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Full text of "Proceedings of the Indian Academy of Sciences- Section B"

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 Communication 9 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+3 c C, 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 CO 2 (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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Hill L 1965 Incorporation of C 14 glycine into the proteins of the fatbody of the desert locust during ovarian 

development; J. Insect PhysioL 111 1605-1615 
Jalaja M and Prabhu V K K 1971 Blood protein concentration in relation to vitellogenesis in Dysdercus 

cingulatus; Experientia 27 639-640 
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 
Possompes B 1956 Development ovarian apres ablation due corpus allatum juvenile chez Calliphora 

erythrocephala Meig. (Diptrere) et chez Sipploidea sipylus W (Pharmoptere); Ann. Sci. Nat. Zool Ser. II 

313-314 
Ramamurthy P S and Engels W 1977 Allatektomie- und juvenilhormon-wirkungen auf synthese und 

einlagetrung von vitellogenin bei der bienenkonigin (Apis mellifica)\ Zool. Jahrb. Physiol 81 165-176 
Strong L 1965a The relationship between the brain, CA and oocyte growth in the Central American locust, 

Schistocerca-sp. I. The Central neurosecretory system, CA and oocyte growth; J. Insect Physiol. 11 

135-146 
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Schistocerca-$p. II. The innervation of the CA, the lateral neurosecretory complex and oocyte growth; J. 

Insect Physiol. 11 271-280 

Telfer W H 1965 The mechanism and'control of yolk formation; Ann. Rev. Entomol. 10 161-184 
Thomas K K and Nation J L 1966 RNA, protein and uric acid content of the body tissue of Periplaneta 

americana L. as influenced by CA during ovarian development; Biol. Bull. 130 442-449 
Thomsen E 1948 The gonotropic hormones in the Diptera; Bull Biol Fr. Belg. Suppl 33 68-80 
Thomsen E 1952 Functional significance of the neurosecretory brain cells and the corpora cardiaca in the 

blow fly Calliphora erythrocephala Meig; J. Exp. Biol 29 137-172 
Thomsen E and Moller 1 1963 Influence of neurosecretory cells and CA on intestinal protease activity in the 

adult Calliphora erythrocephala Meig., J. Exp. Biol 40 301-321 
Tojo S, Kiguchi K and Kimura S 1981 Hormonal control of storage protein synthesis and uptake by the 

fatbody in the silk worm Bombyx mori; J. Insect Physiol 27 491-497 
Turnen S and Chippendale G M 1980 Proteins of the fatbody of non-diapausing larvae of the southwestern 

corn borer, Diatraea grandiosella: Effect of JH; J. Insect Physiol 26 163-169 
Wilkens J L 1969 The endocrine control of protein metabolism as related to reproduction in the fleshfly 

Sarcophaga bullata; J. Insect Physiol 15 1015-1024 
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im Fettkorper von Leucophaea maderae (Insecta); Rev. suisse Zool 73 517-521 
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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. 58C) 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 (KMnO 4 /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 NH 2 groups and lipids. 

The frontal glands contain carbohydrates (1, 2 glycol groups and both acid and 
neutral mucopolysaccharides), proteins (S-H, NH 2 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, NH 2 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 78. 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 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 37C 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 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 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 1 U' (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 5C 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 shath 
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 



I 20 



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 1C 
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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(1979) reported 1721-23 onca.. 

Aleemuddin (1979) observed 26 06 eggs ^pe workers These variatio ns may be due 
the fecundity is at vanance wtth ,thos ot P 16 ^ care durm reari ng. Aleemuddm 

to the substrate used, ^ n ^ ch ^^i n ord er 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 T he 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 , fecund it y , 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 k 1 = ajn^ k 2 = a 2 /n 2 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 P r = (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 





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 







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 

o 


SUMMER 
/ / ^ 




D 


40 



E 


40 



___. C 


E 




X 
1- 


A MONSOON 


B 


^.^^:^.^:.?" v: ^> 


--40 

U 
< 

a? 40 


40 

40 



C 


E 


X. .., A w 1 1 


^TE R 




B 


: ~ ^.-- <s ^:iir:^*^^i-.Tjr. 


.-^. c 


E 


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 (P f ) availability in the tissues. Inorganic phosphate (P t ) 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 MgSO 4 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 37C. 

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 0-5C 
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 





, 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 


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 37C 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' 2 M + + 

EDTAlO~ 2 M + + 

Organophosphate 

10" 4 M + + 

Eserine sulphate 

10' 4 M + + 

AgNO 3 10" 2 M +/- +/- + + +/- +/- 

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 ~ 2 M 

EDTA 10~ 2 M 

Organophosphate 
KT 4 M 


+ + + 
+ + + 
+ + + 


+ + 
+ + 
+ + 


+ + 
+ + 


+ + + + 
+ + + + 
+ + + + 


+ + + 
+ + + 


+ + + 
+ + + 
+ + + 


- 


Eserine 
















sulphate 10 ~ 4 M 
AgNO 3 


+ + + 

+ + 


+ + 


+ + 


+ + + + 


+ + + 
+ + 


+ + + 


+ + 




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 2 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 


J B:/)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 60 Co 
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 60 Co 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 28-30C. 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 
(28~30C), 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 (28-30C). 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. 




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 
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12 



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INSTAR NUMBER 



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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 (28-30C) 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 6 C), 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 162166 
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 (1624N:8033'E). 
After clearing the encrustations on the shells, the snails were acclimatized in the 
laboratory at 27 2C 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 1C) 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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males on different days of starvation. 



