Proceedings of the Indian Academy of Sciences
Volume 91, 1982
CONTENTS (Animal Sciences)
Chromosomal repatterning in drosophila : Dwsophila nasuta nasuta and
•D. kohkoa S R Ramesh and M R Rajasekarasetty 1
Acid phosphatase activity in tissues of Notopterus notopterus chronically
exposed to phenolic compounds
jR C Dalela, Saroj Rani and S R Verma 1
Differences in home ranges of rhesus monkey (Macaca mulatto) groups
living in three ecological habitats
Raghubir Singh Pirta and Mewa Singh 13
Effects of aldrin on serum and liver constituents of freshwater catfish
Clarias batrachus L. Yagana Bano 27
Hepatopancreatic sucrase of Macrobrachium lamarrei (Crustacea, Caridea,
Palaemonidae) ,. Padma Saxena and Ramesh Chandra Murthy 33
Shell selection in the estuarine hermit crab Clibanarius longitarsus (De Haan)
S Ajmal Khan and R Natarajan 39
Evaluation of some organophosphorus insecticides against Dacus cucurbitae
Coquillett on peach N P Kashyap and S. F Hameed 45
Structure and chemical composition of the cuticle of Cirolana fluviatilis,
Sphaeroma walkeri and Sphaeroma terebrans D Leela Vallabhan 57
Effect of some antibiotic compounds in cotton on post-embryonic develop-
ment of spotted bollworm (Earias vittella F.) and the mechanism of resis-
tance in Gossypium arboreum
H C Sharma, R A Agarwal and Munshi Singh 67
Some biometric studies of certain closely related species of the genus Arms
(Pisces : Siluriformes : Ariidae) / R Dhanze and K C Jayaram 79
Electron microscopic study of the spermatheca of Gesonula punctifrons
(Acrididae : Orthoptera) S G Pal and D Ghosh 99
Histology and histochemistry of adrenal glands of Indian mongoose
Herpestes edwardsii edwardsii (Geoffroy)
P Varada Raju and K Hanumantha Rao 113.
Effect of x-rays on the somatic chromosomes of the exotic fish, Tilapia
Q K Manna and # C Som 121
ii ' Contents
Histochemical changes in Setaria cervi caused by certain anthelmintics
Adbul Baqui and Humaira Khatoon 135
Effect of salinity on the survival and growth of Chanda (= Ambassis)
gymnocephalus (Lac.) fry (Pisces ; Centropomidae)
/ Rajasekharan Nair, N K Ealasubramanian and N Balakrlshnan Nair 1 43
A comparative study on the mineral composition of the poultry cestode
Railli&tina tetragona Molin, 1858 and certain tissues of its host
A M Nadakal and K Vijayakumaran Nair 153
A comparison of the electrophoretic haemoglobin pattern of the commensal
rodent species M S Pradhan 159
Studies on egg and nymphal parasites of rice planthoppers, Nilaparvata
lugens (Stal) and Sogatella furcifera (Horvath)
/ S Bentur, Mangal Sain and M B Kalode 165
New natural enemy complex of some fulgoroids (Insecta : Homoptera) with
biological studies of three hymenopterous parasites (Insecta : Hymenoptera)
S Swaminathan and T N Ananthakrishnan 111
Transabdominal migration of ova in some freshwater turtles
P L Duda and V K Gupta 189
Sediment polychaete relationship in the Vasishta Godavari estuary
D Srinivasa Rao and D V Rama Sarma 199
The form-function relationship of vertebrates : A selected review
Hiran M Dutta 207
Metabolic rates and quotients in the cichlid fish, Tilapia mossambica (Peters)
in relation to random activity M Peer Mohamed 217
Microanatomy of the 7th abdominal ganglion and its peripheral nerves in
the scorpion Heterometrus fuhipes -
K Yellamma, K Subhashini, P Murali Mohan and K Sasira Babu 225
Branchial protein metabolism of freshwater fish Tilapia mossambica (Peters)
during acute exposure and acclimation to sublethal alkaline water
M JBhaskar, G Vemananda Reddy, V Krishna Murthy, P Reddanna and
S Govindappa 235
Temperature-related chromosome polymorphism in Drosophila ananassae
D P Dasmohapatra, N K Tripathy and C C Das 243.
Life and facundity tables for the longicorn beetle borer, Olenecamptus bilobus
(Fabricius) (Coleoptera : Cerambycidae) ...;....
'. T N Khan and P K Maiti 24?
Contents m
Behavioural responses of the Indian gerbil, Tatera indica to conspecific
sebum odour of the ventral scent marking gland
Mohd. Idris and Ishwar Prakash 259
Effect of temperature and humidity on the development and fertility-
fecundity of Acrida exaltata Walk Shamshad Alt 267
On some blood flukes (Spirorchiidae : Coeuritrematinae) from freshwater
chelonians in India V Tandon and N K Gupta 275
Life history and behaviour of the cyst nematode, Heterodera oryzicola Rao
and Jayaprakash, 1978 in Rice (Oryza saliva L.)
A Jayaprakash and Y S Rao 283
Sediment-ostracode relationship in the Bimili backwater and the Balacheruvu
tidal stream C Annapurna and D V Rama Sarma 297
Effect of DDT on brain neurosecretory cells of adult Poekilocerus pictus
(Orthoptera : Acrididae) Om Prasad and V K Srivastava 305
Rhythmic oscillations in non-aggressive social behaviour in Bandicota
bengalensis Shakunthala Sridhara and R V Krishnamoorthy 317
Toxicity of certain pesticides found in the habitat to the larvivorous fishes
Aplocheilus lineatus (Cuv. and Val.) and Macropodits cupanas (Cuv. and
Val.)
Sheila Susan Jacob, N Balakrishnan Nair and N K Balasubramanian 323
Histochemical studies on non-specific esterases in epididymis of the bat,
Cynopterus sphinx sphinx L T Mote and M N Nalavade 329
A study of pupal-adult intermediates produced with juvenoid treatment of
Spodoptera litura Fabr. pupae U S Srivastava and S S Prasad 337
A comparative study on certain biochemical aspects of red and white
myotomal muscles of the black skipjack tuna, Euthynnus affinis Cantor
N Gopinathan Pillai and K M Alexander 349
: Circadian basis for the photoperiodic response in the male blackheaded
bunting (Emberiza melanocephala) Vinod Kumar and P D Tewary 357
Steroid metabolism in target related to nuptial plumage production in the
Baya weaver bird V C Kotak and G K Menon 361
. Sex pheromone in a stomatopod crustacean Squilla holoschista
M Deecaraman and T Subramoniam 367
A new species of Argulus Muller (Crustacea : Branchiura), with a note on
tlie distribution of different species of Argulus in India P Natarajam 375
iv Contents
The effect of cephalic transection on the micromorphological changes in the
ventral nerve cord-neurosecretory system of earthworm, Metaphire peguana
(Rosa, 1890) during anterior regeneration
D K Nanda and P S Chaudhun 381
Studies on preference of Callosobruchus maculatus Fabricius to some high
yielding varieties of arhar (Cajanus Cajan L.) Satya Vir 391
Three new species of haematozoans from freshwater teleosts (pisces)
B D Joshi 397
Histological and histochemical studies on the albumen gland and capsular
gland of Thais bufo (Lamarck) (Mollusca : Gastropoda)
R C Rajalakshmi Bhanu, K Shyamasundari and K Hanumantha Rao 407
Effect of temperature on food intake, growth and conversion efficiency of
Eupterote molllfera (Insecta ; Lepidoptera)
S Palanichamy, R Ponnuchamy and T Thangaraj 41 7
Seasonal variations in the phosphorus contents of the muscle of catfish
Clarias batrachus L. Yagana Bano 423
The tannery industrial effluent effect on succinate dehydrogenase activity
pattern in a freshwater snail, Pila globosa
M Guruprasada Rao and N V Nanda Kumar 427
Durational effects of hemispaying on ovarian hypertrophy and estrous cycle
in albino rats Saraswati B Patil and M Appaswamy Rao 433
Structure and seasonal changes in the testes of a freshwater crab, Potamon
koolooense (Rathbun) P C Joshi and S S Khanna 439
Seasonal changes in the ovary of a freshwater crab, Potamon koolooense
(Rathbun) P C Joshi and S S Khanna 451
Evaluation of warfarin against Tatera indica and Merfones hurrianae
R P Mathur and Ishwar Prakash 463
Effects of handling on oxygen consumption and random activity in the
freshwater mullet Rhinomugil corsula (Hamilton) M Peer Mohamed 469
Effects of aqueous and lipoidal extracts of the wall of preovulatory follicles
on the ovary of growing chicks
R K Parshad, G Grewal and S S Guraya 473
Biochemical studies on the haemolymph and heart muscle of normal and
insecticide treated cockroach Periplaneta americana L.
(? Swender Reddy and A Purushotham Rao 481
Contents V
Fecundity of a hillstream minor carp Puntius chilinoides (McClelland)
from Garhwal Himalaya
H R Singh, B P Nauriyal and A K Dobriyal 487
Bionomics of hillstream cyprinids. HI. Food, . parasites and length-weight
relationship of Garhwal mahaseer, Tor tor (Ham.)
Sandeep K Malhotra 493
Effects of sublethal levels of DDT, malathion and mercury on tissue proteins
of Sarotherodon mossambicus (Peters)
K Ramalingam and K Ramalingam 501
Effect of teleostean prey size and salinity on satiation amount, satiation
time and daily ration in the glassy perchlet Chanda (= Ambassis) thomassi
(Day) (Pisces : Centropomidae)
J Rajasekharan Nair and N Balakrishnan Nair 507
Studies on some Tetracotyle Fillipi (1859) metacercariae from fishes of
Lucknow Nirupama Agrawal and Shakila Khan 515
Toxic and sublethal effects of endosulfan on Barbus stigma (Pisces : Cypri-
nidae) T Manoharan and G N Subbiah 523
Interruption of pregnancy by barbiturates in albino rats
Saraswati B Patil and M Appaswamy Rao 533
Observations on the natural history and population ecology of the social
wasp Ropalidia marginata (Lep.) from Peninsular India (Hymenoptera :
Vespidae)
Raghavendra Gadagkar, Madhav Gadgil, N V Joshi and A S Mahabal 539
Ecobiology of Corvospongilla lapidosa (Annandale 1908) (Porifera :
Spongillidae) in the Manjira reservoir, Sangareddy, Andhra Pradesh
/ Seshagiri Rao and M A Khan 553
Seasonal fluctuations in the diet composition of Rhinopoma hardwickei in
the Rajasthan desert Ranjan Advani 563
The annual reproductive cycle of Achaetobonellia maculata Fisher (Echiura :
Bonellidae) R N Singhal 569
Synthesis of 4-methyl (6,7-6-tetrahydrobenzofurano)-coumarin and its
contraception like properties in male rabbits (Oryctolagus cuniculus)
Rakesh Sinha, V P Dixit and Meera Agrawal 577
Cellular sites of steroid synthesis in the oviparous teleost fish (Cyprinus
carpio L.) : A histochemical study
Sardul S Guraya and Surinderpal Kaur 587
vl
Development of the incretory organs in the eyestalk of freshwater pra\vn,
Macrobrachium kistnensis
M S Mirajkar, R Sarojini and R Nagabhushanam 599
Histological observations on tracheal growth during wing development in
Oncopeltus fasciatus (Dallas) (Heteroptera ; Lygaeidae)
Mallela Nivedita 609
The functional demography of adrenal glands in Rattus meltada pallidior
in Indian desert B D Rana 623
Description of three new species of Drosophila (Scaptodrosophila) from
Orissa, India / P Gupta and K K Panigrahy 631
CONTENTS continued
Synthesis of 4-methyl (6,7-fc-tetrahyduobenzofurano) coumarin and its
contraception like properties in male rabbits (Oryctolagus cuntculus)
Rakesh Sinha, V P Dixit and Meera Agrawal 577
Cellular sites of steroid synthesis in the oviparous teleost fish (Cyprinus
carpio L.) : A histochemical study
Sardul S Guraya and Surinderpal Kaur 587
Development of the incretory organs in the eyestalk of freshwater prawn,
Macrobrachium kistnensis
M S Mirajkar, R Sarojini and R Nagabhushanam 599
Histologicai observations on tracheal growth during wing development in
Oncopeltus fasciatus (Dallas) (Heteroptera : Lygaeidae). . . .Mallela Nivedita 609
The functional demography of adrenal glands in Rattus meltada pallidior
in Indian desert B D Rana 623
Description of three new species of Drosophila (Scaptodrosophila) from
Orissa, India / P Gupta and K K Panigrahy 631
Subject index i
Author index , xiii
Volume contents i
Edited and published by S Ramaseshan for the Indian Academy of Sciences, Bangalote 560 080
and printed by him at the Bangalore Press, Bangalore 560 018.
Proc. Indian Acad Sci. (Anim. 3d), Vol. 91, Number 1, January 1982, pp. 1-5.
© Printed in India.
Chromosomal repatteraing in drosophilas Drasophila nasuta nasuta
and D. kohkoa
S R RAMESH and M R RAJASEKARASETTY
Department of Post-Graduate Studies and Research in Zoology, University of
Mysore, M-inasagangotri, Mysore 570 006, India
MS received 24 September 1981
Abstract. Two three-break shifts (transpositions) are detected in a chromosome
comparison between D.n. nasuta and D. kohkoa. Such aberrations are not
reported in studies with chromosome comparisons in Drosophila species. The
probable sequences arc given to explain the occurrence of these transpositions.
Keywords. Nastua subgroup ; transpositions ; inversion?.
1. Introduction
In Drosophila, phylogenetic relationships between species can be established by
way of analysing the banding patterns in the salivary gland chromosomes. Perusal
of the literature reveals that there is notable chromosomal differentia tion in some
groups of Drosophila (Bicudo 1973; Bock 1971; Brncic et al 1971; Hsu 1952;
Kastritsis 1966; Stalker 1965; Stone et al 1961 ; Wasserman 1962a, b, c) while
in others the banding sequences have apparently remained unaltered (Dobzhansky
1972). The Utter is referred to as homosequeijtial species.
