasonal variations and the role of neuro-
secretory hormones on the androgenic gland of
the prawn Macrobrachium lamerrii 503
Eyestalk extract
Seasonal variations and the role of neurose-
cretory hormones on the androgenic gland of the
prawn Macrobrachium lamerrii 503
Fatbody
Endocrine influence on protein synthesis in the
fatbodies of female red cotton bug, Dysdercus
cingulatus Fabr 1
Fecundity
Behavioural responses in terms of feeding and
reproduction in some grasshoppers (Orthoptera:
Insecta) 443
Fecundity of the allochthonous feeder, Rasbora
daniconius (Ham.) and of the autochthonous
feeder, Puntius amphibius (Val.) 481
Feeding
Behavioural analysis of feeding and reproduction
in haematophagous insects 225
Feeding and breeding behaviour
Behavioural analysis of feeding and breeding in
Lamellicorn beetles 303
Feeding behaviour
Feeding and ovipositional behaviour in some
reduviids (Insecta-Heteroptera) 239
Fibroin
Studies on the silk gland of Bombyx mori: A
comparative analysis during fifth instar de-
velopment 413
Index
671
Fiddler crabs
Changes in some biochemical constituents of
the fiddler crabs Uca annulipes Latreille and V.
triangularis (Milne Edwards) in response to eye-
stalk removal 475
Fischoederius elongatus
Topography of nervous system in two pouched
paramphistomes 129
Flehmen response
Recent advances in animal behaviour 173
Food plant chemicals
Role of behavioural studies in the development of
management strategies for forest insect pests
341
Food plants
Influence of food plants on the food utilization
and chemical composition of Henosepilachna sep-
tima (Coleoptera: coccinellidae) 161
Food utilization
Influence of food plants on the food utilization
and chemical composition of Henosepilachna sep-
tima (Coleoptera: coccinellidae) 161
Host selection and food utilization of the red
pumpkin beetle Raphidopalpa foveicollis (Lucas)
(Chrysomelidae: Coleoptera) 547
Foraging behaviour
Behavioural energetics of some insects 219
Forest pest management
Role of behavioural studies in the development of
management strategies for forest insect pests
341
Frog
Tradeoffs in the evolution of frog calls 623
Gastrothylax crumenifer
Effect of starvation on acid phosphatase activity
in Gastrothylax crumenifer 21
Topography of nervous system in two pouched
paramphistomes 129
Genetic relatedness
Kin recognition in animals — a review of recent
findings and a consideration of their relevance for
the theory of kin selection 587
Gluconeogenesis
Effect of hypoxia on tissue metabolism of midgut
gland of the scorpion Heterometnts fulvipes 139
Gonadal response
Extraretinal photoreception involved in photo-
periodic effects on gonadal activity in the
Indian murrel, Channa (Ophiocephalus) punc tatus
(Bloch) 153
Grasshoppers
Behavioural responses in terms of feeding and
reproduction in some grasshoppers (Orthoptera:
Insecta) 443
Groundnut
Bait preferences of rodents in their natural habitat
117
Growth
Laboratory culture of Diaphanosoma Senegal
Gauthier, (Crustacea, Cladocera) from south
India 87
Influence of distillery effluent on growth and
metamorphosis of Rana malabarica (Bibron)
111
Haematophagous insects
Behavioural analysis of feeding and reproduction
in haematophagous insects 225
Haematozoa
Four new species of trypanoplasms from the fresh
water fishes of the genus Mystus in Maharashtra
25
Haemoflagellates
Four new species of trypanoplasms from the fresh
water fishes of the genus Mystus in Maharashtra
25
Hatching
Hormonal rhythm and behavioural trends in
insects 207
Studies on the induced spawning and larval
rearing of a freshwater catfish, Mystus punctatus
(Jerdon) 389
Heliothis armigera (Hubner)
Investigations on Heliothis armigera (Hubner)
in Marathwada XXVIII. Key mortality factors
in regular and overlapping generations on
pigeonpea 463
Heliothis spp.