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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 
O 2 /g/hr in males and 0-47 to 1-88 g and 0-52 to 2-1 8 mgO 2 /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 



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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 
O 2 /g/hr and in females 0-71 mg O 2 /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 

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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 
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*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 (F 2 ) 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 



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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 


























8 


























15 











0-5 






















0-06 










22 





H 


0-8 


1-2 + 


















0-23 


0-20 


0-32 










29 


1-0 


l-8 


l-3 


l-9 











0-2 




0-14 


0-40 


0-26 


0-35 








0-05 


36 


1'2 


2-0 


2-1 


2-5 











0-6 




0-15 


0-06 


0-05 


0-26 








0-10 


43 


l-9 


2-6 


2-4 


2-8 








0-1 


H 




0-12 


0-13 


0-25 


0-05 









0-06 


50 


2-5 


2-7 


2-7 


2-8 








0-6 


l-7 




0-06 


0-05 


0-05 


0-06 






0-15 


0-10 


57 


2-5 


2-7 


2-7 







0-1 


H 






0-06 


0-06 


0-04 









0-12 




64 


2-5 


2-7 


2-7 








0-6 


l-7 






0-06 


0-06 









0-06 


0-10 




71 


2-5 


2-7 







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 7 C 
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 (3056'N, 7552'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 













X 








.x 


* X 










x 






(U 

z 


I * 


\ * 


o 
3) 




z 


* 





o 




X 


a 
z 


X 


c 
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1/5 


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5 


s 


X 


73 
O 
TJ 






t x 










X 


* X 




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 crop fl . 



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. 

b The 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 pairs 






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. 

b The 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 


5-130-18* 



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 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 



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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 

i 



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. 



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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 37C. 

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 O 2 consumption and CO 2 production in 
the midgut gland. 



Regions of 

midgut O 2 uptake CO 2 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 



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S KGarg and S K Jain 



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Extraretinal photoreception in C. punctatus 



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5 K Garg and S K Jain 



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 25C (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 






^ 


25C 






45-^ 




76-5 + 6 


66+4 






(8) 


(5) 


30 \^ 




85 10-2 


69 12 


^^-^ 


^ LD9:15 


(4) 


(6) 


45-^""""^ 


25C 


59 7 


54+12 






(6) 


(5) 


3( K 




428 


62 10 


^\ 




(5) 


(5) 




^ LD 12:12 






^ 


25C 






45""^ 




70 10-4 


67 7 






(6) 


(7) 


30 


LL 25C 


84-2 + 9-6 


71 10 






(5) 


(6) 


30 


DD 25 C C 


634 


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 (S 2 ) oocytes, note the presence of 
peripheral ring of cortical alveoli, (C). Stage III (5 3 ) 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 1C 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 60C 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 

Babu M H, Bhattacharya A K and Rathore Y S 1979 Rate of intake, growth and digestibility of three 

lepidopterous insects on Soybean and green gram; Z. Ang. Entomoi 87 322-327 
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bivittatus and M. femurrubrum; Can. J. Zooi 54 1044-1050 
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four food plants; Bull. Entomoi Res. 71 655-662 



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Ganga G and Meenakshi Nagappan 1983 Feeding and food utilization by the beetle Henosepilachna 

vigintioctopunctata on different host plants; Indian J. Exp. Biol. 21 33-36 
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instar larvae and adults of the migratory locust; Entomol. Exp. appl. 19 1 30-1 38 
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Entomol. 55 800-801 
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Indian J. Anim. Res. 13 98-102 
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grandis, Heliothis zea and H. virescens during growth and development; J. Insect Physiol. 17 657-675 
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on arboreal folivores, Fort Royal, Va (1975). National Zoological Park-Smithsonian Institution, 

Washington, D.C. 
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semilooper, Achaea Janata; Indian J. Entomol. 41 260-266 

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Periplaneta americana M.Sc. Dissertation submitted to Madurai Kamaraj University, Madurai, India 
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Indian J. Ecol. 3 119-124 
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butterfly on wild and cultivated food plants; Ecol. Monogr. 47 209-228 
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polyphagous insect, Prodenia eridania; J. Insect Physiol 12 711-730 
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americana; Comp. Physiol. Ecol. 4 296-298 
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soluble carbohydrate during metamorphosis of the blowfly Phormia regina; Insect Biochem. 4 85-98 
Taylor W E and Bardner R 1968 Leaf injury and food consumption by larvae of Phaeclos coctoleariae and 

Plutella maculipennis feeding on turnip and radish; Entomol. Exp. Appl. 11 177-184 
Waldbauer G P 1964 The consumption, digestion and utilization of solanaceous and non-solanaceous plants 

by larvae of toleacco hornworm, Protoparce sexta; Entomol. Exp. Appl. 1 253-269 
Waldbauer G P 1968 The consumption and utilization of food by insects; Adv. Insect Physiol. 5 229-288 
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STATEMENT ABOUT OWNERSHIP AND OTHER PARTICULARS ABOUT 
PROCEEDINGS OF THE INDIAN ACADEMY OF SCIENCES 

(Animal Sciences) 

1. Place of Publication .. Bangalore 

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Indian Academy of Sciences 
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Bangalore 560 080 

I, C N R Rao, hereby declare that the particulars given above are true to the best 
of my knowledge. 



C N R Rao 
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, 4 copulin' (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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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 187-196, 
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 20C. 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 I 2 (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 


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 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 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 
influence 1 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 (P l and P 2 ) of 15 min duration each and 300 lux 
intensity. P 1 was given at 15-5 cr and P 2 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 P l and P 2 of 1 5 min duration and 10000 lux 
intensity. Pj at 15-5 cr, P 2 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. P l was given at 15-5 cr in the 
first cycle and P 2 at 22 cr of the second cycles. In practice Pj falls 27-5 hr after LD/DD and P 2 
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 -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 l a' and 4 b* 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 4 on' 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 fc on' 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/cm 2 /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 migratorioides y 
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 
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Arch. hal. Biol 106 100-112 
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neurophysiology (New York: Plenum) (eds) A B Borkovec and T J Kelly pp. 93-1 13 
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2027-2031 
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system of the desert locust; Nature (London) 197 56-58 
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Arnold) 2nd ed., 358 pp. 