The members of the nasuta subgroup of the immigrans group of the genus
Drosophila have been studied to establish their evolutionary relationships. The
members are, D. nasuta nasuta, D.n. albomicana, DM. kepulauana, D. kohkoa,
D. putaua, D. sui, D. nixifrons, D. pallidifrons, D. sulfurigaster sulfurigaster,
D.s. neonasiita, D.s. bilimbata and D.s. albostrigata. This is reported in detail by
Rajasekarasetty et al (1980). The present paper deals with the chromosome
relationship between D.n. nasuta and D. kohkoa. The nature of banding in
D. kohkoa\§ studied in comparison with th&t of D.n. nasuta which is taken as the
standard.
2. Materials and methods
As D.n. nasuta (of Coorg, Karnataka, India) and D. kohkoa (of Gulf of Thailand
—University of Texas collection No. 3256-2 # 1) proved to be cross sterile
(Rajasekarasetty et al 1980j, a direct optical comparison of the banding pattern
1
2 5" R Ramesh and M R Rajasekarasetty
of the salivary gland chromosomes of both the species were made. The pro
ccdure of Ranganath aud Krisluianurthy (1975) was used to prepare the salivar
gland chromosomes.
3. Results and discussion
The karyotype of both D.n. nasutazr\& D. kohkoa includes a pair of metacentric
(chromosome 2), two pairs of acrocentrics (sex chromosome md chromosome 3
and a pzir of dots (chromosome 4). The salivary gland chromosome compiemen
of both the species thus ir.clu.des four long arms representing two arms of chrome
some 2 (2Land2#), chromosome 3, X chromosome and a short arm of chromo
some 4.
Comparison of banding patterns of the salivary gland chromosome of D. kohko
with that of D.n. nasiita revealed that the X chromosome and chromosome 2 ar
homosequential but chromosome 3 of the former species differs from that of th
latter by a paracentric inversion named NKLOi and two three-break shifts (trans
positions) named NKo-Sx and NKo-S3 (figures 1, 2);
Structural reorganization of the chromosomes during spcciation involves eithc
paracentric, pericentric inversions, duplications and/or deletions. Chromosomj
different' a tion due to these changes (aberrations) have been reported in differei
groups of Drosophila. The uniqueness of the present report is that, in additio
to a paracentric inversion, two three-break shifts (transpositions) are also invove
in the chromosomal repatterning in D. kohkoa. The existing chromosome
linearity due to transpositions in D. kohkoa could be explained by two successi\
inversions and the probable sequence of which is represented dia grama tically i
figure 3.
Perusal of the literature reveals that the occurrence of such three-break shif
are very rare. Dobzhansky (cf. Patterson and Stone 1952) has expressed tlu
there are no sure cases of three-break rearrangements in Drosophila specie
Similarly White (1973) has opined that chromosomal repatterning due to tiani
positions is rare. As far as we know, this occurrence of transposition* is a maide
report of its kind for species comparisons in Drosophila.
# 1 j. a b c d e f g h i j k 1 m | n
One inversion with two breaks (between— centromere and a, m and n)
# 2 I m 1 k j i h' g f 4- e d c b a j, n
Two inversions with three breaks (between— centromere m, f and e, a and i
# 3 f g h i j k 1m a b c d c n
Figure 3. Diagrammatic representation of the possible/probable rearrangements 1
explain existing linearity of the chromosome 3 in D. kohkoa (when compared wii
chromosome 3 cf D.n. nasuta, taken ?s standard).
( 4£ Centromeric end).
Chromosomal repatterning in Drosophila
...... •„....-
»—i
/. x*.
+ » Centromeric end.
Figures 1-2. 1. Chromosome 3 of D. nasuta nasuta. 2. Chromosome 3 of
D. kohkoa.
Chromosomal repatterning in Drosophila 5
Acknowledgements
The authors are thankful to Pi of. N B Krishnamurthy, Head of the Department
of Zoology and to Dr H A Ranganath, Lecturer in Zoology, University of Mysore,
for their helpful discussions. The financial assistance by the Department of
Atomic Energy, Government of India is gratefully acknowledged.
References
Bicudo H E M C 1973 Chromosomal polymorphism in the saltans group of Drosophila. I. The
saltans subgroup ; Genetica 44 520-552
Bock I R 1971 Intra and interspecific chromosomal inversions in the Drosophila bipectincta
species complex ; Chromosoma 34 206-229
Brncic D, Nair P S and Wheeler M R 1971 Cytotaxonomic relationships within the mesophrag-
matica species group of Drosophila ; Univ. Tex. Publ. 7103 1-16
Dobzhansky Th 1972 Species of Drosophila ; Science 177 664-669
Hsu T C 1952 Chromosomal variation and evolution in the virilis group of Drosophila ; Univ.
Tex. Publ. 5204 35-72
Kastritsis C D 1966 A comparative chromosome study in the incipient species of the Drosophila
paulistorum complex ; Chromosoma 19 208-222
Patterson J T and Stone W S 1952 Evolution in the genus Drosophila (New York : The
Macmillan Co.)
Rajasekarasetty M R, Ramesh S R and Krishnamurthy N B 1980 Interspecific chromosomal
variation among a few members of the nasuta subgroup (Genus : Drosophila) ; Entomon.
5 1-12
Ranganath H A and Krishnamurthy N B 1975 Chromosomal polymorphism in Drosophilal
nasuta. III. Inverted gene arrangements in South Indian populations ; /. Hered. 66
90-96
Stalker H D 1965 The salivary Chromosomes of Drosophila micromelanica and Drosophila
melanura ; Genetics 51 487-507
Stone W S, Guest W C and Wilson F D 1960 The evolutionary implications of the cytological
polymorphism and phylogeny of the vtrttis group of Drosophila ; Proc. Natl. Acad. Sci.
U.S.A. 46 350-361
Wasserman M 1962a Cytological studies of the repleta group of the genus Drosophila. III. The
mercatorum subgroup ; Univ. Tex. Publ. 6205 63-71
Wasserman M 1962b Cytological studies of the repleta group of genus Drosophila. IV. The
hydei subgroup ; Univ. Tex. Publ. 6205 73-83
Wasserman M 1962c Cytological studies of the repleta group of the genus Drosophila. V. The
mulleri subgroup ; Univ. Tex. Publ. 6205 85-117
White M J D 1973 Animal cytology and evolution (Third Edn.) Cambridge University Press
Proc. Indian Acad. Sci. (Anim, Sci.), Vol. 91, Number 1, January 1982, pp. 7-12.
<£) Printed in India.
Acid phosphatase activity in tissues of Notoptems notopterus
chronically exposed to phenolic compounds
R C DALELA, SAROJ RANI and S R VERMA
Pollution Relevant Research Laboratory, Post Box 264, Post-graduate Deportment
of Zoology, DAV College, Muzaffarnagar 251001, India
MS received 10 February 1981 ; revised 13 August 1981
Abstract. Specimens of Notoptems notopterus were exposed to three subl thai
concentrations (l/10tli, l/15th and l/20th of 96 hr LC,0) of phenol (P), 2,4-dinitro-
phenol (DNP), pentachlorophenol (PCP), and their three combinations (PCP +
DNP)/P (highly antagonistic), (DNP + P)/PCP (additive) and (P + DNP)/PCP
(highly synergistic) for 15 and 30 days, and brain, liver, kidney and gills were taken
out separately for determining acid phosphatase activity. In general, inhibition
was maximum (89-32%) and highly significant (P < 0-001) in brain, and minimum
(6-93%) and insignificant in kidney of fish exposed to 1/1 Oth of (P + DNP)/PCP
and P, respectively after 30 days. When P, DNP and PCP were used separately
PCP exerted more inhibitory effects than DNP and P. However, significant stimu-
lation (P < 0-05 ; P < 0-01) at 1/1 5th and l/20th of P and DNP both after 15
and 30 days, and insignificant at l/20th of (PCP + DNP)/P after 15 days was also
observed in kidney.
Keywords. Acid phpsphatases ; Notoptems notopterus, phenolic compounds.
1. Introduction
There is increasing concern today about environmental contamination with
phenolic compounds such as phenol, 2,4-dinitrophenol and pentachlorophenol.
These compounds are the non-specific pesticides (Rappe and Nilson 1972) used
as herbicides, molluscicides and bactericides in industries, wood preservation and
agriculture. As an antiseptic, phenol is also used for medicinal purposes. Inspite
of their extensive use, little attention is paid on their effects on metabolic activities
of freshwater fish (Weinbach and Garb us 1969 ; Desaiah 1978 ; Dalela et al
' 1980 ; Verma et al 1980). Acid phosphatase is a hydrolytic enzyme which takes
part in the dissolution of dead cells and as such is a good indicator of stress condi-
tion in the biological system (Gupta et al 1975 ; Verma et al 1980). This study
was undertaken to evaluate the effects of sublethal concentrations of P, DNP,
PCP and their three combinations— (PCP -h DNP)/P (highly antagonistic),
(DNP + P)/PCP (additive) and (P + DNP)/PCP (highly synergistic) (Verma et al
1981) on acid phosphatase activity (orthophosphoric monoester phosphohydrolase ;
E.G. 3* 1-3' 2) in different tissues of a freshwater fish Notopterus notopterus.
8 R C Dalela, Saroj Rani and S R Venna
2. Materials and methods
Specimens of N. notoptems (16 to 21 cm in length ;. 35 to 60 g in weight) were
collected from Kalinadi and adopted for two weeks to the laboratory conditions.
The technical grades of phenol (C6H5OH), 2,4-diratrophenol ((NO2)2 C6HaOH)
and pentachlorophenol (sodium salt; C6Cl5ONa) manufactured by Thomas Baker
and Co. (London), Thomas and Thomas (India) and Hopkins and William Ltd.
(England), respectively were used. Stock solutions of 1-0 g/L were prepared
separately and the desired concentrations of these chemicals were obtained, using
the table 231 (3) of Standard Methods (1971).
Fifteen fish were transferred in each concentration (l/10th, l/15th and l/20th
of 96 hr LC50) of these chemicals and combinations kept in triplicate for 30 days
(96 hr LC50 ofP, D:NP, POP and (PCP + DNP)/P, (DNP + P)/PCP and
(P4-DNP)/PCP combinations being 12-53 mg/L, 1/34 mg/L, 0-083 mg/L,
24 -00 mg/L, 0-083 mg/L and 0-0065 mg/L, respectively. During acclimatation
and exposure periods, flsh were fed once a day with chilled crustacean diet (contain-
ing cyclops and daphnia) to avoid the starvation effects (Alekseev and Uspendskaya
1974). Solutions were renewed after each 24 hr, to avoid the fouling by food
and excretory matter. Controls were also set side by side for comparison.
Fish were sacrificed after 15 and 30 days, and brain, liver, kidney and gills
were taken out and pooled separately in ice cold petridishes containing 0*25 M
sucrose solution. Tissue homogenates (5%) were prepared separately using 0 -25 M
sucrose solution, with a Potter Elvehjem homogenizer. Homogenates were
centrifuged at 900g under cold conditions (5-0 ± 1-0°C) and supernatants were
used for enzyme study. Acid phosphatase activity was measured by the method
of Shinowara et al (1942). The inorganic phosphate liberated was determined
by Fiske and Subbarow (1925) method. Statistical significance of the difference
between the control and experimental values was calculated by student's ' t ' test
(Fisher 1950).
3. Results and discussion
Average values along with mean ± S.E. of three observations for acid phosphatase
activity in brain, liver, kidney and gills of control fishes, and per cent inhibition/
stimulation in exposed fishes after 15 and 30 days are given in table 1. It is
clear from the table that when fishes were exposed to P, DNP and PCP sepa-
rately, greater inhibition was observed in fishes exposed to PCP and DNP as
compared to fishes exposed to P. This is due to the replacement of hydrogen ty
chloro and nitro groups in PCP and 1>NP, respectively (Kopperman et al 1974).
In general maximum (89-32%) and highly significant (P < 0-001) inhibition as
observed in brain, and minimum and insignificant (6-93%) in kidney at 1/10 th
concentration of (P + DNP)/PCP, respectively after 30 days. However, in
kidney biphasic effects of P, and DNP were observed, i.e., inhibition in enzyme
activity at higher concentrations and stimulation at lower concentrations. Stimu-
lation was significant (P < 0-05) at l/15th of P, l/15th and l/20th of DNP, and
at P < 0-01 in l/20th of P after 15 days, and l/15th and l/20th of P and>DNP
after 30 days while it was insignificant at l/20th of (PCP 4- DNP)/P combination
after 15 days. Inhibition in -acid phosphatase in these tissues was in the order.
AP activity in tissues of N. notopterus
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10 R C Dalela, Saroj Rani and S R Verma
brain > liver > gilts > kidney except after 30 days in DNP and after 15 and 30
days in (PCP + DKP)/P where inhibition was in the order, liver > brain > gills
< kidney, and after 30 days in P where sequence of inhibition was in the order,
brain > gills > liver > kidney. .
Phenols enter in blood circulation offish through gills and skin, and get distri-
buted into different tissues where they affect normal metabolism (Mitrovie et al
1968). Dalela et al (1980) also studied the effect of sublethal concentrations of
P and PCP on hepatic acid and alkaline phosphatases and observed significant
inhibition. Syncrgistic effects of P and DNP on acid and alkaline phosphatases
were also observed by Verma et al (1980). Authors in this investigation observed
that these compounds in combinations showed no definite pattern of toxicity
(i.e., inhibition/stimulation in enzyme activity) as they showed separately. At
l/20th, in brain after 15 and 30 days, at l/10th, l/15th and l/20th in liver after
15 days, at l/15th and l/20th in liver after 30 days, and at l/10th in kidney after
15 days, the per cent inhibition was not significantly different in fish exposed to
PCP, (DNP 4- P)/PCP and (P + DNP)/PCP combinations. At l/15th and
l/20th in kidney after 15 and 30 days, at 1/iOth, l/15th and l/20th in gills after
15 days, and at l/15th and l/20th in gills after 30 days inhibition in fish exposed
to (P + DNP)/PCP was significantly lesser than in fish exposed to PCP alone and
to (DNP+P)/PCP combination. In fish exposed to (PCP + DNP)/P, inhi-
bition in brain and gills was significantly less as compared to the fish exposed to
phenol, in liver inhibition was not significantly different and in kidney significant
stimulation (P < 0-05 ; P < 0-01) was there at l/15th and 1/20 th of P both after
15 and 30 days while in (PCP + DNP)/P insignificant stimulation was observed
at 1/20 th only after 15 days.