Mermithid nematodes as parasites of Heliothis
spp. and other crop pests in Andhra Pradesh,
India 509
Hematology
Functional morphology of air-breathing fishes: A
review 359
Henosepilachna septima
Influence of food plants on the food utilization
and chemical composition of Henosepilachna sep-
tima (Coleoptera: coccinellidae) 161
Heterometrus fulvipes
Effect of hypoxia on tissue metabolism of midgut
gland of the scorpion Heterometrus fulvipes 1 39
Hexamermis spp
Mermithid nematodes as parasites of Heliothis
spp. and other crop pests in Andhra Pradesh,
India . 509
High-energy foragers
Behavioural energetics of some insects 219
Histochemistry
Observations on the histology and histochem-
istry of Penetrocephalus plerocercoid (Pseudo-
phyllidea: Cestoda) 1 1
Functional morphology of air-breathing fishes: A
review 359
672
Index
Histology
Observations on the histology and histochem-
istry of Penetrocephalus plerocercoid (Pseudo-
phyllidea: Cestoda) 1 1
Histophysiology
Recent advances in animal behaviour 173
Hormonal control
Recent advances in animal behaviour 173
Host discrimination
An analysis of the superparasitic behaviour
and host discrimination of chalcid wasps
(Hymenoptera: Chalcidoidea) 325
Host plants
Larval and post-larval development of Spodo-
ptera litura (Fabricius) on some host plants 49
Host selection
Host selection and food utilization of the red
pumpkin beetle, Raphidopalpa foveicollis (Lucas)
(Chrysomelidae: Coleoptera) 547
Hyblaea puera
Role of behavioural studies in the development of
management strategies for forest insect pests
341
Hybrid
Studies on the silk gland of Bombyx mori: A
comparative analysis during fifth instar de-
velopment 413
Hypoglycemia
Changes in some biochemical constituents of
the fiddler crabs Uca annulipes Latreille and U.
triangularis (Milne Edwards) in response to eye-
stalk removal 475
Hypoxia
Effect of hypoxia on tissue metabolism of midgut
gland of the scorpion Heterometrus fuhipes 1 39
Inanition stress
Irradiation effects on the adrenal gland of rats
undergoing inanition stress 79
Inbreeding
Kin recognition in animals — a review of recent
findings and a consideration of their relevance for
the theory of kin selection 587
Induced breeding
Studies on the induced spawning and larval
rearing of a freshwater catfish, Mystus punctatus
(Jerdon) 389
Information and manipulations
On the communication of well-being 575
Insect behaviour
Hormones in insect behaviour 197
Role of behavioural studies in the development of
management strategies for forest insect pests
341
Insect hormones
Hormones in insect behaviour 197
Insect reproduction
Hormones in insect behaviour 197
Irradiation
Irradiation effects on the adrenal gland of rats
undergoing inanition stress 79
Isoenzymes
Effects of DDT and malathion on tissue succinic
dehydrogenase activity (SDH) and lactic dehydro-
genase isoenzymes (LDH) of Sarotherodon
mossambicus (Peters) 527
Isopods
Transpiration rates and acclimation to water
and temperature of the tropical woodlice,
Porcellionides pruinosus Brandt and Porcellio
laevis Latreille 469
Isoptera
Wing microsculpturing in the Brazilian termite
family Serritermitidae (Serritermes serrifer,
Isoptera), and its bearing on phylogeny 399
Wing microsculpturing in the termite genus
Amitermes (Termitidae, Amitermitinae) 517
Juvenile hormone
Endocrine influence on protein synthesis in the
fatbodies of female red cotton bug, Dysdercus
cingulatus Fabr 1
Katelysia
pH and dissolution of crystalline style in some
bivalve molluscs of Porto Novo coastal waters
383
Key mortality factors t
Investigations on Heliothis armigera (Hubner)
in Marathwada XXVIII. Key mortality factors
in regular and overlapping generations on
pigeonpea 463
Kin recognition
Kin recognition in animals — a review of recent
findings and a consideration of their relevance for
the theory of kin selection 587
Kin selection
Evolution of insect sociality — A review of some
attempts to test modern theories 309
Kin recognition in animals — a review of recent
findings and a consideration of their relevance for
the theory of kin selection 587
Kinship discrimination
On the communication of well-being 573
Labeo dyocheilus
Bionomics of hill-stream cyprinids. I. Food, para-
sites and length weight relationship of Labeo
dyocheilus (McClell) 377
Lamellicora beetles
Behavioural analysis of feeding and breeding in
Lamellicorn beetles 303
Larval development
Larval and post-larval development of Spodo-
ptera litura (Fabricius) on some host plants 49
Studies on the induced spawning and larval
rearing of a freshwater catfish, Mystus punctatus
(Jerdon) 389
673
Index
dispersal posture) c _
brinjal crop stalk removal
^BaUreferencesofrodentsintheirnaturalhabitat
^of^-^**^ jggZT^ ^ and
fish Channa punctate OJUxtt^ ^ fa lnfluence of ^^lerv effluent gr .