Johnson G G 1969 Migration and Disposal of Insects by flight. (London: Methuen) 
Loher W and Huber F 1966 Neurons and endocrine control of sexual behaviour in a grasshopper 

(Gomphocerus rufus L); Symp. Soc. Exp. Biol 20 381-400 
Liischer M 1975 Pheromones and polymorphism in bees and termites, 123-141, in: Pheromones and defensive 

secretions in social insects (eds) Ch. Noirot, P E Howse and G Lemasne (Dijon: International Union for 

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 198a 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 

cingulatus (Heteroptera; Pyrrhocoridae); J. Insect Physioi 31 223-227 
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 
Nayar K K 1973 Elements of insect endocrinology (New Delhi: Prentice Hall) 
Odhiambo T R 1966 The metabolic effects of corpus allatum hormone in the male desert locust. II. 

Spontaneous locomotar activity; J. Exp. Biol. 45 51-63 
Pener M P 1974 Neurosecretory and corpus allatum controlled effects of male sexual behaviour in acridids, 

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pp. 264-277 
Rankin M A 1974 The hormonal control of flight in the milk weed bug, Oncopeltusfasciatus In: Experimental 

analysis of insect behaviour (ed.) L Barton-Browne (Berlin, Heidelberg: Springer Verlag) pp. 317 
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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Schafer R 1977 The nature and development of sex attractant specificity in cockroaches of the genus 

Periplaneta. IV. Electrophysiological study of attractant specificity and its determination by juvenile 

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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 3 H 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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Rensing L, Thach B and Bruce V 1965 Daily rhythms in the endocrine glands of Drosophila larvae; 

Experientia 21 103-104 
Roberts S K de F 1966 Circadian activity rhythms in cockroaches. III. The role of endocrine and neural 

factors; J. Cell. Physiol. 67 473-486 
Roberts S K de F 1971 Comment, in Roberts et al In Biochronometry (ed.) M Menaker (Washington: 

National Academy of Sciences) pp. 514-516 
Reynolds S E 1977 Control of cuticle extensibility in the wings of adult Manduca at the time of ecdysis: Effect 

of eclosion hormone and bursicon; J. Exp. Biol. 70 27-39 
Reynolds S E, Taghert P H and Truman J W 1979 Eclosion hormone and bursicon titres and the onset of 

hormonal responsiveness during the last day of adult development in Manduca sexta (L); J. Exp. Biol. 78 

77-86 
Riddiford L M and Williams C M 1971 Role of the corpora cardiaca in the behaviour of Saturniid moths. L 

Release of sex pheromone; Biol Bull Mar. Biol Lab. Woodhole 140 1-7 
Shepard M and Keeley L L 1972 Circadian rhythmicity and capacity for enforced activity in the cockroach, 

Blaberus discoidalis, after cardiacectomy-allatectomy; J. Insect Physiol 18 595-601 
Skopik S Dand Pittendrigh C S 1967 Circadian systems II. The oscillation in the individual Drosophila pupa; 

its independence on developmental stages; Proc. Natn. Acad. Sci. U.S.A. 58 1862-1869 
Thomas R and Finlayson L M Initiation of circadian rhythms in arrhythmic churchyard beetles (Blaps 

mueronata); Nature (London) 228 577-578 
Truman J W 1970 The eclosion hormone: its release by the brain and its action on the central nervous system 

of silkmoths; Am. Zool 10 511-512 
Truman J W 197 la Physiology of insect ecdysis. I. The eclosion behaviour of saturniid moths and its 



Hormonal rhythm and behavioural trends in insects 217 

hormonal release; J. Exp. Biol. 54 805-814 
Truman J W 1971b Orcadian rhythms and physiology with special reference to neuroendocrine processes in 

insects; in Proc. Int. Symp. on Orcadian Rhythmicity (Wageningen, Netherlands: Pudoc Press) pp. 111- 

135 
Truman J W 1972 Physiology of insect rhythms. II. Circadian organisation of the endocrine events 

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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 219-224. 
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 20C 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 40~45C (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 38C, 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 40C 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 35C from the ambient temperature of 20-25C. 
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 (26C), 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 5C or less, forage at an energy cost two or three 
times greater than that at 26C (Heinrich 1972a s b). 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 2C from flowers 
of manzanita Arctostaphylos otayensis, Bombus edwardsii (0-1 g) has a thoracic 
temperature near 37C. 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 4-833 .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 1964a s 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 CO 2 , 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 CO 2 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 CO 2 , water vapour, fatty acids and derivatives, 
ammonia, amines, amino acids etc. have been tested on mosquitoes for release of 
host-attack reaction (Brown 1966). CO 2 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 
index 1 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 F 1 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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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 239-247. 
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 <. /T J 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 ,es are usua lly ,d in ,he pleura, m e mbrane 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). 

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246 ETHaridass 

Edwards J S 1966 






Gillette J D and Wigglesworth 



sial 



T N 



89 






p- 

* 







A33 



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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 
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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 23-27C 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 : 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 









ijj 


on 






CL 


CD 






cr 


I 

o 


n n 







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 



PO 


n 




cr 


fN 






o 


CNJ 
1 
O 


n 


n 




ro 






00 


00 


n 


n 


UJ 









*- 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 





1 


1 





1 








1 


1 


2 














2 













2 




















2 







3 











1 


2 











2 


4 





3 








1 













1 




















1 


































3 


2 





2 














































































4 































1 











1 











































5 



























































* 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 g Ageof 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. 

References 

Bhattacharya A K and Pant N C 1965 Development of insect physiology in India. In Entomology in India 

(supplement) (ed.) S K Prasad (New Delhi: The Entomological Society of India) 103-126 
Chakravorty S and Das H C 1983a Sexual behaviour of the stored grain pest; Environ. Ecol. I 83-87 
Chakravorty S and Das H C 1983b Egg laying behaviour of the rice moth Corcyra cephalonica; Environ Ecol 

I 169-174 
Deseo K V 1976 The oviposition of the Indian meal moth (Piodia interpunctella Hbn., Lep., Phyticidae) 

influenced by olfactory stimuli and antennectomy. In The host-plant in relation to insect behaviour and 

reproduction (ed.) T Jermy (New York and London: Plenum Press) 61-65 
Engelmann F 1970 The physiology of insect reproduction (New York: Pergamon Press) pp. 307 
Good N E 1 933 Biology of the flour beetle Tribolium confusum Duv., and Triboliumferrugenium Fab.; J. Agric. 