Loomis and Lipmann (1948) and Simon (1953) after DNP exposure, and Yap
etal (1975) and D^saiah (1978) after PCP exposure, pointed out that uncoupling
of oxidative phosphorylation is the main cause for inhibition of phosphatases.
Uncoupling of oxidative phosphorylation was also pointed out by Dalela et al
(1980) and Verma et al (1980) for the inhibition of acid and alkaline phosphatases.
Simon (1953) stated that concentrations higher than those needed to prevent
oxidative phosphorylation injured the mitochondrial system so greatly as to
block the action of enzymes concerned with oxidative metabolism. Action of
uncouplers of oxidative phosphorylation has been pointed out on the basis of
chemical (Pressman 1963) and chemi-osmotic (Mitchell 1961) interactions.
According to (Pressman 1963), uncouplers promote the conductivity of protons
within mitochondrial membranes and subsequently prevent the formation of a
gradient across the membrane. According to Mitchell (1961), uncouplers promote
the splitting of an energy rich intermediate compound prior to ATP production.
Weinbach and Garbus (1969) suggested that uncouplers traverse through lipo-
protein layer of mitochondrial membrane and interact with protein groups that then
undergo structural changes. It is generally assumed that major changes in mito-
chondria! function are reflected in morphological alterations and that normal
mitochondrial profiles are dependent on the continuing supply of energy rich
intermediates produced by oxidative phosphorylation. Weinbach and Garbus
(1969) indicated that these uncouplers bind tightly with mitochondrial proteins
which are involved in amino acid metabolism. However, authors of this investi-
gation, assumed that all these interactions and processes held simultaneously when
AP activity in tissues of M riotopterus H
ih were exposed to these chemicals and their combinations, causing the
icoupling of phosphorylation and finally affect the activity of phosphatases.
Gxing of chemicals enhances toxicity (synergism) in some cases aoid decreases
.ntagonism) in other cases but the actual mechanism of combination effects on
nd phosphatase activity is not well-known.
Acknowledgement
!SIR (New Delhi) is thankfully acknowledged for financing the research programmes
f which the present work is a part.
References
Jekseev V A and Uspenskaya N E 1974 Toxicological characteristics of acute phenol poisoning
of some fresh water worms ; GidrobioL Zh. 10 48-55
>alela R C, Rani S and Verrna S R 1980 Physiological stress induced by sublethal concentrations
of phenol and pentachlorc phenol in Notopterus notopterus : Hepatic acid and alkaline
phosphatases and succinic dehydrogenase ; Environ. Pollut. 21 3-8
3esaiah D 1978 Effect of pen-tachlorophenol on the ATPases in rat tissues ; Pentachlorophenol
12 277-283
Fisher R A 1950 (Statistical methods for research workers ; llth ed. (London : Oliver and
Boyd).
Fiske G H and Subbarow K 1925 The colorunetric estimation of phosphorus ; /. BioL Chem*
66 375-381
jrupta P K, Dhar U and Bawa S R 1975 Effect of malathion and radiation separately and
jointly upon rat enzymes in vivo ; Environ. PhysioL Biochem. S 49-53
Copperman H L, Carlson R M and Caple R 1974 Aqueous chlciination and ozonatiop studies.
Structure — toxicity correlations of phenolic compounds to Daphnia magna ; Chem.Biol.
Interaction 9 245-251
Loomis W F and Lipmann F 1948 Reversible inhibition of the coupling between phosphorylation
and oxidation ; /. Biol Chem. 173 807-814
Mitchell P 1961 Coupling of phosphorylation to electron and hydrogen transfer by a chemi-
osmotic type of mechanism ; Nature (London) 191 144-148
Milrovic V V, Brown V M, Shurben D G and Barryman M H 1968 Some pathological effect
of subacute and acute poisoning of rainbow trout by phenol in hard water ; Water Res.
2 249-254
Pressman B C 1963 In, Energy linked functions of mitochondria (ed.) B Chance (New York :
Academic Press) pp 188-191
Rappe C and Nilsson C A 1972 An artifact in the gas chromatographic determination of impu-
rities in pentachlorophenol ; /. Chromatogr. 67 247-253
Shinowara G Y, Johns L M and Reinhart H L 1942 The estimation of serum inorganic phos-
phate and acid and alkaline phosphatase activity ; /. BioL Chem. 142 921-928
Sinon E W 1953 Mechanism of dinitrophenol toxicity ; BioL Rev. Cambridge Philos. Soc. 28
453-479
Standard methods for the examination of water and waste water 1971 13th ed. Am. Publ. Hlth.
Assoc. Inc. New York, N.Y.
Verma S R, Rani S and Dalela R C 1980 Effects of phenol and dinitrophenol on acid and
alkaline phosphatases in tissues of a fish (Notopterus notopterus) ; Arch. Environ. Con tarn.
Toxicol 9 451-459
11 & C Dateta, Saroj Rani and S R Vermd
Verma S R, Rani S and Dalela R C 1981 Synergism, antagonism and additivity of phenol,
pentachlorophenol and dinitrophenol to a fish (Notopterus notopterus) ; Arch. Environ.
Contam. Toxicol. 10 365-371
Weinbach E C and Garbus J 1969 Mechanism of action of reagents that uncouple oxidative phos-
phorylation ; Nature (London) 221 1016-1018
Yap H M, Desaiah D, Cutkomp L K and Koch R B 1975 In vitro inhibition of fish brain*
ATPase activity by cyclodiene insecticides and related compounds ; Bull Environ. Contam.
Toxicol. 14 163-167
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 1, January 1982, pp. 13-26.
<£J) Printed in India.
Differences in home ranges of rhesus monkey (Macaca mulatto)
groups living in three ecological habitats
RAGHUBIR SINGH PIRTA and MEWA SINGH*
Department of Psychology, Utkal University, Bhubaneswar 751 004, India
* Department of Psychology, University of Mysore, Mysore 570006, India
MS received 13 July 1981 ; revised 20 October 1981
Abstract. Field observations were carried out on rhesus monkeys living in Asarori
Forest, Chakia Forest, and temples. Data on group size, group composition and
socionomic sex-ratios were obtained. An average home range size in these three
habitats was found to be 5 -18 km2, 1-52 km2 and 0-01 7 km2 respectively. A posi-
tive correlation was fouad between group size and home range size in the Asarori
Forest. Core areas were absent inside the home ranges in Chakia Forest. The
average core area in other 2 habitats was 0-48 km2 and 0-009 km2 in Asarori Forest
and temples respectively. The variability in home ranges and core areas is analysed
in terms of differences in ecological conditions.
Keywords. Home range ; phylogenetic adaptation ; adaptive modification ; rhesus
monkey.
1. Introduction
In our earlier studies (Pirta and Singh 1978, 1980) we have emphasised the phylo-
genetic adaptivcncss of home ranges in rhesus monkeys. The nature of a phylo-
genetically adaptive behavioural system varies from an extremely environmentally
labile to a highly environmentally stable one (Lorenz 1965). The reviews by
Glutton-Brock and Harvey (1977) and Southwick and Siddiqi (1974) indicate
that home range size is an environmentally labile behavioural system and varies
greatly both within and between the species of non-human primates. Although
the home ranges of Hanuman langur (Vogel 1977) and bonnet monkey (Rahman
and Parthasarthy 1978) have, been studied in various ecological habitats in India,
such comparative data on rhesus monkey are lacking. Such information helps
in understanding the adaptive modifications going on in the behaviour of a species.
They result from the interaction of phylo genetically acquired blueprints and the
environment. In the present study our observations on the home ranges of
rhesus monkey inhabitii g 3 natural environmental conditions are reported.
2. Method
During the exploratory phase we became thoroughly acquainted with the geo-
graphical features of all 3 habitats. Our main emphasis was to record the loca-
11
14 Raghubir Singh Pirta and Mewa Singh
tion of a monkey group as accurately as possible on a map, whenever and wherever
it was encountered. A group was followed from a few minutes to several hours
at a stretch on an observation day. Occasionally, a group was followed from
dawn to dusk and durii g late evening and early morning hours. The period of
study and tims devoted to observations in different habitats are given in table 1.
Behavioural observations were started after the monkeys became acquainted
with the observer. We observed the monkeys by standing at the periphery of
the group. All recordings were made on notebooks and maps ad lib. The main
variables measured are given in table 2. However, qualitative notes of the eco-
logical characteristics of a habitat and behaviour Of monkeys were also taken.
3. Study areas
3.1. Asarori forest
The study site (32 km2) included blocks of Laldhang, Chandrabani, Asarori, Maho-
bawala and Mohamadpur (compartments 1, 2 and 3 only), which form a major
portion of Asarori forest range in the Western division of Dehra Dun forest
(figure 1). The Asarori forest is on the northern slope of the Siwalik Hills, \vith
elevations ranging from 425 m at the valley floor to 950 m at the Siwalik crest.
Detailed description of the Asarori forest has been reported by Lindburg (1971).
The major area of the forest part studied was covered by Shorea robusta which
was interspersed with other tree species, grassland and eroded stream beds or
raos.
Table 1. Period of Study and groups observed m different habitats.
Habitat
Area explored
Year
Days
Groups
Asarori Forest
32km3
Jan. 1974 to Dec. 75
400
13
Chakia Forest
24km3
Aug. 1977 to July 78
70
3
Urban area
40km3
do
150
2
Table 2. Sampling variables and their measures
Variable?
Measures
1. Group size
2. Group composition
3. Home range size
4. Core area size
Number of individuals which regularly associate together
and share a common home range.
Number of individuals in each age-sex class, i.e. adult males,
adult females, juveniles and infants.
Total area (km2) over which the group was seen moving and
foraging during one year period.
Area (km2) within the home range most frequently used for
night resting.
Differences in home ranges of rhesus monkey
15
N
Forest road
Forest quarter
Edge of forest
Ravine
Figure 1. Map showing the principal features of forest habitat in Asarori.
3.2. Chakia forest
The study site (24 km3) was an isolated part of Chakia forest range of Varanasi
division, and included four blocks : Sapahi, Sherpur, Amlahwa and Garhar
(figure 2). It was mainly a scrub forest covered by interspersed trees of Tama^
rindus indica, Azadirachta indica, Mangifera indica, Syzygium cumini, Semicarpu$
cuiacaridum, Tectona grandis, Bombax malabaricum, Sutea monosperma and
young plantations of bamboo and acacia. Shrubs of Smilax indica, Carissa
spinarum, Abrus precatorius and Ziziphus mauritiana formed a thick vegetation
along the ravines. The whole area was surrounded by cultivated land. In the
southern part were 2 hillocks while the remaining area was plain but interspersed
with deep ravines. The river Karamanasha flows in the middle of this forest
part from south to north, accompanied by its 2 canals.
3.3. Urban area
The Varanasi city and its surrounding area, covering approximately 40 kma,
was explored for urban monkey population. Finally, two temples inside the
city, each with a resident monkey group were selected for long term observations.
3-3a. Sankat Mochan temple : This temple was surrounded by a boundary
wall and covered approximately 1-2 hectares. On both sides of the main temple
16
Raghubir Singh Pirta and Mewa Singh
CHAKIA
Figure 2. Map showing the principal features of forest habitat at Chakia.
Black circles show rhesus groups and empty circles langur groups.
building was a thick vegetation of trees and sfrrubs. The tree species in this
temple included Ficus religiosa, Ficus bengalensis, Azadirachta indica, Phyllanthus
emblica, Semicarpus anacaridum etc. There were shrubs of Carissa spinamm,
Smilax indica and Ziziphus mauritiana. Various kinds of vegetables and grasses
were also grown in the temple and its adjacent gardens. The Sankat Mochan
temple is the temple of the monkey god Hanuman. People visited this temple
specifically on Tuesdays and Saturdays to feed the monkeys. Othci features of
the temple are shown in figure 3.
3.3b. Durga temple : This temple was in the midst of buildings and covered
approximately 0-6 hectares. On one side of it was a big pond. Except for a
few trees in the compounds of adjacent buildings there was no vegetation* in
Differences in home ranges of rhesus monkey
17
meter
Figure 3. Principal features of Sankat Mochan temple.
Durga temple. There was less open space for monkeys in this temple incomjpa*
rison to the Sankat Mochan temple. Because the Durga temple was located just
on the side of the main road, the monkeys of this temple had more contact with
human beings than those of the Sankat Mochan temple. Other habitat features
of Durga temple are sho^n in figure 4.
A comparison pf Asarori forest, Chakia forest and temple habitats is given ifa
table 3?
18
Raghubir Singh Pirta and Mewa Singh
Figure 4. Principal feature of Durga temple.
4. Observations
Rhesus monkeys live in groups which comprise of adult males, adult females>
juveniles and infants. A group occupies a circumscribed area of a particular
niche, the home range.
4.1. Group size
In all 13 bisexual groups, 3 temporary all-male associations and 2 solitary males
were observed in Asarori forest. A total of 598 monkeys lived in an area of
32 km2. The number of monkeys in bisexual groups varied from 11 to 127
an average group size of 45 (table 4).
Differences in home ranges of rhesus monkey
Table 3. Comparison of the three habitats.
19
Characteristics Temples, Varanasi
Chakia forest,
Varanasi
Asarori forest,
Dehra Dun
Temperature
Annual rainfall
Vegetation
7°C-41°C
1088mm
A few trees in Sankat
Mochan temple
High
Human influence
Other wild mammals Jackal (in Sanfcat
Mochan temple)
7°C-41°C
1088mm
Scrub forest
Medium
Jackal, pig, leopard ( ?)
0° C-40° C
1600mm
Moist Deciduous
forest
Very less
Jackal, pig, deer
Species, antelopes,
elephants, leopard
Sleeping trees
Roof (Durga temple)
Few trees
Numerous trees
and trees (Sankat
Mochan temple)
Water sources
Many
Many
Few
Food sources
Good, localised
Poor, scattered
Good, scattered
Predators
Man, dog, hawk
Man, dog, hawk,
Man, dog, weasel,
leopard (?)
hawk, leopard
Other primates
None
Langur
Langur
Table 4. Numerical data on group size, group composition, home range size and
core area of rhesus groups in Asarori forest.