ascorbic acid
L. mauritii x^aHras 557
dras
.mdwitti •„ Madras 557 rearing u» ««"-—- 38*
Distribution of earthworms m Maar (jerdon) themid(5Ut of the silkworm
iori, Mr^r«to '^fcfce"^ro(!en treatment on Alkaline Prot^an^uSg metamorphosis
^"
groto lydekkerianus (Cabra)
Macrobrackium lamerrii
prawn M^fcr^ «— Midgut gland metabolism of
Macrobrachium nooim ^ development of Effect of hypoxia on t Heterometrus
egg in MacrofcrflcMum not"/" en e 421 ^^
Mathai) Midgut protease . the mjdgut of the silkworm
Matingu • «i assessment of the impact of some Alkaline protease ^m e rf metamorphosis
A behavioural assessmeni factors on the gombyx man L. changes uu 6 J6?
environmental and ^f**, cepfta/omM
S±S;LSSe-°U^) swaj^ M1^es in insect behaviour
S^^^^^ll^ "preferencesofrodentsintheirnaturalhabitat
M-thai) ^Smorphologyofair-breathingnshes^A
Matured tissue preference^;nBoreferenceand review ^
^SSorphologyofair-breathingflshes.A
Functional morpnoiugj - «» - 359
brinjal crop review
Mortality . o^tirnaffulant treated
Gray
Gray management
Atonal rhythm and behavioural trends^
insects
N
of Heliothis
velopment
India
674
Index
Multivoltine
Alkaline protease in the midgut of the silkworm
Bombyx mori L.: changes during metamorphosis
567
Muscle
Effect of carbaryl on esterases in the air-breathing
fish Channa punctatus (Bloch) 73
Mutualism
Evolution of insect sociality — review of some
attempts to test modern theories 309
Mystus punctatus
Studies on the induced spawning and larval
rearing of a freshwater catfish, Mystus punctatus
(Jerdon) 389
Nervous system
Topography of nervous system in two pouched
paramphistomes 129
Neurophysiological activity
Hormones in insect behaviour 197
Neurosecretory cells
Endocrine influence on protein synthesis in the
fatbodies of female red cotton bug, Dysdercus
cingulatus Fabr 1
Development, endocrine organs and moulting
in the embryos of Dysdercus cingulatus Fabr
(Heteroptera: Pyrrhocoridae) 489
Nilaparvata lugens
Mechanism of resistance in rice varieties showing
differential reaction to brown planthopper 37
No-choice tests
Laboratory evaluation of anticoagulant treated
baits against Indian field mouse, Mus booduga
Gray 93
Nutrient stores
Effects of starvation on respiration and major
nutrient stores of the prosobranch snail Bellamy a
bengalensis (Lamarck) 99
Olfactory cues
Recent advances in animal behaviour 173
Operculum
Ultrastructure of the eggs of Reduviidae: I. Eggs
of Piratinae (Insecta-Heteroptera) 533
Organochlorine
Effects of DDT and malathion on tissues succinic
dehydrogenase activity (SDH) and lactic dehydro-
genase isoenzymes (LDH) of Sarotherodon
mossambicus (Peters) 527
Organophosphorous
Effects of DDT and malathion on tissue succinic
dehydrogenase activity (SDH) and lactic dehydro-
genase isoenzymes (LDH) of Sarotherodon
mossambicus (Peters) 527
O, serrata
Distribution of earthworms in Madras 557
Osmetrichia
Recent advances in animal behaviour 1 73
Overlapping of species
Community and succession of the round-head
borers (Coleoptera: Cerambycidae) infesting the
felled logs of White Dhup, Canarium euphyllum
Kurz 435
Ovipositional behaviour
Feeding and ovipositional behaviour in some
reduviids (Insecta-Heteroptera) 239
Oviposition
A behavioural assessment of the impact of some
environmental and physiological factors on the
reproductive potential of Corcyra cephalonica
(Stainton) (Lepidoptera: Pyralidae) 249
Behavioural strategies of emergence, swarming,
mating and oviposition in mayflies 351
Ovomermis albicans
Mermithid nematodes as parasites of Heliothis
spp. and other crop pests in Andhra Pradesh,
India 509
Oxidative metabolism
Effects of DDT and malathion on tissue succinic
dehydrogenase activity (SDH) and lactic dehydro-
genase isoenzymes (LDH) of Sarotherodon
mossambicus (Peters) 527
Palpation
Physico-chemical factors in the acridid feeding
behaviour (Orthoptera: Acrididae) 283
Paramphistome
Topography of nervous system in two pouched
paramphistomes 129