Res. 46 327-334 
Karnavar G K 1972 Mating behaviour and fecundity in Trogoderma granarium (Coleoptera: Dermestidae); J. 

Stored Prod. Res. 8 65-69 
Kehat M and Gordon D 1975 Mating,. longevity, fertility and fecundity of the cotton leaf-worm, Spodoptera 

littoralis Boisd. (Lepidoptera: Noctuidae); Phytoparasitica 3 87-102 
Kehat M and Gordon D 1977 Mating ability, longevity and fecundity of the spiny bollworm, Earias insulana 

(Lepidoptera: Noctuidae); Entomol. Exp. Appl. 22 267-273 
Krishna S S and Narain A S 1976 Ovipositional programming in the rice moth, Corcyra cephalonica 

(Stainton) (Lepidoptera: Gelechidae) in relation to certain extrinsic and intrinsic cues; Proc. Indian Natl. 

Sci. Acad. B42 325-332 
Krishna S S and Sinha A K 1 969 Chemosensory relationship between Epilachna vigintioctopunctata and its 

host plant, Luffa aegyptiaca; Ann. Entomol. Soc. Am. 62 928-929 
Lee J S and Smith L B 1976 Effect of different sex ratios on the oviposition and fertility of the red flour beetle, 

Tribolium castaneum (Herbst); The Man. Entomol. 10 38-41 
Mammen K V and Vishalakshi A 1973 Studies on the effect of crowding of larvae on the development, sex 

ratio and fecundity of Corcyra cephalonica (Staint.); Agric. Res. J. Kerala 1 1 86-87 
Mani H C and Krishna S S 1984 Follow up studies concerning effects of certain environmental variables on 

oviposition and egg viability in Earias fabia Stoll (Lepidoptera: Noctuidae); Mitt. Zool. Mus. Berl. 60 9-1 6 
Mishra S N and Krishna S S 1978 Influence of some specific time- and age-related mating schedules on 

oviposition and egg fertility in Corcyra cephalonica (Lepidoptera: Galleridae); Entomon 4 197-199 
Mishra S N and Krishna S S 1980 Oviposition and egg fertility in Corcyra cephalonica Staint. (Lepidoptera: 

Galleridae) affected by programmed manipulations in larval ecology during rearing or in adult light 

regimes; Z. Angew. Zool. 67 101-105 
Mishra S N and Krishna S S 1981 Variation in the reproductive potential of Corcyra cephalonica Staint. 

(Lepidoptera: Galleridae) following antennectomy or alactomy in males or females; Z. Angew. Zool. 68 

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. 

(Lepidoptera: Noctuidae) under different sex ratios and population processes of the larvae; AppL Entomol. 

Zool 1 190-198 

Paterson D D 1939 Statistical technique in agricultural research (New York: McGraw-Hill) pp. 263 
Piltz H 1977 Corcyra cephalonica (Staint.). In Diseases, pests and weeds in tropical crops (eds) J Kranz, H 

Schmutterer and W Koch (Berlin and Hamburg: Verlag Paul Parey) 439-440 
Reynolds J M 1 944 The biology of Tribolium destructor (Uytt). I. Some effects of fertilisation and food factors 

on fecundity and fertility; Ann. Appl. Biol. 31 132-142 
Russel V M, Schulten G G M and Roorda F A 1980 Laboratory observations on the development of the rice 

moth Corcyra cephalonica (Lepidoptera: Galleridae) on millet and sorghum at 28C and different relative 

humidities; Z. Angew. Entomol. 89 488-498 



264 S S Krishna and S N Mishra 

Sehgal S S and Chand A T 1978 Mating competitiveness of adult rice moths 60 CO irradiated as pupae; Indian 

J. Entomol. 40 303-307 
Seshagiri Rao D 1954 Notes on rice moth Corcyra cephalonica (Stainton) (Lepidoptera: Gaileridae); Indian J. 

Entomol 1695-114 
Shahi K P and Krishna S S 1 979 Effects of the presence of male with unmated or mated female and some light 

regimes on the programming of oviposition in Eariasfabia Stoll (Lep., Noctuidae); Z. Angew. Entomol. 88 

175-181 

Shorey H H 1976 Animal communication by pheromones (New York: Academic Press) pp. 307 
Singh D and Sidhu H S 1975 Effect of some physiological and environmental factors on the response of males 

to the female sex pheromone of the rice moth Corcyra cephalonica (Stainton); Indian J. Ecol. 2 62-67 
Singh D and Sidhu H S 1976a Demonstration, extraction and trapping efficiency of female sex pheromone of 

the rice moth Corcyra cephalonica (Stainton); J. Res. Punjab Agric. Univ. 13 85-90 
Singh D and Sidhu H S 1976b Effect of some physiological and environmental factors on the production and 

release of sex pheromone by the females of the rice moth, Corcyra cephalonica (Stainton); Indian J. Ecol 3 

49-55 
Singh R N and Krishna S S 1980 Studies on some specific biological and temporal factors affecting mating 

and/or oviposition in Tribolium castaneum (Hbst.) (Coleoptera: Tenebrionidae); Bolm. Zool Univ. S. 

Paulo 5 19-29 
Srivastava A K and Krishna S S 1976 Studies on the utilisation of food in the larva of the rice moth, Corcyra 

cephalonica Stain t I. The hydrogen-ion concentration of the gut contents of the larva; Z. Angew. Zool 63 

71-76 
Srivastava A K and Krishna S S 1978 Studies on the utilisation of food in the larva of the rice moth, Corcyra 

cephalonica. II. Metabolism of some carbohydrates in vivo and in vitro; Z. Angew. Zool 65 1 1-19 
Wigglesworth V B 1960 Nutrition and reproduction in insects; Proc. Nutr. Soc. 10 18-23 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 265-282. 
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 (1973 y 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 




* 

M 



) .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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X 






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ri 


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5 




4 


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55 




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270 



S Y Paranjape 



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. 