Group composition *
VJlimtl VI 1 <JU\J
size
MM
FF
JJ
II
jnomc t^orc oocwnouuc sex-
range area ratio (MM : FF)
(km2) (km2)
G 1
30
3
8
12
7
5-06
0-56 I:
2-66
G2
127
11
35
61
20
14-06
1-81 :
3-18
G 3
11
1
3
4
3
1-1?
0-04 :
3-00
G 4
77
8
25
29
15
9-56
G-88
3-12
G5
70
6
21
33
10
11-25
1-13 :
3-50
G6
37
2
10
19
6
1-75
0-07 :
5-00
G7
33
2
9
15
7
2-75
0-14 :
4-50
G 8
37
3
9
18
7
3-93
0-32 :
3-00 '
G9
28
3
9
12
4
3-93
0-22 1:
3-00
G 10
32
4
10
12
6
2-25
0-11 1:
2-50!
Gil
37
3
9
18
7
5-06
0-56 1:
3-00
G12
37
3
10
17
7
3-93
0-32 1:
3-33
G13
28
2
7
13
6
2-81
0-14 1:
3-50
Mean
44-92
3-92
12-69
20-23
8-07
5-18
0-48 1:
3-33
S.E.M.
db 8-36
±0-78
± 2-45 ±
3-97
db 1'28
±1-09 ±0-20
* Based
on census in
June-July
1974;
MM— adult males
; FF— adult
females |
JJ— juveniles ; II— infantf .
20
Raghubir Singh Pirta and Mewa Singh
In the Chakia forest, 5 bisexual groups and 1 isolated male lived in an area of
24 km2. Three groups counted ranged from 27 to 38 ; an average group had
31-6 individuals (table 5).
In the city of Varanasi 9 bisexual groups were located in an area of 40 km2
approximately. Two temple groups were counted, providing an average group
size of 98-5 (table 6).
Table 5. Numerical data on group size, group composition and home range size
of rhesus groups in Chakia forest.
Group
Group -
Group Composition
#
Home
Socionomic
size
MM
FF
JJ
II
range
sex-ratio
km2
(MM : FF)
Group 1
38
3
17
12
6
3-00
1:5-66
Group 2
27
?
11
7
7
0-56
1:5-50
Group 3
30
3
17
5
5
1-00
1:5-66
Mean
31-66
2-66
15-00
8-00
6-00
1-52
1:5-0)
S.E.M.
± 3-31
±0-35
± 2-00.
± 2*08
±0-57
±0-75
* Based on census in December 1977
JJ— juveniles ; II — infants.
MM— adult males; FF— adult females;
Table 6. Numerical data on group size, group composition, home range size and
core area of temple monkeys.
Group composition *
Group
TJT~«~.~
Core
Socionomic
Size MM FF
JJ
II range
area
sex-ratio
km2
km2
(MM:FF)
Sankat
Mochan
temple
group
129 13 40
46
30 0-020
0-012
1:3-07
Durga
temple
group
68 7 20
28
13 0-015
0-006
1:2-85
Mean
98-50 10-00 30-00
37-00
21*50 0-017
0-009
1:2-96
S.E.M.
± 30-50 ± 3-00 ±10-00
± 9-00
i 8-50 0
0
* Based on census in December 1977 ; MM— adult males ; FF — ad It females
JJ— juveniles ; II — infants.
Differences in home ranges of rhesus monkey 21
4.2. Group composition
An average group in Asarori comprised of 3-9 adult males, 12-7 adult females,
20*2 juveniles and 8 infants. In Chakia forest an average group size comprised
of 2*6 adult males, 15 adult femiies, 8 juveniles and 6 infants. There were 10
adult males, 30 adult females 37 juveniles and 21*5 infants in an average temple
group. Socionomic sex-ratios (adult males : adult females) in Asarori, Chakia
forest and temples were 1:3, 1:5 and 1 :3 respectively.
4.3. Home range size
An average home range size of 13 bisexual groups in Asarori forest was 5- 18 km2.
The horn) rang^ remained the same for 2 years except for some minor changes
in the case of som? groups. There was extensive overlapping of home ranges
among 13 groups of Asarori forest. A group shared its home range with at least
4 other groups (figure 5). The largest gioup (G2) shared the home range of 10
groups. A relationship was found between the group size and home range size.
As the number of individuals increased the size of the home range also increased
(product-moment coefficient of correlation, r =0-934 ; df = 11 ; p < -01).
In Chakia forest the average home range size was 1-52 km2 for the 3 rhesus
groups. The overlapping of home ranges was less in the Chakia forest when
compared to the Asarori forest. The home ranges in Chakia forest were also
smaller in size (figure 6).
The temple group lived in an average home range of 0-0 17 km2. There wat
no overlapping among the home ranges of Durga temple group and Sankas
Mochan temple group (figures 7 and 8). Whenever another group was seen on
the periphery of Durga temple group home range, the latter group immediately
chased the former group away.
Figure 5. Home ranges of rhesus munkeys occupying the Asarori forest.
22
Raghublr Singh Pirta and Mewa Singh
CHA
Km.
Figure 6. Home ranges of rhesus monkey groups occupying the Chakia forest.
4.4. Core area size
In Asarori forest, each group had one or two core areas, which were preferred
to other parts of its home ran^e. More than 60% of the sleeping sites converged
in this area (s). The size of core areas varied from 0-04 km2 to 1-81 km2 with
a mean of 0-48 km2. There was no overlapping among core areas of different
groups (figure 9). Deep ravines, high ridges, dense shrubs, tall trees of Shorea
robusta and Terminalia tomentosa and presence of water were characteristic features
of these core areas. The size of the core areas increased with the size of group
in Asarori forest (product-moment coefficient of correlation, r =0-942 ; df = 11 ;
p <0'Q1). : * * - •
Differences in home ranges of rhesus monkey
23
Figure 7. Home range (thin line) and core area (thick line) of the Sankat Mochan
temple group.
Core areas were not discernible in the home ranges of Ghakfe forest monkeys.
The temple monkey had permanent places to sleep during nights. Ths average
size of the core area was 0-009 km2. The Sankat Mocha * temple monkeys slept
on the trees during nights while Dursa temple monkeys slept on the temple roof.
5. Discussion
We found variability in home ranges of rhesus monkeys in different habitat?.
This variability can be understood partially in terms of some conclusions drawn
by Glutton-Brock and Harvey (1977) for primates in general.
(i) "Populations living in areas of lo\v food availability tend to have larger
home ranges than those living m areas where food is more abundant." The
Asaiori forest groups lived in larger home ranges (mean 5-18 km2) than those
of temple groups msan 0-0 17 km2). Souths ick and Siddiqi (1974) have also
reported similar differences in the home ranges of forest and temple rhesus groups.
However, conditions in Chakia forest are different due to scarce food resources,
fe\? night lodging trees and restricted space. These conditions are almost similar
to those reported by Lindburg (1971) for the 4 rhesus groups living ia Forest
Research Institute, Deftra JHtti. Although the rhesus groups at Chakia forest
24
Raghubir Singh Pirta and Mewa Singh
N
urga Temple,,
Group (
A si Group .**"••• (
8. Home range (thin line) and core area (thick line) of the
O'j" Within Populat o
Purred habita? tend
tetion of rhesus moneys, the ho
than the hom
4 groups at porest
ran^es «<>r the cote aieas.
" Pr°P°rtion ^
Wre rauch
the home
also observed a
f
^
season
S^up size the
' Mak*a>* (1978)
'*** is
Differences in home ranges of rhesus monkey
N
25
Figure 9. Core areas of rhesus monkey groups occupying the Asarori forest.
range (Lindburg 1971). Our observations also support this, however, it seems
true only for those groups \\hose home ranges are faiily large, approximately
above 5 km2.
Acknowledgements
This work was done in the Institute of Advanced Studies, Meerut University,
Meerut, and Kashi Vidyapith University, Varanasj. RSP is thankful to UGC
for Postdoctoral Fellowship at the Centre of Advanced Study in Psychology
Utkal University, Bhubancswar.
References
Glutton-Brock T H and Harvey P H 1977 Primate ecology and social organization ; /. Zool.
(London) 183 1-30
Lindburg D G"l971 The rhesus monkey in North India ; an ecological and behavioural study ; in
Primate Behaviour : Developments in field and laboratory research II (cd.) L A Rosenblum
(New York : Academic Press) pp. 1-106.
Lorenz K Z 1965 Evolution and modification of behaviour (London : Methuen)
Makwana S C 1978 Field ecology and behaviour of rhesus macaque (Macaca mulatto) : 1. Group
composition, home range, roosting sites and foraging routes in the Asarori Forest ; Primates
19 4S3-492
Pirta R S and Singh M 1978 Establishment of home range, intraspecific and interspecific rela-
tions in rhesus monkeys (Macaca mulatto) under infant-infant rearing conditions ; Proc.
"Indian Acad. ScL B87 267-278
Pirta R S and Singh M 1980 Changes in home ranges of rhesus monkey (.Macaca mulatto)
groups living in natural habitats ; Proc. Indian Acad. ScL B89 515-525
26 Raghubir Singh Pirta and Mewa Singh
Rahman H and Parthasartliy M D 1978 Behavioural variants of bonnet macaque (Macaca radiatd)
inhabiting cultivated gardens ; J. Bombay Nat. Hist. Soc. 75 406-425
Southwick C H and Siddiqi M F 1974 Contrasts in primate social behaviour ; Biomence 24
398-406
Vogel C 1977 Ecology and sociology of Presbytis cntellus ; in Use of non-human primates in
biomedical research (eds.) M R N Prasad and T C Anand Kumar (New Delhi : Indian National
Science Academy) pp. 24-45.
Ptoc. Indian Acad. Sci. (Anim. Sci), Vol. 91, Number 1, January 1982, pp. 27-32.
© Printed in India.
Effects of aldrin on serum and liver constituents of freshwater
catfish Clarias batrachus L.
YAGANA BANG
Department of Zoology, Aligarh Muslim University, Al'garh 202 001, India
MS received 20 August 1980 ; revised 21 November 1981
Abstract. The changes in total protein, total phosphorus, calcium and cholesterol
contents were observed in serum and liver of treated and control fish Clarias batra-
chus L. Aldrin caused more pronounced effect in liver than in serum. Values
remained significantly low in liver of experimental fish than in control, while the
serum constituents fluctuated widely. The variations observed in different values
are explained as transfer or development of tolerance. A constant increase of
cholesterol in serum corresponding to a regular decrease in liver is interpreted aS
deportation of cholesterol from liver to serum as a result of liver damage by aldrin
poisoning.
Keywords. Aldrin effects ; scrum ; liver constituents ; C. batrachus.
1. Introduction
Investigations have proved that chlorinated hydrocarbons are highly toxic to fish."
Acute toxicity causes damage to the central nervous system, resulting in instability,
respiratory difficulties and sluggishness. Other chronic effects are residue accumu-
lation in fats, damage to liver and kidneys, reduced reproduction and restricted
growth (Donald 1968). Besides, many alterations have also been reported to occur
in blood and tissue chemistry as a result of toxicants. A haemoglobin decrease
without a change in erythrocyte count was observed due to the effect of DDT
(Rudd and Genelly 1956). Exposure to endrin resulted in increased concentration
of sodium, potassium, calcium, and cholesterol in serum with a lower values of
sodium, potassium, calcium and zinc in the liver of northern puffer Sphaeroides
macidatus than control (Eisler and Edmunds 1966). Changes in serum proteins
and free amino acids were reported in Chamia punctatus after exposure to mala-
thion, endrin and dieldrin (Shakoori et al 1976). In the same fish Lone and
Javaid (1976) observed the variation in blood caused by the effect of DDT and
dieldrin.
But so far no such investigation has been reported on aldrin. We report here
the changes in chemical constituents of serum and liver tissue of a catfish Clarias
batrachus which was exposed to various concentrations of aldrin .
28 Yagana Sana
J. Material mi methods
C. batrachus (23-25 cm in length and weighing 80-100 g) were collected from t
lo.val pond and acclimatized for two weeks in a large size aquarium. Fish meal
was provided daily UJL to 24 hr before the aldrin administration.
15 fish were maintained in each of the 5 buckets, containing 20 litres of tap water
(pH 6-7, water temperature 25-30° C). Appropriate quantity of technical grade
aldrin was dissolved in acetone and the final concentrations of 0-1, 0*2, 0-5 and
1 ppm were added to each of four buckets. The control fish (5th bucket) received
only 1 ml of acetone. No / sh meal was provided during the experiment. 5 fish
from each treatment \vere sacrificed after 12, 60 and 132 hr of aldrin
exposure.
Blood from each fish was collected in a clean test tube after serving the caudal
peduncle and was placed in a refrigerator at 10° C for 24 hr. After ccntrifuga-
tion at 3500 r.p.m. for 15 min, serum was drained out and returned to the refrige-
rator for storage at 4° C. The liver was removed, washed with physiological saline
solution and kept in refrigerator till use. The total cholesterol in serum and liver
was estimated using the method of Zaltikis et al (1953). Other chemical consti-
tuents were measured using methods as described by Oscr (1965).
3. Results
The 24 hr lethal concentration (LC100) for C. batrachus was observed to be 1 ppm
aldrin. At lower concentrations, no mortality occurred in experimental fish except
for two additional counts at 0*5 ppm.
3.1. Observations on serum
As indicated in figure 1, the serum constituents varied markedly with doses and
exposure time. At 12 hr exposure the serum protein showed a gradual fall (P <
0-01) in all concentrations. Longer exposure (60 and 132 hr) exhibited a rapid
faII(P > 0-01)in total protein for 0-lppm, the constituent rose higher for 0*2ppm
and declined again for 0 • 5 ppm (P > 0-01).
The value of total phosphorus was found increasing \vith increasing concentra-
tion at different exposure times. Total phosphorus, which showed a continuous
increase with concentration of 12 hr exposure, declines slightly for 60 hr exposure
at 0*2 ppm and more markedly at 0-5 ppm. At 132 hr exposure a small but
constant decline of trend was observed for concentration higher thai 0-1 ppm.
The cholesterol level showed a continuous increase with aldrin concentration
and exposure time, teing maximum in fish exposed for 132 hr and minimum in
12 hr exposed fish (P> 0-01).