Parasites
Bionomics of hill-stream cyprinids. I. Food, para-
sites and length- weight relationship of Labeo
dyocheilus (McClell)* 377
Parasitization index
Bionomics of hill-stream cyprinids. I. Food, para-
sites and length weight relationship of Labeo
dyocheilus (McClell)* 377
Parental manipulation
Evolution of insect sociality — A review of some
attempts to test modern theories 309
Penetrocephalus
Observations on the histology and histochem-
istry of Penetrocephalus plerocercoid (Pseudo-
phyllidea: Cestoda) 1 1
Pentatomimermis sp
Mermithid nematodes as parasites of Heliothis
spp. and other crop pests in Andhra Pradesh,
India 509
Phagostimulant
Physico-chemical factors in the acridid feeding
behaviour (Orthoptera: Acrididae) 283
Phase response curves
The Drosophila circadian clock 1 87
Index
Pheromones
Recent advances in animal behaviour l /3
Pheromones in insect control
Role of behavioural studies in the development ot
management strategies for forest insect pests
34 1
photoreception involved in photo-
effects on gonadal activity in the Indian
Channa (Ophiocephalus)
(Bloch)
effects on gonadal actwity
the
Post embryonic development
Behavioural responses in terms i»!
reproduction in some grasshopper
ptera: Insecta)
Post-lar\'al development
Larval and post-larval development oi \^*M*
ptera litura (Fabricius) on some host p!anis
Prostate glands
Male reproductive system of seme uigentu
trematodes
Protein
Endocrine influence on protein s>
fatbodies of female red cotton bug.
cingulatus Fabr
Some biochemical changes in the r
cycle of a hill stream teleosi Puntu*
(Bloch)
icroscuipturng in the Brazilian tenge
Serritermitidae (Serritemes sernfer
and its bearing on phytogeny 399
physiological factors
Aevonl Kmcort
Prothoracic $ands
Development, endocrine orgaB. art
in the embryos of Dysdera* ^u!
(Heteroptera: Pynhocondae)
-
feeder,
of the
l
ngcoup~ flrm^erfl VnuDuw 0^^ ethology
Investigations on J«« w ^^ factors ^ tioli of insect sociality A !
in Marathwada XXV^-^ ions on to test modem Aeon*
in regular and overl PP ^ ^^^ food utstotion
pigeonpea Behavioural responses it
pi ratinae ^ Reduviidae: I . Eggs reproduction in some grass nopp
. /"r.^fw-tfl-T-leteropteraj Insecia;
*r oirfttmae (insecui IAV r
ative ethology f ^^
ltion of insect soaalrt>-A«tM* ^
_._ lpts to test modem theories
Quantitative ^u^^^ of f<cdwg and
uiw**"'— -- nc_ta.Heteroptera) — Insecta)
of Piratinae (Insecta ncic F
Pituitary extract spawning and larval
SS-.-S^*1"1^
(Jerdon)
Plerocercoid ^ K.QfoloeY and histochem-
=
phylHdea: Cestoda)
utdi»t««« oi the
review
lteh,M
feeder, R«lww
laevis Latreille
469
is Latreille
676
Index
Reproduction
Laboratory culture of Diaphanosoma Senegal
Gauthier, (Crustacea, Cladocera) from south
India 87
Behavioural analysis of feeding and reproduction
in haematophagous insects 225
Reproductive investment in insects
Recent advances in animal behaviour 173
Reptiles
Recent advances in animal behaviour 173
Rhythm
Hormonal rhythm and behavioural trends in
insects 207
Rice moth
A behavioural assessment of the impact of some
environmental and physiological factors on the
reproductive potential of Corcyra cephalonica
(Stainton) (Lepidoptera: Pyralidae) 249
Rice varieties
Mechanism of resistance in rice varieties showing
differential reaction to brown planthopper 37
Riverine ecosystems
Bionomics of hill-stream cyprinids. I. Food, para-
sites and length-weight relationship of Labeo
dyocheilus (McClell)* 377
RNA
Studies on the silk gland of Bombyx mori\ A
comparative analysis during fifth instar
development 413
Rodents
Bait preferences of rodents in their natural habitat
117
Sceliphron violaceum
Behavioural energetics of some insects 219
Scent marking
Recent advances in animal behaviour 173
Schooling and alarm responses
Recent advances in animal behaviour 173
Second generation anticoagulants
Laboratory evaluation of anticoagulant treated
baits against Indian field mouse, Mus booduga