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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. 



References 

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219-229 
Bei-Bienko G Y A and Mischenko L L 1951 Acridoidea of the USSR and adjacent countries Part I (Russian) 

English Translation IPST, Jerusalem 1963 pp. 8, 16-17, 23-27, 37-53 
Bernays E A and Chapman R F 1974 The regulation of food intake by Acridids in Experimental analysis of 

insect behaviour (ed.) L B Browne (Berlin, Heidelberg, New York: Springer- Verlag) pp. 48-59 
Bhalerao A M and Paranjape S Y 1982 Comparative studies on Tetrigid eggs Abstract: Afro- Asian 

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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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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 295-301. 
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 






J1 2 


18-33 


13-66 










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 










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 




J 2 


151-66 


11-40 


7-00 


+ 1-50 




fl 


188-88 


9-21 


3-35 


7-63 


** 


F 2 


247-22 


8-90 


3-35 


15-19 


0-689 


M, 


244-99 


9-75 


1-65 


0-05 




M 2 


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 
S 



( %) 30U3ppUl 



Behaviour response of Aphis gossypii 



299 




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. 



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300 T K Banerjee and D Raychaudhuri 

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. 



References 

Agarwala B K and Raychaudhuri D N 1 979 Biotic potential of weeds in respect of Aphis gossypii GL infesting 

some economic plants in Kalimpong, West Bengal; Indian Agric. Sci. 23 25-29 
Bodenheimer F S and Swirski E 1947 Studies on the physical ecology of the wooly apple aphis (Eriosoma 

lanigenan] and its parasite Aphelinus mali in Palestine; Bull. Agric. Exp. Sta. Rehovoi. 41 20 
Dadd R H 1968 Dietary aminoacids and wing determination in the Aphid Myzus persicae; Ann. Entomol. Soc. 

Am.'6l 1201-1210 
Dickson R C and Laird E F 1962 Green peach aphid populations on desert sugarbeats; J. Econ. Entomol 55 

501-504 
Evans AC 1938 Physiological relationships between insects and their host plant I. Effect of chemical 

composition of the plant on reproduction and production of winged forms in Brevicoryne brassicae L; 

Ann. Appl Bioi 8 49-64 



Behaviour response of Aphis gossypii 301 

Gibson R W 1971 The resistance of three Solan um species to Myzus persicae, Macrosiphum euphorbiae and 

Aulocorthum solani (Aphididae: Homoptera); Ann. Appl. Bioi 68 245-251 
Ghosh M R and Mitra A 1979 Population of Lipaphis erysimi (Kalt) on mustard and radish; Proc. Symp. 

aphids. Zoo. Soc. Orissa Bhubaneswar pp. 1-440 
Johnson B 1 966 Wing polymorphism in aphids. III. The effect of temperature and photoperiod. Entomol. 

Exp. Appl. 9 301-313 

Kennedy J S and Booth CO 1954 Host alternation in Aphis fabae Scop; Ann. Appl. Bioi 41 88-106 
Lees A D 1967 The production of apterous and alate forms in aphid Meg our a viciae Buckton with reference 

to the role of crowding; J. Insect Physiol. 13 289-318 
Mittler T E and Kunkel H 1971 Wing production by grouped and isolated apterae of the aphid Myzus 

persicae on artificial diet; Entomol. Exp. Appl. 14 83-92 
Mittler T E 1973 Aphid polymorphism as affected by diet. In Perspectives in aphid biology (ed.) A D Lowe 

(Auckland New Zealand: Ent. Soc. New Zealand) pp. 65-75 
Mueller H J and Unger K 1951 Uber die ursachen der unterschiedlichen Resistenz von Vicia faba L. 

gegenuber der Bohnenblattlaus Doralis fabae Scop; Zuechter 21 1-30, 76-89 
Odum E P 1971 Fundamentals of Ecology (London: W B Saunders Company) 1-574 
Rattanlal 1957 Effect of water content of aphid and their host plants on the appearance of alatae; Indian J. 

Entomol. 17 52-62 
Raccah B, Tahori A S and Applebaum S W 1971 Effect of nutritional factors in synthetic diet on increase of 

alate forms in Myzus persicae; J. Insect Physiol. 17 1385-1390 
Rajagopal S and Abdul Kareem A 1979 Studies on certain factors affecting alatae production green peach 

aphid; Proc. Symp. on aphids (ed.) B K Behura (Bhubaneswar: Zoological Society of Orissa) pp. 17-22 
Roy D K and Behura B K 1979 Seasonal variation in the population of Aphis gossypii Gl. on brinjal; Proc. 

Symp. on aphids (ed.) B K Behura (Bhubaneswar: Zoological Society of Orissa) pp. 60-64 
Schaefers G A and Judge F D 1971 Effect of temperature, photoperiod and host plant on alasy, 

polymorphism in the aphid Chaetosiphonfragaefolii; J. Insect. Physiol 17 365-69 
Turner R B 1971 Dietary aminoacid requirements of the cotton aphid, Aphis gossypii Gl. the sulphur 

containing aminoacids; J. Insect Physiol. 17 2451-2456 
Van Emden H F, Eastop V F, Hughes R D and Way M J 1969 Ecology of Myzus persicae; Ann. Rev. Entomol. 

14 197-270 
Wadley F M 1923 Factors affecting the production of alate and apterous forms of Myzus persicae; Ann. 