Calcium content declined steadily in fish treated with 0-1 ppm after 12 hr and
increased later with rising concentration. At 60 hr exposure the level rose for
0- 1 and 0-2 ppm and thereafter declined a little, remaining all the time higher than
the control value as well as the corresponding values for 12 hr exposure. For
132 hr exposure the slight increase observed at 0 • 1 ppm (higher than 60 hr values)
\vas followed by a continuous declining trend for higher concentrations.
Effects of aldrin in catfish
29
JJ
O
3. a
18
I 8Q
I6O
I4O
120
IOO
80
60
II
5OO
4OO
\
\
\
O O.I ppm O-Zpptn O.3Ppm
ALDRIN CONCENTRATION
Figure 1. Scrum constituents of Clarias batmchus for different exposure time.
3.2. Observations on liver
The liver constituents exhibited a morereguUr pattern of variation (figure 2).
In all experimental fish the values of protein, calcium and cholesterol content
decreased gradually with increasing aldrin toxicity at different exposure times.
The value of total protein and calcium \vas significantly higher for 60 hr exposure
(P > 0-05) than 12 and 132 hr. The decline of protein content for 12 hr exposure
\vas steep and steady up to 0-2 ppm. Thereafter values remained lo\v at higher
30
Yagana Bono
9.0
§, 88
C 8.6
I
(L
. 8-4
* ..8
8-0
23.5
21-5
S »*5
O
U J3.S
13-5
.»« 1 85*0
F
, 1450
65O
?50
O Olppnn O-2 ppm O-5 ppw
ALDRIN CONCENTRATION
Figur* 2. Liver constituents of Clarias batrachus for dijfiferent exposure time.
concentrations. "Whereas at this exposure calcium COM tew t decreased only up to
0* 1 ppm and after that the values ^ere found uniform \vith rising concentrations,
A slow and steady decrease in cholesterol level \vas ot served both with exposure
time and concentration, values for 12 hr exposure being highest and those for
132 hr the lowest (P > 0-001).
4. l>js€0ssion
Boyle Bt al (1966) reported that in a fish tissue aldrin starts converting into dial-
dtin 8 hr after the fish is exposed to it, and the conversion reaches 94% in about
32 days. In our observation also, this conversion must have taken place a little
in 12 hr and considerably more for 60 and 132 hr in increasing proportion.
The median tolerance limit of four species of fish has been reported 24 hr at
aldrin concentrations ranging from 0-089 to 0*018 ppm and for dialdri* 0*062
Effects of aldrin in catfish 31
to 0-014 ppm (Gakstatter 1968). The present paper describes time dependence
of tolerance towards aldrin and dialdrin as shown by liver and serum and other
tissues by inference.
The level of protein decreased 25 to 50% in serum and 8 to 10% in liver
correspondingly (figures 1 and 2). Low levels in serum protein have also been
reported in goldfish (Grant and Mehrle 1970). The protein graphs for serum and
liver show quite a different pattern of variation with concentration and time. In
serum for a 12 hr exposure, the effect of aldrin is rather small at 0-1 and 0-2 fpm
and becomes more marked at 0-5 ppm in a systematic and expected manner.
The effect is found to be markedly enhanced at longer exposure thereby showing
an important time-dependence of the aldrin affect. At 0*1 fpm, the protein
values are the same after 60 and 1?2 hr. From this it is inferred that at this dose
aldrin and its byproduct cease to effect protein after 60 hr exposure. The upward
bend of graphs for higher concentrations indicates fish tolerance to the chemical
\vhich is time dependent as well as concentration dependent. For a longer
exposure time and higher concentration, the tolerance starts declining.
In liver the protein graph showed no evidence of tolerance developed at 0 • 1 and
0*2 ppm doses for 12 hr exposure, therefore steep constant decline. At a higher
dose, sufficient amount of antibodies appear to be formed to check a further
decline of protein to ar appreciable extent. For 60 hr exposure, the time is long
enough to permit the interference by produced antibodies even at 0- 1 ppm level.
This raises the observed protein values at this concentration as compareid to 12 hr.
This tendency continues up to 0-2 ppm indicating a maximum tolerance near this
point after which the toleraaace declines resulting in low protein values. For 132 hr
exposure the tolerance cycle appears to have come down to zero again at 0- 1 ppm
drug level to permit a little more decline of protein value than for 12 hr. At 0-2
and 0-5 ppm, the tolerance appears to have decreased appreciably below its maxi-
mum value, but it remains effective enough to place the 0-2 and 0-5 ppm points
for 132 hr above those for 12 hr.
The higher level of total phosphorus in the serum of aldrin-treated fish appears
to be related to the changes in liver produced under the effect of toxicant. Indue-
tion o'f serum aminotransferases (SGOT and SGPT) lactic dehydrogenase and
alkaline phosphatase are reported to be as a result of hepatic changes caused by
pesticides (Matsumura 1975).
The continuous increase in total phosphorus for 12 hr exposure means a large
concentration is almost proportionately more effective in causing the damage that
is releasing phosphorus. Regarding the time factor it is noticed that at 0- 1 ppm
the released phosphorus increases with period of exposure though not in proportion
to the time but at a reduced rate. At 0 • 2 ppm, an increase in the released quantity
of phosphorus is observed with time, but slightly less in quantity from 12 to
60 hr exposure, and very little in going from 60 to 132 hr. At 0- 5 ppm the trend
is reverse and the released phosphorus decreases from 12 to 60 and then from
60 to 132 hr.
Figures 1 and 2 reveal that for a 12 hr exposure the calcium value declines
to about 36% in serum and 29% in liver at 0-1 ppm dose. The level remains
constant in liver for higher concentrations up to 0-5 ppm but rises steadily in
serum correspondingly. This precludes a direct transfer from liver to blood or
vice versa for this exposure due to the abrupt liver damage. The constant level
32 Yagana Bano
in liver can be interpreted as the stoppage to further liver damage due to the pro-
duction of antibodies under higher doses called tolerance. The rise of calcium
content in serum may be at least partly also due to a similar reason but a sizeable
part of the excess calcium should result from various tissues, other than liver
because no loss of calcium at the corresponding point is seen (figure 2). For 60 hr
exposure the values of liver calcium are related to about maximum tolerance and
132 hr values associated to tolerance that comes down again to almost zero.
In serum the calcium values were observed to be higher than the control value
at certain concentrations and exposure times. This peculiarity is hard to explain
on tolerance basis and the transfer of calcium appears evident in these conditions
from other tissues to serum. The decline for longer period will then indicate a
greater excretion.
On comparing figure 1 with figure 2 it is seen that the trends of cholesterol varia-
tion in liver and serum are opposite to each other. Almost a regular increase
observed in serum at different concentrations and exposures, contrasts to a regular
decrease in liver. This is a clear case of transfer of cholesterol from liver to serum
and from other organs and confirms the observations of Eisler and Edmunds (1966)
on puffers with ertdrin.
Acknowledgement
The author thanks Prof. Shah Mashhood Alam, Head of the Department for pro-
viding necessary laboratory facilities. Thanks are also due to the Council of
Scientific and Industrial Research, New Delhi, for a fellowship.
References
Boyle H W, Burttschell R and Rosen A A 1966 Infrared identification of chlorinated insecti-
cides in tissues of poisoned fish. In organic pesticides in the environment ; Advan. Chem.
Ser. 60 American Chemical Society Chap. 17
Donald W J 1968 Pesticides and fishes— A review of selected literature ; Trans. Amer. Fish. Soc.
97 398-424
Eisler R and Edmunds H 1966 Effect of endrin on blood and tissue chemistry of marine fish.;
Trans. Amer. Fish. Soc. 95 153-159
Gakstatter J H 1968 Rates of accumulation of 14 C— dieldrirt residues in tissues of gold-fish
exposed to a single sublethal dose of 14C— aldrin ; /. Fish. Res. Board. Can. 25 1797-1802
Grant B F and Mehrle P M 1970 Chronic endrin poisoning in gold-fish Carassius auratus ; J.
Fish Res. Board Can. 27 2225
Lone K P and Javaid M Y 1976 Effect of sublethal doses of DDT and dieldrin on the blood
of Channa punctatus (Bloch) ; Pak. J. Zool. 8 143-9
Nfatsumura F 1975 Toxicology of insecticides (New York and London; Plenum Press)
Oser B L 1965 Hawks physiological chemistry 14th ed. (New York : McGraw-Hill)
Rudd R L and Genelly 1956 Pesticides their use and toxicity in relation to wildlife ; Calif.
Fish. Gama. Bull. 7 1-309
Shakoori A R, Zaheer S A and Ahmad M S 1976 Effect of malathion dieldrin and endrin on
blood serum proteins and fiee amino acids pool of Channa punctatus (Bloch) ; Pak. J.
Zool. 8 125-34 . •' •
Zaltikis A, Zak B and Bayl A J 1953 A new method for the direct determination of serum
cholesterol ; /. Lab. Clin. Med. 41 436
Proc. Indian Acad. Soi. (Anim. Sci.), Vol. 91, Number 1, January 1982, pp. 33-38.
©Printed in India.
Hepatopancreatic sucrase of Macrobrachium lamarrei
(Crustacea., Caridea, Palaemoiiidae)
PADMA SAXENA and RAMESH CHANDRA MURTHY
Department of Zoology, University of Lucknow, Lucknow 226007, India
MS received 20 February 1981 ; revised 7 December 19S1
Abstract. The effect of seven factors was studied on the activity of hepatopan-
creatic sucrase of Macrobrachium lamarrei. Its optimum pH is 6-0 and optimum
temperature 50° C and Us activity was suppressed by the two end products,
glucose and fructose. On prolonging incubation period sucrase activity remained
constant up to 8 hr, declined thereafter, finally becoming zero. Increasing enzyme
concentration produced a similar effect. Its Km value is 4-5 x 10~2M. Dialysis
suppressed its activity by 17*9%.
Keywords. Crustacea ; caridea ; palaemonidae ; Macrobrachium lamarrei ;
sucrase ; hepatopancreatic sucrase.
1. Introduction
The enzyme sucrase (/?~fructofuranosidase, EC 3.2.1,26) is known to occur in
the digestive system of a wide variety of crustaceans (Mansour-Bek 1954; van
Weel 1970; Vonk 1960). Yet information on its kinetic properties is scanty,
being confined to its response to pH (Agarwal 1963, 1964; Martin 1966;
Newcomer 1956; Nicholis 1931; Wiersm'a ai;d van Ween 1928). The present
work was conducted to determine the properties of this enzyme by studying the
effect of seven factors on hepatopancreatic sucrase of Macrobrachium lamarrei,
a freshwater shrimp, reported to be rich in this enzyme (Murthy 1978).
2. Materials and methods
Hepatopancreatic glands from 100 Macrobrachium lamarrei Milne Edward were
pooled in ice cold distilled water> dried between filter-paper sheets, weighed and
homogenized in distilled water. The homogenate was centrifuged at 3000 xg for
15 min at 4°C and the supernatant diluted to a concentration of 10 mg (wet
weight)/ml (or 0-1 ml = 1 mg) of hepatopancreas. The assay system consisted
of: appropriate buffer 0 • 3 ml, 0 • 3 M sucrose 0 • 2 ml and enzyme extract 0 • 1 ml ;
in controls a heat denatured enzyme was added. After incubating the mixtures
at 37° C for 4 hr, following Bsrnfleld's (1955) colorimetric method for estimating
hexose sugars, the reaction was stopped by adding 0*5 ml of 3,5-dinitrosalicylic
reagent ; thereafter the mixtures were wanned for 15 min, the volume raised sto ;
34 Padma Saxena and Rarnesh Chandra Murthy
6 ml with distilled water and readings taken at 540 nm. Under conditions similar
to those for the enzyme assay, direct reaction of glucose with dinitrosalicylic reagent
gives 1 extinction unit = 0-6 mg glucose. Of the seven factors — pH, temperature,
end products, incubation period, enzyme and substrate concentration- and dialysis—
pH was the first factor to be studied, in order to ascertain its optimum value, at
which the effect of the six remaining factors was investigated. Some experimental
details are given in §3. The presented values of each factor represent the mean
of five replicates.
3, Observations
3.1. Determination of optimum pH
According to the results using two buffer series (0 • 1 M sodium citrate buffer from
pH 3-5-6-5 and 0-1 M Sorenson's phosphate buffer from pH 5-5-8-0), sucrase
remained quite active from pH 4-5-7-0 and its optimum activity occurred at pH
6-0 (figure 1). While its optimum pH with both buffers coincided, enzymic
activity at this pH (6-0) with phosphate buffer was 9-7% greater than that using
citrate buffer.
3.2. Effect of temperature
Enzymic activity was tested at eleven temperatures ranging from 10°-80°C
(figure 2). Sucrase showed optimum activity at 50° C9 although remaining quite
active from 20°-6°C. The activity increased slowly from 1Q°-40°C but steeply
ftom 40°-50°C; above 50° C it fell sharply becoming almost zero at 80° C.
3.3. Effect of end products
Pour tubes, A to D, of the assay system were prepared and to three, S to D9 the
end product(s) were added before their incubation. No end product was added
to tube A> being meant to serve as the blank for calculating normal enzymic acti-
vity. In the parallel controls of 3 to D, the end product(s) were added after
their incubation. The details are tabulated belo\v :
Tube
A
Tube
B
Tube
C
Tube
D
1% Glucose, in ml
0-0
0-1
0-0
0-05
1% Fructose, in ml
0-0
0-0
0-1
0-05
Distilled water, in ml
0-2
0-1
0-1
0-1
The solutions of glucose and fructose when added separately caused inhibition
of aucrase activity to the same extent, by 7-2%; whereas a mixture of both caused
inhibition (figure 3).
Hepatopancreatic sucrose of M. lamarrei
• 0.8
O.2
Citrs** bufftr
4,O 5.O 6.O 7.O
.-? 0.8
0.6
B Glue...
C Fructwst
D GiucMt + P'ue!»i»
I 0.9
D C D
Figures 1-4. 1. Effect of pH o» hepatopancrcatic sucrase. 2. Effect of temperature
on hepatopancreatic sucrase. 3. Effect of end products on hepatopancreatic sucrase.