Gray 93
Secretions of specialised skin glands
Recent advances in animal behaviour 173
Secretory activity
Development, endocrine organs and moulting
in the embryos of Dysdercus cingulatus Fabr
(Heteroptera: Pyrrhocoridae) 489
Sensilla
Physico-chemical factors in the acridid feeding
behaviour (Orthoptera: Acrididae) 283
Sensillae
Behavioural responses in terms of feeding and
reproduction in some grasshoppers (Orthoptera:
Insecta) 443
Serritermes serrifer
Wing microsculpturing in the Brazilian termite
family Serritermitidae (Serritermes serrifer,
Isoptera) and its bearing on phylogeny 399
Serritermitidae
Wing microsculpturing in the Brazilian termite
family Serritermitidae (Serritermes serrifer^
Isoptera) and its bearing on phylogeny 399
Sex-investment ratios
Evolution of insect sociality — A review of some
attempts to test modern theories 309
Sex pheromones
Application of sex pheromones in sugarcane pest
management 333
Sexual selection
Tradeoffs in the evolution of frog calls 623
Sigmoid growth rate
Behavioural response (feeding preference and
dispersal posture) of Aphis gossypii Glover on
brinjal crop 295
Silk gland
Studies on the silk gland of Bombyx mori: A
comparative analysis during fifth instar de-
velopment 413
Slender loris
Effect of castration and androgen treatment on
the androgen dependent parameters in the ac-
cessory glands of the slender loris, Loris tardi-
gradus lydekkerianus (Cabra) 427
Social animals
Kin recognition in animals — a review of recent
findings and a consideration of their relevance for
the theory of kin selection 587
Social insects
Hormones in insect behaviour 197
Evolution of insect sociality — A review of some
attempts to test modern theories 309
Social postures in mice
Recent advances in animal behaviour 173
Social stress
Biochemical correlates of agonistic behaviour in
Bandicota bengalensis: Hepatic cholesterol and
ascorbic acid 123
Spawning
Studies on mating, spawning and development of
egg in Macrobrachium nobilti (Henderson and
Mathai) 421
S-phenetyl formamidino 4 (N-ethyl isothioamide)
morpholine dihydrochloride
Effect of two graded doses of x-irradiation on the
rat adrenal gland and its protection by S-phenetyl
formamidino 4 (N-ethyl isothioamide) morph-
otine dihydrochloride 523
Spodoptera litura
Larval and post-larval development of Spodo-
ptera litura (Fabricius) on some host plants 49
Index
677
Starvation
Effect of starvation on acid phosphatase activity
in Gastrothylax crumenifer 21
Starvation effects
Effects of starvation on respiration and major
nutrient stores of the prosobranch snail Bellamya
bengalensis (Lamarck) 99
Style dissolution
pH and dissolution of crystalline style in some
bivalve molluscs of Porto Novo coastal waters
383
Sugarcane
Application of sex pheromones in sugarcane pest
management 333
Sugars
Some biochemical changes in the reproductive
cycle of hill stream teleost Puntius chilinoides
(McClelland) 67
Superparasitism
An analysis of the superparasitic behaviour
and host discrimination of chalcid wasps
(Hymenoptera: Chalcidoidea) 325
Susceptibility
Laboratory evaluation of anticoagulant treated
baits against Indian field mouse, Mus booduga
Gray 93
Swarming
Behavioural strategies of emergence, swarming,
mating and oviposition in mayflies 351
Synchronization
Communication and synchronization of cir-
cadian rhythms in insectivorous bats 655
Temperature
Transpiration rates and acclimation to water
and temperature of the tropical woodlice,
Porcellionides pruinosus Brandt and Porcellio
laevis Latreille 469
Termites
Wing microsculpturing in the termite genus
Amitermes (Termitidae, Amitermitinae) 517
Thoracic ganglion
Seasonal variations and the role of neurose-
cretory hormones on the androgenic gland of the
prawn Macrobrachium lamerrii 503
Tissue metabolism
Effect of hypoxia on tissue metabolism of
midgut gland of the scorpion Heterometrus
fulvipes 139
Total protein
Studies on the silk gland of Bombyx mori: A