Entomol. Soc. Am. 16 279-303 
* Wearing C H 1972 Selection of Brussels Sprouts of different water status by apterous and alate Myzus 

persicae and Brevicoryne brassicae in relation to age of leaves; Entomol. Exp. Appl. 15 139-154 
*Wolfenberger DO 1946 Disperson of small organism; Am. Midi Nat. 35 1-152 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 303-308. 
(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 23C 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 
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13 231-253 
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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 4 the 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 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 





Sterility 


B 


Classical individual fitness 


10 





Will not be selected 




Inclusive fitness 


10 


5 






Number of offspring 


21 





Parental 


C 


Classical individual fitness 


10-5 





manipulation 




Inclusive fitness 


10-5 


5-25 






Number of offspring 


31 





Kin selection 


D 


Classical individual fitness 


15-5 









Inclusive fitness 


15-5 


7-75 






Number of offspring 


21 orO 


Oor 21 


Classical 


E 


Classical individual fitness 


10-5 or 


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 ^Plation 
Gadagkar (1985) have modd^^^^"" 1 "^ ****** Joshi and 
investment ratios under different let s of ir, ^ C mpUted the P timum s 
(ngure2). This has been done b con? P "^^ and loc *l mate competition 

S^cotonieswhereaoerSn^S'rft; haplodi P loid Population with 
outbreed while the remaining fraction H K reproductive offspring disperse 
be summarised as follows (nT?n^f brothei " siste r 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 multi P le ^ting and local 
, y (qU6en contro1 ) P red ^ts a 1:1 
( fem ^H Local 
in the frame work of 
mate com P etitio " however, the 

^ le biased than 

the prediction of 
tmui " investment ratio under 

adng ^ We h P e ^ 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 +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. 



References 

Alexander R D 1974 The evolution of social behaviour Ann. Rev. Ecol. Syst. 5 325-383 

Alexander R D and Shannann P L 1977 Local mate competition and parental investment in social insects; 

Science 196 494-500 

Barash D P 1982 Sociobiology and behaviour (New York: Elsevier) pp. xiv 4- 426 
Brian M V 1983 Social insects (London: Chapman and Hall) 
Charnov E L \982The theory of sex allocation (New Jersey: Princeton Univ. Press) 
Darwin C 1859 On the origin of species by means of natural selection or the preservation of favoured races in the 

struggle for life (London: John Murray) ix + 502 pp. 

Evans H E and West-Eberhard M J 1970 The wasps (Ann Arbor: Univ. of Michigan Press) 
Edwards R 1980 Social wasps; Their biology and control East Grinstead-W Sussex England Rentokil 
Gadagkar R 1980 Dominance hierarchy and division of labour in the social wasp Ropalidia marginata (Lep.) 

(Hymenoptera: Vespidae); Curr. Sci. 49 772-775 
Gadagkar R, Gadgil M and Mahabal A S 1982a Observations on population ecology and sociobiology of the 



324 Raghavendra Gadagkar 

paper wasp Ropalidia marginata (Lep.) (Family Vespidae); Proc. Symp. Ecol. Anim. Pop. Zoo/. Surv. India 

Part 4 pp. 49-61 
Gadagkar R, Gadgil M, Joshi N V and Mahabal A S 1982b Observations on the natural history and 

population ecology of the social wasp Ropalidia marginata (Lep.) from Peninsular India (Hymenoptera: 

Vespidae); Proc. Indian Acad. Sci. (Anim. Sci.) 91 539-552 
Gadagkar R and Joshi N V 1982a Behaviour of the Indian social wasp Ropalidia cyathiformis (Fab.) on a nest 

of separate combs (Hymenoptera: Vespidae); J. Zoo/. London 198 27-37 
Gadagkar R and Joshi N V 1982b A comparative study of social structure in colonies of Ropalidia. In The 

biology of social insects (ed.) M D Breed, C D Michener and M E Evans Proc. IX Cong. Int. Union for the 

study of social insects Boulder, Colorado, USA, August 1982 (Boulder: Westview Press) pp. 187-191 
Gadagkar R and Joshi N V 1983 Quantitative ethology of social wasps: Time-activity budgets and caste 

differentiation in Ropalidia marginata (Lep.) (Hymenoptera: Vespidae); Anim. Behav. 31 26-31 
Gadagkar R and Joshi N V 1984 Social organisation in the Indian wasp Ropalidia cyathiformis (Fab.) 

(Hymenoptera: Vespidae); Z. Tierpsychol 64 15-32 

Gadagkar R and Joshi N V 1985 Colony fission in a social wasp; Curr. Sci. 54 51-62 
Hamilton W D 1964a The genetical evolution of social behaviour I. J. Theor. Biol. 1 1-16 
Hamilton W D 1964b The genetical evolution of social behaviour II. J. Theor. Biol 7 17-52 
Hamilton W D 1967 Extraordinary sex ratios; Science 156 477-488 

Hamilton W D 1972 Altruism and related phenomena mainly in social insects; Ann. Rev. Ecol. Syst. 3 1 92-232 
Hermann H R 1979-82 Social Insects (New York and London: Academic Press) Vols I-IV 
Jeanne R L 1980 Evolution of social behaviour in Vespidae; Ann. Rev. Entomol. 25 371-396 
Joshi N V and Gadagkar R 1985 Evolution of sex ratios in social hymenoptera: Kin selection local mate 

competition polyandry and kin recognition J. Genet. 64 41-58 
Lakatos I and Musgrave A 1970 Criticism and the growth of knowledge (Cambridge: Cambridge University 

Press) pp. 282 

Lin N and Michener C D 1972 Evolution of sociality in insects; Q. Rev. Biol. 47 131-159 
Michener C D 1974 The social behaviour of the bees (Cambridge Mass: Harvard Univ. Press) 
Metcalf R A and Whitt G S 1977 Relative inclusive fitness in the social wasp Polistes metricus, Beh. Ecol. 