4. Effect of incubation period on hepatopancreatic sucrase.
1.4. Effect of incubation period
Results of incubating 13 tubes for progressively longer duration by one hour
that from 2-8 hr the rate of increase of formation of end products was more
or less linear (figure 4). Thereafter, the build-up of hexose sugars decreased
becoming constant after 14 hr of incubation. This pattern indicates a somewhat
constant rate of enzymic activity up to 8 hr, followed by decreased activity,
reaching finally close to zero at 14 hr.
. ,5. Effect of enzyme concentration
Enzyme extracts of ten concentrations, ranging from 2-5mg/ml to 25mg/ml of
hepatopancreas, were tested (figure 5). The liberation of hexose sugais increased
more or less linearly up to an enzyme concentration of 20 mg/ml. Above it the
increase was considerably slower, becoming almost nil at 25 mg/ml.
36
Padma Saxena and. Ramesh Chandra Murthy
f.O
£ 0.6
O.a
0.2
1.4
10
20 25
.03 .06 .09 .12 J5
Substrate concunlration (M) /I
2.0
Figures 5-7. 5. Effect of sucsrase concentration on its activity. 6. Effect of substrate
concentration on hepatopancreatic sucrase. 7 Lineweaver-Burk plot for Michaelis
constant (Km value) of hepatopancreatic sucrase.
3 . 6. Effect of substrate concentration
Sucrose solutions of 12 concentrations, ranging from 0*0075 to 0-15M,
ested. The rate of liberation of hex ose sugars was almost linear up to 0*06 M
concentration; after which it gradually slowed down (figure 6). The Michaelis
constant (Km value) of hepatopancreatic sucrase as calculated from the collected
data is 4-5 x 10~2M (figure 7).
3.7. Effect of dialysis
An enzyme preparation dialysed for 24 hr at 4° C against double distilled water
suffered a 17*9% loss ui activity.
tJepatopancfeatic sucrose of M. lamarrei 37
4. Discussion
In Astacus fluviatilis the optimum activity of gastric juice sucrase ws reported to
occur at pH 6-0 (Wiersma and van Ween 1928), as well as over a small pH range
4-5-5*0 (Kruger and Graetz 1928). The optimum activity of hepatopaucreatic •
sucrase of Marinogammarus obtusatus (4-2-6-4; Martin 1966), Porcellio laevis
(5-5-6-5; Newcomer 1956) and of Thalamita crenata (7-74-7-87; van Wsel 1960)
takes place over a narrow p>H raage. In contrast, that of Corophium volutator
and Orchcstia gammaretta (5-8, 6-0 respectively; Agarwal 19635 1964), Ligia
oceanica (5-8; Nicholls, 1931) occurs at a sh?rp pH. The optimum pH of
hepatopaucreatic sucrase of M. lamarrei being 6-0 is a sharp peak. Being lower
than the pH of the stomach contents (6-4-6-7; Murthy 1978), sucrase activity
in vivo would therefore be about 66-6-72*25% of its full activity in vitro.
The effect of the six remaining factors apparently remains unmvesti gated on
crustacean sucrase. However, as the effect of three of them, temperature, end
products and dialysis has been studied on insectan sucrase, the findings on
M. lamarrei have been compared with the available iosectan data. The optimum
temperature of hepatopancreatic sucrase of M. lanwrrei corresponds to that of
Sarcophaga ruficornis and Musca domestica (50° C; Sinha 1976), but is higher than
that ofBlatella germanica (25° C; Day and Po\\ning 1949), Sesamia inferens (30°C
for gut, 35°C for salivary glands; Agarwal 1976), Acy rtho siphon piston (35°C;
Srivastava and Auclair 1962), Chrysomphdus aonidum and Aonidiella aurantii
(37° C; Ishaaya and Swirski, 1970) and Lygus disponsi (37° C; Hori 1971).
Inhibition by the end products of sucrase activity, as occurs in M. lamarrei, has
been recorded L.I insects like Bombyx mori (Horie 1959), Dysdtrcus fasciatus (Khan
and Ford 1967), A pisum (Srivastava end Auclair 1962) and S inferens (Agarwal
1976); no effect \vas recorded in Aedes aegypti (Yang and Davies 1968) ard
L* disponsi (Hori 1971) by their accumulation.
The results in the case of three factors, (i) prolonged incubation, (ii) increasing
concentration of enzyme aaad (iii) substrate are similar, as an initial fast hydrolysis
of the substrate undergoes slowing down. The_slowing down after prolonged incu-
bation can be due either to the inhibitory effect of glucose and fructose formed
or to a depletion of sucrose or to a combination of both. The retardation by
relatively higher concentrations of the substrate can be due to the conversion cf
the total enzyme into ES-^omplex, as postulated by Karlson (1969). However,
accumulation of formed hexose sugars can be a contributory factor. That by
stronger enzyme extracts can be attributed either to rapid exhaustion of the sub-
strate due to excessive enzyme or to the inhibitory effect of the formed end products
or to a combination of both,
At present, reduced activity of hepatopancreatic sucrase of M. lamarrei after
dialysis- can neither be explained nor compared. However, dialysed sucrase from
the gut and salivary glands of lepidopterous larvae showed 8% activation and
37*5% inhibition respectively after dialysis (Agarwal 1976).
38 fadma Saxena and Rdmesh Chandfa Uwthy
References
Agarwal A K 1976 Effect of various factors an the activity of sucrase from tft<5 larva* of
Sesamia inferens ; Ent. Exp. AppL 20 19-30
Agarwal V P 1963 Determination of optimum pH for the activity of caecal inv«rtasc of
Corophium volutator Pallas ; /. Anim. Morphol Physiol. 10 86-88
Agarwal V P 1964 Determination of optimum pH for the activity of caecal carbdhydrases of
the amphipod Orchestic* gammarella \ J. Zool. 143 545-551
Bcrnfield P 1955 Amylases a- and ^- Methods in ertzymology (eds.) S R Colowick and N O
Kaplan (New York : Academic Press) 1 149-158
Day M F and Powning R F 1949 A study of process of digestion in certain insects ; Aust.
J. Sci. Res. B2 175-215
Hori K 1971 Nature of gut invertase of Lygus disponsi (Hemiptera : Miridae); Res. Bull of
Obihiro Zootech. Ur.iv. Ser. 1 666-675
Horie Y 1959 Physiological studies on the alimentary canal of the silk worm Bombyx mori.
II. Carbohydrases in the cigestivc fluid and in the midgi't tissue ; Butt. Serkult. Exp. Sta.
Jpn. 15 365-381
Ishaaya I and Swirski E 1970 Invertase and amylase activity in the armour scales Chrysomphalus
aonidum and Aonidiella aurantii ; J. Insect Physiol. 16 1599-1606
Karlson P 1969 Introduction to modern Biochemistry 3rd edition (New York : Academic Press)
Khan M A and Ford J B 1967 The distribution and localisation of digestive enzymes in the
alimentary canal and salivary glands of the cotton stainer, Dysdercus fascfatus ; /. Insect
Physiol 13 1610-1628
Kriiger P and Graetz E 1928 Die Permeate des Flusskrebsmagensaftes ; ZooL Jahrb. Abt, AUg.
Zool. Physiol. Ttere 45 463-514
Mansour-Bek J J 1954 Digestion in Invertebrates ; Tabulae Biologicae 21 173-195
Martin A L 1966 Feeding and digestion in two intertidal gammarids Marinogammarus obtusatus
and M. pirloti ; /. ZooL 148 515-525
Murthy R C 1978 Study of carbohydrases in the digestive system of Macrobrachium lamarrei
Edwards (Crustacea : Decapoda) ; Comp. Physiol. EcoL 3 13-16
Newcomer W S 1956 Digestive carbohyorases of the wood louse, Porcellio laevis (Crustacea :
Isopoda) ; Physiol Zool 29 157-162
Nicholls A G 1931 Studies on Ligfa oceanica. Part II, Process of feeding, digestion and
absorption with a description of the foregut ; /. Mar. Biol Ass. U.K. 17 675-707
Sinha M 1976 Invertase activity in the midgut of Sarcophaga ruficornis and Musca domestica
(Diptera : Insecta) ; Experientia 32 341-342
Srivastava P N and Auclair J J 1962 Characteristics of invertase from the alimentary canal of
the pea aphid, Acyrthosiphon pisum (Harr.) (Homoptera : Aphtdidae) ; /. Insect Physiol.
8 527-536
van Weel P B 1960 On the secretion of digestive enzymes by the marine crab Thalannta
cremta ; Z. Vgl Physiol 43 467-536
van Weel P B 1970 Digestion in Crustacea. In Chemical Zoology (eds.) M Florida and 8 T
Scheer 5 (A), 97-113 (New York : Academic Press)
Vonk H J 1960 Digestion and Metabolism. In Physiology of Crustacea (oi>) T. H. Watenfiaa
(New York : Academic Press) i 291-311
Wiersma C A G and van der Ween R 1928 Die Kohlcnhydratverdaiwna bci Asteetu fluvtatiltj ;
Z. Vgl. Physiol. 7 269-278
Yang Y J and Davies D M 1968 Occurrence and nature of iavertasc activity in adufc black-
flies (Simuliidae) ; J. Insect Physiol. 14 1221-1232
fcoc. mdhn A*d. Sci. (Anim. Sd), Vol. 91, Number i, January i982, pp 39-44
© Printrd in India.
Shell selection in the estaarine hermit crab Clibanarius
(De Haan)
S AJMAL KHAN and R NATARAJAN
of Advanced Study in Marine Biology, Annamalai University
Pawngipettai 608 502, India
MS received 13 April 19S1 ; revised 11 December 1981
Abstract. In the laboratory, under choice situation, C. longitarsus preferred shells
with greater shell width and shell weight. The correlation coefficient values calcu-
lated between the crab and shell parameters in the laboratory sample showed that
this hermit crab selects a shell of suitable dimension for its occupation. In the
multiple regression equations calculated with carapace length against the three shell
variables aperture width, shell width and shell weight which are deemed to he
important for hermit crabs, the regression coefficient of the variables varied much.
The unsealed first principal component explained 84-9% of the total variability
of the shell parameters and scaled first principal component explained 95 -8% of shell
parameters. The scaled first principal component was found to be a reliable esti-
mator of shell size and the hermit crab, if given a choice, selected a shell of suitable
dimension which fits its body quite closely.
Keywords. Clibanarius longitarsus ; shell selection in laboratory ; multivariate
analysis.
1. Introduction
Empty gastropod shells constitute an important resource for hermit crabs and a
hermit crab in order to protect itself from the environment and from its predators
must enter a gastropod shell. This behaviour of hermit crabs living in empty
gastropod shells intrigued naturalists down through the ages from Aristotle (Reese
1963) to the present (Ajmal Khan et al 1980). Hermit crabs do not enter gastro-
pod shells at random but prefer shells according to shape, shell covering, dimen-
sion and weight (Grant and Ulmer 1974). Under choice situation, the shell factors
influencing the estuarine hermit crab Clibanarius longitarsus in shell selection
weie studied presently through multivariate analysis.
2. Materials and methods
pive measures of the gastropod shells, i.e., shell length, shell width, aperture length,
aperture width and shell weight and three measures of hermit ciabs, i.e., carapace
length, dactylus length of second left walking leg and weight of the crab were used
39
40 S Ajmal Khan and R Natarajan
in the present study. The length of the shell was measured from the tip of spire
to the other end of the shell, the shell width was the greatest distance at right angles
to the length of the shell, aperture length was measured parallel to shell length and
the aperture width was the greatest distance from the margin of the outer lip of the
inner wall of the inner lip at 90° to aperture length (parietal callosities were
excluded from aperture width). The shells were completely dried before weighing.
Carapace length of the crab was measured from the tip of the rostrum to the
posterior notch and the live body weight was taken after blotting the specimen dry
with a blotting paper. Correlation coefficient values between crab and shell
variables were calculated for 50 specimens collected from the field and for 50 speci-
mens of hermit crabs allowed to select shells of their choice in the laboratory. For
shell selection experiments in the laboratory, the crabs were divided into 4 size
groups (I group 4-6-9 mm, II group 7-10 mm, III group 10- 1-14-9 mm and IV
group 15 mm and above) and the gastropod shells inhabited in the field by crabs
in each length were identified and the empty gastropod shells represented in each
length group were collected and used in the shdl selection experiments. Percen-
tage distribution of molluscan shell types, occupied by the crabs of the four size
groups is given elsewhere (Ajmal Khan and Natarajen 1981). Shell selection
experiments in the laboratory were done following Grant and Ulmer (1974).
3. Results
Shell preference of the hermit crab was found to vary from one size group to
another. While the first size group crabs showed preference for the shells of Nassa
jacksoniarta> second, third and fourth size groups showed preference for the shells
of Nassa dofsata, Babylonia splrata and 3ursa spinosa respectively.
The mean values of carapace lengths of crabs and variables of gastropod shells
occupied by the hermit crabs in the field and selected in the laboratory experiments,
are given in table 1. Since the standard errors of the shells selected ia the labo-
ratory were noticeably larger than the corresponding values in field sample, a
modified form of the t test, the statistic d (Bailey 1959) was used to determine
?f the carapace length and shell parameters of the two categories \vere sigaificantly
different from each other. It was found that the carapace length of hermit crabs
in the above two situations were not significantly different. In the same way
among the shell variables, the aperture width in these two categories was not
significantly different but the shell width and shell weight differed significantly.
The correlation coefficient values calculated between the crabs and shell para-
meters in field samples and laboratory selected samples are given in table 2. It
\*as found that there was no large scale difference in the r values of aperture vwdth
and shell width calculated against carapace length in the two situations and in
both the cases the values were highly significant (P < -001). Eventhougfr the r
values in the first category (field sample) were statistically significant in all cases
except one (carapace length /shell length), the r values in the second category were
highly significant. By looking at the r values in the second situation, it could be
inferred that a single factor alone was not influencing this hermit crab ' m shell
selection and this hermit crab normally selected a shell suitable in 'general <Jimen-
sion. But the diffi:ulty. in- putting* forward tW Mj-crve*infefence*only^*6y* tooMng
Sliell selection in C. longitarsus
41
Table 1. Results of crab carapace lengths and shell variables of shells occupied
and shell selected by C. longitwsus. Table values are means ± standard errors. The
"<T* values are those given from the formula of Bailey (1959) for comparing two
means. P : Probability.