comparative analysis during fifth instar de-
velopment 413
Transpiration
Transpiration rates and acclimation to water
and temperature of the tropical woodlice,
Porcellionides pruinosus Brandt and Porcellio
laevis Latreille 469
Trematoda
Topography of nervous system in two pouched
paramphistomes 129
Trematodes
Male reproductive system of some digenetic
trematodes 145
Trophic and reproductive behaviour
Behavioural analysis of feeding and breeding in
Orthopteran insects 265
Trypanoplasma
Four new species of trypanoplasms from the fresh
water fishes of the genus Mystus in Maharashtra
25
Ultrasonics
Communications and synchronization of cir-
cadian rhythms in insectivorous bats 655
Varietal resistance
Mechanism of resistance in rice varieties showing
differential reaction to brown planthopper 37
Ventral nerve cord
Electrical stimulation — Effects on the protein in
the ventral nerve cord of cockroach, Periplaneta
americana 407
Vertebrates
Recent advances in animal behaviour 1 73
Visual and vocal signals
Recent advances in animal behaviour 1 73
Vocalisation in birds and mammals
Recent advances in animal behaviour 173
Vocalization
Tradeoffs in the evolution of frog calls 623
Walking low-energy foragers
Behavioural energetics of some insects 219
Warfarin
Laboratory evaluation of anticoagulant treated
baits against Indian field mouse, Mus booduga
Gray 93
Wheat
Bait preferences of rodents in their natural habitat
117
Wing microsculpturing
Wing microsculpturing in the Brazilian termite
family Serritermitidae (Serritermes serrifer,
Isoptera) and its bearing on phylogeny 399
Wing microsculpturing in the termite genus
Amitermes (Termitidae, Amitermitinae) 517
X-rays
Effect of two graded doses of x-irradiation on the
rat adrenal gland and its protection by S-phenetyl
formamidino 4(N-ethyl isothioamide) morph-
oline dihydrochloride 523
AUTHOR INDEX
Ahmad Nafis
Bait preferences of rodents in their natural habitat
117
Alexander K M
Recent advances in animal behaviour 173
Ananthakrishnan T N
Behavioural responses in terms of feeding and
reproduction in some grasshoppers (Orthoptera:
Insecta) 443
Annadurai R S
see Raman K 547
Arunachalarn S
Effect of carbaryl on esterases in the air-breathing
fish Channa punctatus (Bloch) 73
Babu G Ramesh
Effect of hypoxia on tissue metabolism of midgut
gland of the scorpion Heterometrus fulvipes 139
Balasubramanian M P
see Arunachalam S 73
Balasubramanian N K
see Kumar K Prem 481
Balasubramanyam M
Laboratory evaluation of anticoagulant treated
baits against Indian field mouse, Mus booduga
Gray 93
Balasundaram C
Studies on mating, spawning and development of
egg in Macrobrachium nobilii (Henderson and
Mathai) 421
Balasundaram K
see Maheswari S L 407
Banerjee T K
Behavioural response (feeding preference and
dispersal posture) of Aphis gossypii Glover on
brinjal crop 295
Bhatnagar V S
Mermithid nematodes as parasites of Heliothis
spp. and other crop pests in Andhra Pradesh,
India 509
Bilapate G G
see Garad G P 49
Investigations on Heliothis armigera (Hubner)
in Marathwada XXVIII. Key mortality fac-
tors in regular and overlapping generations on
pigeonpea 463
Chandra K J
Observations on the histology and histochem-
istry of Penetrocephalus plerocercoid (Pseudo-
phyllidea: Cestoda) 1 1
Chandrashekaran M K
The Drosophila circadian clock 187
see Marimuthu G 655
Chaturvedi P K
see Hasan S S 79
Effect of two graded doses of X-irradiation on the
rat adrenal gland and its protection by S-phenetyl
formamidino 4(N-ethyl isothioamide) morph-
oline dihydrochloride 523
Chetty J Sulochana
see Ganga G 161
Christopher M J
see Balasubramanyam M 93
David H
Application of sex pheromones in sugarcane pest
management 333
Davies J C
see Bhatnagar V S 509
De Souza S J Stephen T
see Haniffa MA 1 1 1
Dhileepan K
see Ananthakrishnan T N 443
Dutta M Hiran
Functional morphology of air-breathing fishes: A
review 359
Easwaramoorthy S
see David H
333
Gadagkar Raghavendra