Sociobiol. 2 353-360 

Michod R E 1982 The theory of kin selection; Ann. Rev. Ecol. Syst. 13 23-55 
Noonan K M 1978 Sex ratio of parental investment in colonies of the social wasps Polistes fuscatus; Science 

199 1354-1356 
Noonan K M 1981 Individual strategies of inclusive fitness maximizing in Polistes fuscatus foundresses. In 

Natural selection and social behaviour (eds) R D Alexander and D W Tinkle (New York: Chiron Press) 

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Orlove M J 1975 A model of kin selection not involving coefficients of relationships; J. Theor. Biol. 49 

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Trivers R L and Hare H 1976 Haplodiploidy and the evolution of the social insects; Science 191 249-263 
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, No. 3, June 1985, pp. 325-331. 
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 = Nx Cp (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 C UcU 

in one host (Van Lenteten and 



whe two more fences oviposit 
Bakkerl975). m v,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 exact ly the 

scarcity of healthy hosts. However ins ^not undouD y ^ ^ ^ 

parasite recognise the hosts Wlth j?f ^ some species. Hence the 

known clearly is that such an abl1 

-s Wlt h d iff erent 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 unp arasit 1S ed 
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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Pro, Indian Acad. SeL (Anin, Sci.), Vol. 94, No. 3, June 1985, pp. 333-339. 
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 P r ^ at ^ y ltionbecomes difficult. This warrants 
insecticides. Hence the suppression ol t n ^ P"?.^ ^ tQ ^ integra t e d harmoniously 
different methods of suppression strateg ^ injuring level. Among the modern 
in order to manage them below the eco _ Qmones offers SCO pe for monitoring 
technologies for pest suppression, use o F ^ fertility o f W ud females thereby 
the activity of these borers and also in _reau ^ ^^ This method C an also be 
bringing down the fertile egg PJP""! . in the pes t 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 ^ f n the niht in sho ot 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 to 107 in stalk borer, to 5 in top borer, to 10 in 
internode borer and 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 










Z 7 :Z 8 :Z 9 :Z 10 


component 


1 


31 


0.70 B 


1:1:1:1 










Z 7 :Z 8 :Z 9 :Z 10 


component 


0-1 


25 


060 B 


4:8:4:1 










^7 ^8 ^9 " ZIQ 


component 


1 


1643 


16-5 A 


4:8:4:1 










Z,:Z 8 :2 9 :Z 10 


component 


0-1 


54 


121 B 



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 






Z 7 :Z 8 :Z 9 :Z 10 component 


751 


1O80 BC 


Z 7 component CA 83/86 


21 


0-31 C 


Z 8 component CA 83/37 


38 


055 C 


Z 9 component CA 83/38 


39 


0-56 C 


Z 1Q component CA 83/39 


275 


3-94 c 


Z 8 :Z 7 mixture 2: 1) CA 83/40 


536 


12-70 5 


Z 8 :Z 7 mixture 2:1) CA 83/41 


1585 


22-75* 


Z 8 + Z 10 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* 


5-80 B 


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-49 A \-29 A 

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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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 7C (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). 

References 

Boerger H and Clifford H F 1975 Emergence of mayflies (Ephemeroptera) from a northern brown-water 

stream of Alberta, Canada; Verh. Int. Ver. Theor. Angew. Limnol 19 3022-3028 
Brinck P 1956 Reproductive system and mating in Ephemeroptera; Opusc. Entomol. 22 1-37 
Brittain J E 1975 The life cycle ofBaetis macani Kimmins (Ephemeridae) in a Norwegian mountain biotype; 

Entomol. Scand. 6 47-51 
Brittain J E 1980 Mayfly strategies in a Norwegian subalpine lake. In Adv. Ephemeroptera Bio/., Proc. Int. 

Conf. Ephemeroptera 3rd, Winnipeg, Can. pp. 179-186 
Brittain J E 1982 Biology of mayflies; Ann. Rev. Entomol 27 119-147 

Corbet P S 1964 Temporal patterns of emergence in aquatic insects; Can. Entomol. 96 264-279 
Degrange C 1959 L'ovolarviparite de Cloeon dipterum (L). (Ephem Baetidae); Bull. Soc. Entomol. Fr. 64 

94^100 
Edmunds G F Jr and Edmunds C H 1980 Predation, climate and emergence and mating of mayflies. In Adv. 

Ephemeroptera Bio/., Proc. Int. Conf. Ephemeroptera, 3rd, Winnipeg, Can. pp. 277-285 
Edmunds G F, Jensen S L and Berner L \976The mayflies of North and Central America (Minneapolis: Univ. 

Minnesota) pp. 330 
Elliott J M 1972 Effect of temperature on the time of hatching in Baetis rhodani (Ephemeroptera: Baetidae); 

Oecologia 9 47-51 
Elliott J M 1978 Effect of temperature on the hatching time of eggs of Ephemerella ignita (Poda) 

(Ephemeroptera: Ephemerellidae); Freshwater Bio/. 8 51-58 
Fremling C R 1973a Factors influencing the distribution of burrowing mayflies along the Mississippi River. 

In (eds) W L Peters and J G Peters Proc. 1st Int. Conf. Ephemeroptera, pp. 12-25 
Fremling C R 1973b Environmental synchronization of mass Hexagenia bilineata (Ephemeroptera) 

emergences from the Mississippi River; Verh. Int. Ver. Theor. Angew. Limnol 18 1521-1526 
Friesen M K, Flannagan J F and Laufersweiler P M 1980 Diel emergence patterns of some mayflies 

(Ephemeroptera) of the Roseau River (Manitoba, Canada) In Adv. Ophemeroptera Bio/., Proc. Int. Conf. 

Ephemeroptera 3rd, Winnipeg, Can. pp. 287-296 
Hartland-Rowe R 1958 The biology of a tropical mayfly Povilla adusta Navas with special reference to the 

lunar rhythm of emergence; Rev. Zoo/. Bor. Afr. 58 185-202 



Behavioural strategies in Mayflies 357 

Horridge G A 1976 The ommatidium of the dorsal eye of Cloeon as a specialization for photo 

reisomerization; Proc. R. Soc. London BI93 17-29 
Humpesch U 1971 Zur Faktorenalyse des Schlupfrhythmus der Fiugstadien Von Baetis alpinus Pict. 