Variable
Shells occupied Shells selected d value
Carapace length
13-13 mm
±0*91
13-12 mm
±0-99
0-01
>0'10
Shell width
20-98 mm
±0*66
28-26 mm
±2-39
2*94
<0'01
Aperture width
9-68 mm
±0-29
12-04 mm
±1-10
1-90
>0-05
Shell weight
8-88 mm
±0-78
18-31 mm
±3-12
2-93
<o-oi
Table 2. Correlation coefficient values between the crab and shell parameters of
shells occupied and shells selected by C. Icmgf tarsus. P : Probability ; Number
of sample 50 each.
Parameters
r value in
field sample
r value calculated
after shell selection
P experiment in the
laboratory.
P
Carapace length/Aperture length
+0-413
<o-oi
+0-960
< o-ooi
Carapace length/Aperture \vidth
+0/636
< 0-001
+0-890
< o-ooi
Carapace length/Shell length
+1-356
(Spurious)
+•7-922
< 0*001
Carapace length/Shell width
+0-464
< o-ooi
+0*962
<0-Q01
Carapace length/Shell weight v
+0-397
<0-01
+0-819
<o-ooi
Dactylus kitgth/Aperture length
+0-301
<0*05
' 0-924
< 0*001
Dactylus length/Aperture width
+0*331
<0-02
+0-902
<o-ooi
Dactylus length/Shell length
+0-318
<0-05
+0-904
< 0-001
Dactylus length/Shell width
+0-326
<0-02
+0-962
< 0-001
Dactylus length/Shell weight
+0*328
<0*02
+0-837
< o-ooi
Crab body weight/Shell weight
+0-352
<0-02
+0-815
< o-ooi
at the r values was tlv.it shell variables are interrelated \*ith each other and simple
regression and correlation techniques fail to show the ir dividual influence of a
shell parameter during selection. So, for this type of study multivariate analysis
seems to fre preferable, * - " * * ' *
42 S Ajmal Khan and R Natarajan
Table 3. Results of multiple regression of carapace bitgth (F) against aperture
width (xj), shell width (#2) and shell weight fc3) of gastropod sholls occupied in the
field and selected in the laboratory by C. longitarsus.
Regression Coefficient of varia-
equation tion of shell para-
meters
Sheila occupied
in the field
y-y = 0-012
-l-Q'023
if
Xi = 63-07
#2= 60*08
x3=56-78
Shells selected in
the laboratory
y-y = 0-395
4-0-351
-H-Q-O^
fo-Xt)
v^2 ^2'
Xl = 64-87
*2= 59-76
*3= 82-96
Through studies on the sh-11 selection behavioui of heimit crabs, it became clear
that, among different shell variables, three variables, viz., shell volume, shell aper-
ture width and shell weight \vere more important. The volume of shell interior
is certainly of prime importance to hermit crabs, but volume estimate is difficult
time consuming and often inaccurate and imprecise (Kuris and Brody 1976). So
a change of shell var iable was made and instead of shell volume, shell width in
addition to the other two variables was used in the multivariate analysis of the
present study.
The multiple regression equation of carapace length against the three shell
variables in the field situation and by choice in the laboratory are given in table 3.
The coefficient of variation in both the equations for aperture and shell widths did
not differ widely, but the coefficient of variation for shell weight differed widely.
Moreover, in the laboratory choice situation, even though the r values used in
the multiple regression equation did not vary much, regression coefficient for weight
differed widely from the other two. The main problem in using multiple regres-
sion to distinguish the effects of separate parameters was that there was high
correlation between the three shell variables which obscured their separate effects.
To overcome this difficulty principal component analysis \vas used.
For principal component analysis all the shell variables have to be expressed
in the same units. Here the shell width and aperture width were measured in
millimeters and shell weight in grams. To overcome this defect caused by the
three shell variables measured in different units, scaling of shell variables has to
be done and both the unsealed and scaled principal component analyses have to
be compared. Scaling can be done by dividing the shell parameters by the square
root of the sums of squares of their deviation (Mitchell 1976). Logarithmic
transformation of the variables also approximately satisfies these conditions (Kuris
and Brody 1976). In the present study scaling was done by the second method.
The results of the unsealed and scaled principal component analyses are given in
tables 4 and 5 respectively. Multiple regression equation of the carapace length
of the crabs against the unsealed and scaled principal components are given in
table 6.
The first principal component in the unsealed situation explained 84-9% of the
total variability of the shell parameters. The second component explained only
Shetl selection in C. tongitarstt$ 43
Table 4. Principal components* of Shells Selected by C. tongitarsus in the labora-
tory. No Scaling has been performed on them.
Xi — Aperture width ; ^—shell width and *3 — shell weight.
Percentage contributions
to total variability of
Principal components shell parameters
1 Component 1-000000*! 84-9
+0-964919^
II Component -Q • 1 396QQ*X 1 • 5
+1-000000^3
Table 5. Principal components of Shells selected by C. longitarsm in the labo-
ratory. Scaling was performed by logarithmic transformation of Shell and crali
variables, ^-aperture width ; x2— stall width and ^3~Sli€ll weight.
Percentage contributions
to total variability of
Piincipal components shell peramttcrs
I Component 1*000000 *i 95-800
+0-999043 *3
+0-992294 x9
II Component 1-000000 x1 0'013
+ -0-50000C *2
+0-500000 x3
Table 6. Regression equations of carapace length against unsealed and scaled
principal components for shells selected in the laboratory by C. longitarsus.
K— carapace length ; Xi—I principal component ; #2— II principal component.
Coefficient of variation
Regression equation of principal components
Unscahd y-~y = 0-054 to -&) *a « 77- 1 J
+7-550 v*2-Xa> *a « 74'OJ
Scaled Y-^Y = Q* 1 54 to -jcx) xs = 61 • 39
+0*005 (#2—^ *2 ^ negligible
1-5% of the total variability. In scaled componejit analysis, the results were still
more significant; the first principal component explained 95-8% of the total
variability while the percentage of variability of the shell parameters explained
by the second component \vas very low (0-013%). Moreover both in unsealed and
scaled first principal components, the values attached to the three variables were
neatly equal; in the scaled first principal component the values were more OF less
the same. This indicates that in the fust prijicipalcouiponeBt the three shell vari-
ables play equal roles in shell selection. TJie coeffiiceirt of variation of the fits-i
and second principal components in the ut scaled situation was very close .to the
44 $ Ajmal than and A Natarajan
multiple regression equation. But in the scaled situation the coefficient of variation
of second principal component was negligible.
4, Discussion
The first principal component seems to be a more reliable estimator of shell size
for hermit crabs than selection by multiple regression of the best pair of shell and
crab size correlates (Kuris and Brody 1916). In the present study also, both in
scaled and unsealed conditions, the variability of the shell parameters could be
mostly explained by the first principal component. Similar results were also
reported by Mitchell (1976). When a multiple regression equation was calculated,
with first principal component and second principal component against carapace
length, the coefficient of variation of the two components in unsealed situation
was more or less equal. In scaled condition, the coefficient of variation for first
principal component was high and it was negligible in the case of second compo-
nent- It therefore seems to be advisable to look at the scaled analysis and inter-
pret the first principal component as the main guideline which influences C. longi-
tarsus to select its shell. In Pagurus bernkardus, the percentage contributions to
the total variability of the shell parameters of the first principal component was
slightly less in the case of scaled analysis than in the unsealed analysis (Mitchell
1976). Even then, the percentage of variation in crab weight explained by the
scaled first principal component was more than that of unsealed first principal
component. But in the present study, the percentage contribution to the total
variability of shell parameters was more in the scaled first principal component
thm in the unsealed first principal component. So it is probable that the scaled
first principal component will explain more percentage of variation in crab length
than the second principal component. This first principal component cai be very
easily interpreted* as, in all the cases, the length concerned \vitb. all the three shell
variables is almost equal. This means that all the three shell variables play equally
important roles in the selection of a shell.
Acknowledgements
The authors are grateful to CSIR and UGC for financial support-
References
Ajmal Khan S Mercy Thomas and Natarajan R 1980 Principal component analysis in the shell
selection behaviour of the land hermit crab Coenobita cavipes Stimpson ; Indian J, Mar. Set,
9 293-294
Ajmal Khan S and Natarajan R 1981 Coexistence in hermit crabs of Vellar estuary ; Indian /,
Mar. ScL 10 201-204
Bailey R T J (ed.) 1959 Statistical methods in biology (London : English Universities Press) p. 51
Grant W C and Ulmer K M 1974 Shell selection and aggressive behaviour in two sympatric
species of hermit crabs ; Biol Bull. 146 32-43
Kuris M A and Brody M S 1976 Use of principal component analysis to describe the snail shell
resources for hermit crabs ; /. Exp* Mar. BhL Ecol 2Z 69-77
Mitchell K A 1976 Shell selection in the hermit crab Pagurus bemhar&s , Mar. BioL 35 335-343
Rcesa B S 1963 The behavioural mechanisms underlying shell selection by hermit crabs ; Behaviour
21 78-126 .-
Proc. Indian Acad. Soi. (Anim. Sci.), Vol. 9J, Ho. 1, January 1982, Pp. 45-55
© Printed in India.
Evaluation of some organophosphorus insecticides against Dacus
cucurbitae Coquillett on peacht
N P KASHYAP* and S F HAMEED**
Department of Entomology- Apiculture, Himachal Pradesh Krishi Vishva Vidyalaya,
Palampur 176062, India
* Assistant Professor, College of Agriculture, Palampur 176062, India
** Present address : Department of Entomology, Rajendra Agricultural University,
Academic Complex, Pusa (Samastipur), 848 125, Irdia
MS received 25 September 19&0 ; revised 20 March 19.81
Abstract Toxicity and persistence of fenitrothion, fenthion, malathion, methyl
parathion, and trichlorphon applied at 0*05% (400 g/ ha) were evaluated on peach
fruits (Prunus persica L.) against the noonate larvae of Dacus cucurbitae Coquillett
in two seasons (1977-78). Fenitrothion and methyl parathion were highly toxic
materials followed by fenthton and malathion, while trichlorphon was the least
toxic. Fenitrothion was highly persistent (12 days) followed by methyl parathion
(7 days). All the insecticide residues were within the acceptable limits at the time
of harvest.
Keywords. Organophosphorus insecticides ; toxicity ; persistence.
1. Introduction
Fruit fly, Dacus cucurbitae Coquillett, is a serious insect pest of peach (Prunus
persica L.) in Himachal Pradesh. The crop sustains severe injuries by the larvae
when the fruits are about to ripen and render these unfit for human consumption.
There is a possibility of preventing the ovipositioa of the fruit fly on the peach
fruits by giving a protective cover spray of an effective insecticide. Pruthi (1969)
Myburgh (1961), Samp- io et al (1966), Peretz et al (1966), Nagappan et al (1970)'
Anonymous, (1975) and Sharma et al (1973) reported that the pest could be
controlled by a number of less persistent insecticides by such sprays. None of
these reports are, however, based on detailed experimentation of intrinsic toxi-
city to the neonate larvae of fruit fly, persistence of effective toxicity or consumers'
safety following their applications. Taking these as objectives in view, the
present contribution reports the results of evaluation of the effectiveness of tWo
spray schedules of fenitrothion, fenthion, malathion, methyl parathion and
tricblorphon on peach against the neonate larvae of fruit fly.
t Part of Ph.D. thesis submitted to the Himachal Pradesh Krishi Vishva Vidyalaya, Palampur,
by the senior author under the guidance of the second author.
45
46 N P Kashyap and S F Hameed
2. Materials and methods
Commercial formulations of five organ ophosphorus (OP) insecticides viz. fenitro-
fyiion, fenthion mathyi parathion, malathion and trichlorphon were sprayed on
peach trees (cv * Webcock') in an orchard of the Department of Horticulture,
Himachal Pradesh Agricultural University, Palampur, with the help of a foot
sprayer (Maruti. make) to 'run-off' at the recommended rates of 0 -05% cowcentra-
tion(40C £/ha). The experiment was conducted in a randomized block design taking
single-tree plots for a replication. There were 18 trees for 5 treatments and a
control. which were replicated 3 times. The trees -were 6-7 years of age with 6 m
planting distance in a hexagonal system. All the other horticultural operations
and foitiliz'*r applications followed in the orchard were according to the recom-
mendation of the package of practices for horticultural crops, Himachal Pradesh
(Anonymous 1975). The trees were first sprayed on 21 May 1977 and again
sprayed on 4 June 1977. Samples were collected at 0-day (immediately after the
fruits were dried), 1, 3, 5, 7 and 14 days following treatments. The samples
^ere later processed for estimating the deposits. The experiment was repeated
with two sprays of the above insecticides c.t the same concentration in 1978 at
the sain? location with another set of 18 trees having similar age and bearing.
The first spray was given on 18 May 1978, and the Second on 1 June 1978.
The sampling intervals were same as in the first year.
Eight fruits were randomly collected from all .around the periphery of each
treated tree per treatment at each interval for residue. analysis. Before extrac-
tion, the weight of the sample was recorded and the surface area of each fruit was
determined by Turrell's (1946) method. The deposits of the respective fruit
sampbs of each insecticide were extracted in redistilled solvents (berzene for
fenitrothion, fenthior., methyl parathion and tridalorphon, and CC14 for mala-
thion), by taking a sample of 8 fruits in a wide mouthed stoppered glass bottle
(capacity 1L) and sufficient solvent \vas added so as to cover the fruUs. Volume
of the solvent was recorded and the bottle was labelled before shaking on a shaking
machine for one hour. The extract was filtered into another container to which
sufficient quantity of anhydrous sodium sulphate was added to remove the mois*
tore and stored in a refrigerater until analysed. The extracts were then subjected
to bio- and chemical assays. For bioassay (table 1), adult males of Drosophila
melanogaster Meig. were taken as test insects and the rest of the procedure
was the same as reported by Thakur and Hameed (1980). For chemical assays,
the extracts of fenitrothion and methyl parathion were cleaned up by the method
of Thornburg (1963); for malatbion, Hameed and Rattan Lai's method (1971), and
for fenthion an dtrichlorphon, the method of Jain et al (1974a) were used. Results
of the quantitative estimations obtained by bioassay were verified by standard
chemical methods. For fenitrothion and methyl parathion, the procedure o
Averell and Morris (1948) was used and for malathion the method of Sutherland
(19.64) was used. Fenthion and trichlorphon residues were assayed by the method
of Jain et al (1974a), Standard calibration curves were dra\vn separately for each
insecticide. Recovery of each insecticide was studied in two different ways.