Evolution of insect sociality — A review of some
attempts to test modern theories 309
Kin recognition in animals — a review of recent
findings and a consideration of their relevance for
the theory of kin selection 587
Gadgil Madhav
On the communication of well-being 575
Gadgil Sulochana
see Gadgil Madhav 575
Ganga G
Influence of food plants on the food utilization
and chemical composition of Henosepilachna sep-
tima (Coleoptera: coccinellidae) 161
Garad G P
Larval and post-larval development of Spodo-
ptera litura (Fabricius) on some host plants 49
Garg S K
Extraretinal photoreception involved in photo-
periodic effects on gonadal activity in the Indian
murrel, Channa (Ophiocephalus) punctatus
(Bloch) 153
Index
679
Gopal K Satya
Male reproductive system of some digenetic
trematodes 145
Gyananath G
see Sarojini R 503
Hameed P Shahul
pH and dissolution of crystalline style in some
bivalve molluscs of Porto Novo coastal waters
383
HaniflFa M A
Influence of distillery effluent on growth and
metamorphosis of Rana malabarica (Bibron)
111
Haridass E T
Feeding and ovipositional behaviour in some
reduviids (Insecta-Heteroptera) 239
Ultrastructure of the eggs of Reduviidae: I. Eggs
of Piratinae (Insecta-Heteroptera) 533
Hasan S S
Irradiation effects on the adrenal gland of rats
undergoing inanition stress 79
see Chaturvedi P K 523
Hegde Malati
see Gadgil Madhav 575
Ismail A Sultan
Distribution of earthworms in Madras
557
Jacob Mariamma
Development, endocrine organs and moulting in
the embryos of Dysdercus cingulatus Fabr
(Heteroptera: Pyrrhocoridae) 489
Jadhav D R
see Bhatnagar V S 509
Jain S K
see Garg S K l53
John Elizabeth
see Sridhara Shakunthala 123
John P A
see Kumar K Prem 481
Joshi N V
see Gadgil Madhav 575
Jyothirmayi G N
see Babu G Ramesh 139
427
37
Kadam K M
see Manjula A
Kalode M B
see Reddy K Venugopala
Khan T N
Community and succession of the round-head
borers (Coleoptera: Cerambycidae) infesting the
felled logs of White Dhup, Canahum euphyllum
v 435
Kurz
Krishna S S
A behavioural assessment of the impact of some
environmental and physiological factors on the
reproductive potential of Corcyra cephalomca
(Stainton) (Lepidoptera: Pyralidae) 249
Krishnaswamy S
see Venkataraman K 87
Kumaraguru A K
see Balasundaram C 421
Kumari K Veena
see Veeresh G K 303
Kumar K Prem
Fecundity of the allochthonous feeder, Rasbora
daniconius (Ham), and of the autochthonous
feeder, Puntius amphibius (Val) 48 1
Maheswari S L
Electrical stimulation — Effects on the protein in
the ventral nerve cord of cockroach, Periplaneta
americana 407
Malhotra K Sandeep
Bionomics of hill-stream cyprinids. I. Food, para-
sites and length-weight relationship of Labeo
dyocheilus (McClell) 377
Manjula A
Effect of castration and androgen treatment on
the androgen dependent parameters in the ac-
cessory glands of the slender loris, Loris lardi-
gradus lydekkerianus (Cabra) 427
Manoharan T
see Ganga G 161
Marimuthu G
Communication and synchronization of cir-
cadian rhythms in insectivorous bats 655
Mishra Neerja
Topography of nervous system in two pouched
paramphistomes 129
Mishra S N
see Krishna S S 249
Munshi J S Datta
see Dutta M Hiran 359
Muraleedharan D
see Raghavan Raji 1
Hormonal rhythm and behavioural trends in
insects 207
Muralirangan M C
Physico-chemical factors in the acridid feeding
bahaviour (Orthoptera: Acrididae) 283
Muralirangan Meera
see Muralirangan M C 283
Murthy V A
see Ismail A Sultan 557
Murugesan A G
5*eHaniffaMA 1U
Nair G Achuthan
Transpiration rates and acclimation to water
and temperature of the tropical woodlice,
Porcellionides prumosus Brandt and Procellio
laevis Latreille ^9
Nair K S S
Role of behavioural studies in the development oi
management strategies for forest insect pests
341
680
Index
Nair N Balakrishnan
see Nair G Achuthan 469
Nandagopal V
see David H 333
Nanjundiah Vidyanand
The evolution of communication and social be-
haviour in Dictyostelium discoidewn 639
Narendran T C