(Baetidae, Ephemeroptera); Qecologia (Berl) 1 328-341 
Humpesch U H and Elliott J M 1980 Effect of temperature on the hatching time of eggs of three Rhithrogena 

spp. (Ephemeroptera) from Austrian stream and river; J. Anitn. Ecol. 49 643-661 
Langford T E 1975 The emergence of insects from a British river, warmed by power station cooling-waters; 

Part II. The emergence of some species of Ephemeroptera, Trichoptera and Megaloptera in relation to 

water temperature and river flow, upstream and downstream of the cooling- water outfalls; Hydrobiologia 

4791-133 
Muller-Liebenau I 1960 Eintagsfliegen aus der Eifel (Insecta, Ephemeroptera); Gewaesser Abwaesser 27 

55-79 
Pescador M L and Peters W L 1974 The life history and ecology of Baetisca rogersi Berner (Ephemeroptera: 

Baetiscidae); Bull. Fla. State Mus. Biol Sci. 17 151-209 
Peters W L and Peters J G 1977 Adult life and emergence of Dolania americana in Northwestern Florida 

(Ephemeroptera: Behningiidae); Int. Rev. Ges. Hydrobiol 62 409-438 

Pleskot G 1953 Zur Okologie der Leptophebiiden (Ephemeroptera); Oesterr. Zool 4 45-107 
Savolainen E 1978 Swarming in Ephemeroptera: the mechanism of swarming and the effects of illumination 

and weather; Ann. Zool Fenn. 15 17-52 
Sivaramakrishnan K G 1984 A new genus and species of Leptophlebiidae: Atalophlebiinae from southern 

India (Ephemeroptera); InL J. Entomoi 26 194-203 
Sutcliffe D W and Carrick T R 1 973 Studies on mountain streams in the English Lake District. I. Calcium and 

the distribution of invertebrates in the River Duddon; Freshwater Biol. 3 437-462 
Thibault M 1971 Ecologie d'un ruisseau a'truites des Pyrenees - Atlantiques, le Lissuraga. II. Les 

fluctuations thermique de 1'eau; repercussion sur les periodes de sortie et la taille de quelques 

Ephemeropteres, Plecopteres et Trichopteres; Ann. Hydrobiol. 2 241 -274 
Tjonneland A 1960 The flight activity of mayflies as expressed in some East African species; Arbok Univ. 

Bergen Mat. Naturvitensk. Ser. I 1-88 
Tjonneland A 1970 A possible effect of obligatory parthenogenesis on the flight activity of some tropical 

larvo-aquatic insects; Arbok Univ. Bergen Mat. Naturvitensk. Ser. 3 1-7 
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90 145-165 



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 CO 2 , 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 
AgNO 3 /HNO 3 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 NO 3 /HNO 3 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 O 2 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) 

F0 2 =K(A P0 2 )/T. 

where FO 2 = O 2 uptake in ml O 2 /min; A is the area in cm 2 for gaseous exchange; PO 2 is 
the mean difference between the oxygen tensions of water and blood; K is the 
permeation coefficients (rnl O 2 /m/cm 2 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 O 2 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 CO 2 tension in the blood. Air-breathing fishes have much higher CO 2 tension 
in the blood and the arterial CO 2 tension of water breathers is generally below 5 torr 
(Rahn 1966). Whereas, in the air breathers CO 2 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 CO 2 which is the causative factor for efficient 
hemoglobin function to counteract the increased CO 2 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 CO 2 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 CO 2 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 CO 2 affinity. Third, whether or not there is a correlation 
between Bohr effect and effect of carbamino CO 2 on the O 2 affinity of hemoglobin. He 
also found that the blood CO 2 content of air-breathing fish and amphibians is much 
higher than that of water breathers, but hemoglobin showed no adaptation to an 
increased CO 2 load. But the drop in oxygen affinity of hemoglobin caused by CO 2 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 O 2 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 O 2 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 (VO 2 ) 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 CO 2 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 O 2 affinity, more 
hemoglobin (Hb) and can maintain a higher aquatic oxygen consumption rate (VO 2 ) 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 
(VO 2 ) 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 O 2 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 VO 2 by aquatic respiration 
down to a PO 2 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 PO 2 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 u the 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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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 = aL n 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 0-25/0-18C (13-5-14C) 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 1324L 2 - 2103 


Male 


= 15998 + 2-1396 


0-00251304L 2 - 1396 


Pooled 


= 18199 + 2-0871 


0-00151391L 2 - 0871 


<17cm 


= 1-7670+1-7559 


0-00171002L 1 ' 7559 


> 17-1 cm 


= 3-0889 + 2-2850 


0-000814892L 2 - 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, F 1% = 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, F 5% = 3-91 d/1; 151) and between < 17cm and 

> 17-1 cm (F = 0-139, F 5 / = 3-91, d/= 1; 151) were not significant while those 
between sexes and < 17 cm (F = 141-521, F t o, = 4-71, d/2; 226) and between sexes and 

> 17.1 cm (F = 10-693, F l% = 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; t 1% = 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 


P 2 


0-7590 


0-6245 


0-6792 


0-3077 


0-7986 


r 2 


0-7572 


0-6233 


0-6874 


0-3035 


0-7979 



N = numer of observations; p 2 = proportion of correlated variance; r 2 : 
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 (p 2 ) 
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 (F 1;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= aL n used in the present analysis to the cubic formula W~ CL 3 (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 (1 129' N 
lat. and 7946' 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 


Mean 


Style pH 
Range 


Mean 


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. 

References 

Aiyakrinskaya I O 1977 On the dissolution of crystalline style in some bivalve molluscs; ZooL Zh. 56 23-27 
Alyakrinskaya I O 1979 On the properties and size of crystalline style in bivalves; in: Respiratory proteins oj 

some groups of animals (Moscow: Nauka) 142-150 

Kristensen J H 1972 Structure and function of crystalline style of bivalves; Ophelia 19 91-108 
Langton R W and Gabbott P A 1 974 The tidal rhythm of extracellular digestion and response to feeding ir 

Ostrea edulis L: Mar. Bioi 24 181-187 

Langton R W 1977 Digestive rhythms in the mussel Mytilus edulis; Mar. Bioi 41 53-58 
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 1-8C, 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 + 1-8C. 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 1-8C. 
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 1-8C. 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 
1-8C. 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 694g; $ 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 -8C 28-5 1 -8C 



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/mm 2 . 

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 

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(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/mm 2 . 

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. 



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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/mm 2 , 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: 



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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. 



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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. 



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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. 



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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