An analysis of the superparasitic behaviour and
host discrimination of chalcid wasps (Hymeno-
ptera: Chalcidoidea) 325
Natarajan P
see Ramanathan N 389
Nauriyal B P
Some biochemical changes in the reproductive
cycle of a hill stream teleost Puntius chilinoides
(McClelland) 67
Nesbitt B F
see David H 333
Padmanaban B
. see Ananthakrishnan T N 443
Palanichamy S
see Arunachalam S 73
Pandian T J
Behavioural energetics of some insects 219
Paranjape S Y
Behavioural analysis of feeding and breeding in
Orthopteran insects 265
Parshad V R
see Ahmad Nafis 117
Pawar C S
see Bhatnagar V S 509
Prabhu V K K
Hormones in insect behaviour 197
see Jacob Mariamma 489
Prasad N L N S
Activity-tune budget in blackbuck 57
Prasad R S
Behavioural analysis of feeding and reproduction
in haematophagous insects 225
Purushotham K R
see Balasubramanyam M 93
Raghavan Raji
Endocrine influence on protein synthesis in the
fatbodies of female red cotton bug, Dysdercus
cingulatus Fabr 1
Ramalingam K
Effects of DDT and malathion on tissue succinic
dehydrogenase activity (SDH) and lactic dehydro-
genase isoenzymes (LDH) of Sarotherodon
mossambicus (Peters) 527
Ramanathan N
Studies on the induced spawning and larval
rearing of a freshwater catfish, Mystus punctatus
(Jerdon) 389
Raman K
Host selection and food utilization of the red
pumpkin beetle Raphidopalpa foveicollis (Lucas)
(Chrysomelidae: Coleoptera) 547
Rand A Stanley
Tradeoffs in the evolution of frog calls 623
Rao D G V Prasada
see Rao G V Krishna 475
Rao G V Krishna
Changes in some biochemical constituents of
the fiddler crabs Uca annulipes Latreille and U.
triangularis (Milne Edwards) in response to eye-
stalk removal 475
Rao K Hanumantha
see Chandra K J il
see Gopal K Satya 145
Rao M Balaparameswara
see Reddy D Satya 99
Rao P Venkateswara
see Babu G Ramesh 139
Rao Y Prabhakara
see Rao G V Krishna 475
Rathore N S
see Roonwal ML 399
see Roonwal ML 517
Raychaudhuri D
see Banerjee T K 295
Reddy D Satya
Effects of starvation on^respiration and major
nutrient stores of the prosbbranch snail Bellamy a
bengalensis (Lamarck) 99
Reddy K Venugopala. .
Mechanism of resistance in rice varieties showing
differential reaction to brown planthopper 37
Roonwal M L
Wing microsculpturing in the Brazilian termite
family Serritermitidae (Serritermes serrifer,
Isoptera) and its bearing on phylogeny 399
Wing microsculpturing in the termite genus
Amitermes (Termitidae, Amitermitinae) 517
Sarangi S K
Studies on the silk gland of Bombyx mori: A
comparative analysis during fifth instar
development 413
Alkaline protease in the midgut of the silkworm
Bombyx mori L: changes during metamorphosis
567
Sarojini R
Seasonal variations and the role of neurose-
cretory hormones on the androgenic gland of the
prawn Macrobrachium lamerrii 503
Sathyanarayana M C
Effect of starvation on acid phosphatase activity
in Gastrothylax cnanenifer 21
Selvarajan V R
see Maheswari S L 407
Index
681
Selvi R Senthamil
see Ganga G 161
Shivpuje P R
see Garad G P 49
Shyamasundari K
see Chandra K J 11
Singh H R
see Nauriyal B P 67
Sivaramakrishnan K G
Behavioural strategies, of emergence, swarming,
mating and oviposition in mayflies 351
Somasekhar T
see Sridhara Shakunthala 123
Sridhara Shakunthala
Biochemical correlates of agonistic behaviour in
Bandicota bengalensis: Hepatic cholesterol and
ascorbic acid 123
Subramanyam M V V
see Sridhara Shakunthala 123
Sukumaran N
see Ramanathan N 389
Sundarabai A
see Sridhara Shakunthala 123
Tandon Veena
see Mishra Neerja
129
Veeresh G K
Behavioural analysis of feeding and breeding in
Lamellicorn beetles 303
Venkataraman K
Laboratory culture of Diaphanosoma Senegal
Gauthier, (Crustacea, Cladocera) from South
India 87
see Sivaramakrishnan KG 351
Vijayalakshmi C
see Gopal K Satya 145
Wahul M A
Four new species of trypanoplasms from the fresh
water fishes of the genus Mystus in Maharashtra
25
Xavier Barnabas
see Haniffa M A
111
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