Firstly, the insecticides were recovered from the surface deposits and secondly,
as total residues. Insecticides were more satisfactorily recovered from the surface
of the fruits thain from the whole fruits (total residues), Fenitrothion recovered
Evaluation of insecticides against Coquillett 47
Table 1. D osfagc-mortalify response of insecticides to Drosophila melanoga&ter.
Insecticide Regression equation LI>ia &&}
Fiducial
limits
FenitrotkioflL Y =* 1. • 6038* -1- 2 • 5 246 0 • 0035
0-0030
0-0041
Feiithiou y = 1 • 7825* + 1-6888 0 • 0720
0-0624
0*0832
Malathion Y= 1-7758* +0-7684 0*2415
0-2098
0-2780
Methyl paratkion Y = I . • 865 O.v + 1 • 0484 0-1315
0-11.66
0-1482
Trioblorphon Y = 2 • 2 240* + 0 • 9507 0 • 4739
0-4204
0-5342
IE none of these cases the data were found to be *lgn;ifictMitJy heterogeneous at P = 0*05.
y = Probit kill ; x — log cone. (/u-g/2ml) x 104 for fenitrothion aitd 10:l for rest of the insecticides.
to the extent of 85 -9-89 -6% (surface deposit) and 83-3-86-0% (total residue),
fenthion 82-6-83-1% (surface deposit) and 82-5-83-5% (total residue), nulathicn
93-3-96-2% (surface deposit) and 90-3-91-7% (total residue), methyl parathicn
92-3-93 -3% (surface deposit) and 91-1-92-4% (total residue), and trichlorphon
80-2-80-8% (surface deposit) and 78-9-80-7% (total residue).
Peach fruits were harvested on 23 June 1977 arid 23 June 1978 in the 1st and 2nd
seasons respectively. Residues were later extracted by macerating them in a
Waring blender with equal quantity of anhydrous sodium sulphate in a known
volume of solvent. The slurry so obtained was decanted, filtered and cleaned
up as per the method reported by Thakur and Hameed (1980). Intrinsic toxi-
city of the deposits of the 5 OP insecticides to the neonate larvae of fruit fly
(table 3) was also determined by bioassay and the actual amount of insecticides
in the deposits so formed from their commercial formulations, giving the desired
toxicity, was determined chemically and by bioassay.
Half-life values of each insecticide on peach were worked out on the basis of
the formula of Hoskins (1961). Safety interval (days) was determined on the
basis of formula given by Thakur and Hameed (1980). Effective life of each
insecticide was found out by substituting the value of log LD90 to the If of time
deposit regression equations.
1. Results and discussion
Residue-film method of bioassay was very satisfactory (table 2) as it could detect
residues of fenitrothion as low as 0-50^g/cm2 x 10~3 compared with 416//g/
cm2 x 10~3by the colorimetric method of Averell and Norris (1948). Fenitrothion
48
IT P Kashyap and S F Hameed
Table 2. Sensitivity of bio and chemical assays.
,„»«««—- -™
Bicassay fig/cm3
X 10-Ji
Chemical assay
fig/cm2 x 10~3
Insecticides
Surface
Total
S urface
Total
deposit
residue
deposit
residue
Fonitrothiou
0-5005
0-5160
399-9553
416-3180
Fenthion
16-6366
16-7454
714-6489
707-2002
Malathion
49-0888
49-9509
495-4271
527-6732 '
Matliyl parathioa
29-0868
29-4828
191-0425
193-0698
Trichlorphon
155-4194
159-3025
1052-4938
1046-2320
Table 3. Toxicity of insecticide deposits to the ncoiiatc larvae of Dacus cucwbitae.
Insecticide
Regression
equation
LD50
(jag/cm8)
Fiducial
limits
( /ig/cm2 )
LD90
(jig/cm2)
Fiducial
limits
(^g/cms)
Fenitrothiou
y = 1-8837* + 1-4404
0-0776
0-0594
0-1014
0/3715
0-2090
0-6758
Fentiiion
y= 1-7465* + 1-2891
0-1333
0-0997
0-1780
0-7219
0-3755
1 • 3868
Malathion.
y= 1-4011* + 1-8860
0-1669
0-l.ltfQ
0-2403
1-3715
0-5777
3-2562
Methyl parathion
y = 1-4353* + 2- 1853
0-0914
0-0637
0-1312
0-7145
0-3455
1-4784
Trichlorphon
7 = 1-7913* + 0-8080
0-2189
0-1631
0-2938
1 - 1366
0-6112
. 2-1135
y = Probit kill In none of these cases the data werp found to be significantly heterogeneous
at P = 0-05.
x = log cone, x 10*
was a highly toxic material to the vinegar flies (table 1) which increased the sensi-
tivity of the m-sthod. Fejjtrothion and methyl parathion were also highly toxic
(table 3) to the larvae of D. cucurbitae followed by fenthion and malathion, while
trichiorphon was the least toxic material Toxicity of deposits on the basis of
minimum effective level (m.e.L), i.e., LD^ value (Gratwick and Tew 1966) were ;
fenitrothion > methyl parathion > fenitrothion > trichlorp hon > malathion.
Evaluation of insecticides against Coquillett
49
Table 4. Extent and magnitude of insecticide deposits in relation to their toxicity
to tho neonate larvae of Daeus citcitrbitae.
Initial deposit
Insecticide
(at 0-05% cone.)
First season 1977
Second season 1978
First Spray
(21-5-1977)
Second spray
(4-6-1977)
First spray
(18-5-1978)
Second spray
(I -6-1978)
Fenitrothion
1.
6-570
6-967
7-404
7-748
2.
84-66
89-78
95-41
99-84
3.
1,7- 68
18-75
19*93
20-85
(1 :5)
(1 :5)
(1 :5)
(1 ;5)
Fenthion
1.
7-105
7-011
7-205
7-356
2.
53-30
52-59
54-05
55-18
3.
9-84
9-71
9-98
10-19
(1 :5)
0:5)
(1 :5)
(1 :5)
Malathion
1.
6-946
7-088
7-1.22
7-165
2.
41-62
42-47
42-67
42-93
3.
5-06
5-17
5-19
5-22
(1 : 8)
(1:8)
(1 :8)
(I :8)
Methyl para thion
1.
7-1,67
7-157
9-014
8-939
2.
78-41
78-30
98-62
97-80
3.
10-03
10-02
12-61
12-61
(1 :8)
(1 :S)
(1 :8)
(I :8)
Trichlorphon
1.
6-060
6-086
7-198
7-170
2.
27-68
27-80
32-88
32-75
3.
5-33
5-35
6-33
6-31
(1:5)
0 :c)
0:5)
(1 t5>
1. Initial deposits, (average of bioassay and chemical assay).
2. Number of times the initial deposit exceeding the U>60 value vide table 3.
3. Number of times the initial deposit exceeding the m.c.1. (Lt>50) value vide table 3.
Figures? in parentheses are ratios between m.e.l. and intrinsic toxicity of deposits.
The results were in agreement with the findings of Hameed et al (1980). The
deposits of all the insecticides in general were high, much in excess to their respec-
tive m.e.1. (table 4). The deposits of fenitrothion, for example, OB peach fruits
gave deposits of 6 -57- 7- 75 jug/cm* (tables 6 and 7) in two sprays of the two
seasons which were in excess of its m.eJ. (0'3715/*g/cm2, table 3).
Similarly, the deposits of fenthion and methyl parathion were about 10-11
times io excess of their nxe.ls. But the deposits of malathiorj and trichlorphon
were minimum i.e. only about 5-6 times in excess of m.e.ls. whe?n compared with
their respective LD50 values. The deposits of fenitrothion were far in excess
(92 times) of their intrinsic toxicity (LD50) followed by methyl parathion (86 times),
fenthion (54 times), malathion (43 times) and trichlorphon (30 times). These
observations showed that allthese chemicals provided adequate deposits for t}-«e
50 N P Kashyap and S F Hameed
Table 5. Threshold of toxic action of insecticide deposits to the neonate larvae
of Dacus cucurbitae.
Insecticides recommended
dose 0*05 (a,i.)
Deposits of insecticides on 14th day fag/cm2)
First season (1977) Second Season (1 978)
First spray Second spray First spray Second spray
Fenitrothion
0-224
0-267
0-300
0-288
(80-8)
(84-4)
(86-6)
(85-8)
Fenthion
0-062
0-052
0-111
0-121
(28-1)
(23-8)
(44-5)
(47-1)
Malathion*
0-108
0-159
0-14(5
0-202
(39-6)
(48-9)
(46-8)
(54'6)
Methyl parathion
0-102
0-095
0-115
0-113
(52-7)
(51-0)
(55-7)
(53-3)
Trichlorphon
0-049
0-038
0'066
0-056
02-2)
(8-7)
(17-6)
(14-4)
* Deposits of insecticides on 7th day.
Figures in parentheses are corresponding probable kill of Dacus cucurbitae larvae vide regression
equations given in table 3.
control of insects. Although the initial deposits of fenitrothion and methy
parathion in general were in excess of their intrinsic toxicity, the margin
between m.e.1. and intrinsic to>icity (LD50) of ths deposits (mentioned in ratios)
was the highest only with methyl parathion and malathion followed by fentitro-
thion, feuthion and trichlorphon. Thus, it can be concluded that the deposits
of fenitrothion followed by methyl parathion provided comfortable margins
when compared either with their LD90 of LD50.
The residues of insecticides on the 14th day (tables 6 and 7) when subjected
to the respective regression equations (table 3), corresponding per cent kill of
the neonate larvae of fruit fly was obtained (table 5). The deposits of 0-30 ^g/
cm2 of fenitrothion in the 1st spray (2nd season) on the 14th day following
treatment (table 5) gave 87% mortality of the larvae, followed by methyl
parathion, fen thion and trichlorphon . ' Malathion, however, after 7 days did
not afford more than 55% mortality of the insect.
Results of the field experiments on the persistence of 5 OP insecticides are
summarized in tables 6 a-nd 7. Chemical estimations in ell the cases
approximately agreed well with the data obtained from the bioassay of field-sample
extracts. D^ta of the two estimations of each insecticide in both the seasons were
also positively correlated to each other. During the 1st season, bioassay and
chemical estimation of the deposits of 5 OP insecticides sprayed twice at an
interval of 15 days on peach fruits showed the higest deposit \vith methyl parjathioj\
on 0-day (i.e. 7 • 17 and 7 • 16 ^g/cm2) for the 1st and 2nd spray, respectively followed
Evaluation of insecticides against Coqulllett $\
Table 6. Persistence of insecticide deposits in peach fruits (first season, 1977),
two sprays.
Method of No. of
Insecticide estimation sprays!
Depor
rit (ftg/c'm
2) following treatment at
0-day
1-day
3-day
5-day
7-day
1 4-day
Fenitrothion Bioassay
6-46
2-64
1-89
0-87
0-58 -
0-21
Chem. assay I
6-68
2-84
I- 82
0-86
0-60
0-24
r r= c-9994
(6-57)
(2'74)
(1-85)
(0-86)
(0-59)
(0-22)
Bioassay
6-94
2-64
1-73
0-83
0-69
0-28
Chem. assay II
6-99
2-69
1-89
0-79
0-60
0-25
r =0-9995
(6-97)
(2-66)
(1-81)
(0-81)
(0-64)
(0-27)
Fenthion Bioassay
7-30
2-61
2-00
1-22
0-73
a-08
Chem. assiy I
6-91.
2-71
2 '00
1-07
0-78
0-04
r = 0-9603
(7- 10)
(2 -66)
(2-00)
(1.-14)
(0-75)
(0-06)
Bioassay
7-16
2'84
1-91
1-12
0-71
0-07
Chem. assay II
6-86
2-74
.1-94
1-24
0-66
0-03
r= 0-9994
(7'0l)
(2-79)
(1-93)
(1-18)
(0-68)
(0-05)
Malathion Bioassay
7-02
2-02
0-86
0-38
0-10
BDL
• Chem. assay I
6-91
1-92
0-76
0-33
0-11
BDL
r = 0-9999"
(6-95)
(1-97)
(0-81)
(0-35)
(0-11)
Bioassay
7-12
1-81,
0-85
0-37
0-16
BDL
Chem. assay II
7-06
1-73
0-91
0-38
0-15
BDL
r =0-9998
(7-09)
(1-77)
(0-88)
(0-38)
(0-16)
Methyl parathion Bioassay
7-13
3-38
2-35
1-04
0-79
0-11
Chem. assay I
7-21
3-31
2-29
1-08
0-79
0-09
r =0-9998
(7-17)
(3-34)
(2-32)
(1-06)
(0/79)
(0-10)
Bioassay
7-1.8
3-07
2-12
0-86
0-63
0-09
Chem. assay II
7-13
3-10
2-23
0-85
0-64
0-09
r= 0-999?
(7-16)
(3-09)
(2-18)
(0-85)
(0-63)
(0-09)
Trichlorplion Bioassay
6-03
2-62
1-59
0-84
0-27 -.
0-05
Chem. assay I
6-09
2-63
1-67
0-83
0-27
BDL
/•= 0-9884
(6-06)
(2-63)
(l'63>
(0-83)
(0-27)
(0-05)
Bioassay
6-07
2-54
1-54
0-71
0-20
0-04
Chem. assay II
6-10
2-67
1-51
0-63
0-21
BDL
r =0-9996 '
(6-09)
(2-61)
(1-52)
(0-67)
(0'21)
(0-04)
S ill-face area (sq. cm) of I
30-59
31-31
32-06
32-67
34*13
36-60
one peach fruit** IT
37-97
39-01
39-62
40-72
42-30
45-65
Percentage increase in I
0
2-35
4-80
6-80
11-57
19^65
fruit size over zero II
24-12
27-52
29-49
33-11
38-28
49-23
day sample
r =s Coefficient of correlation significant at P «= 0/01 ,
* Average of 3 replications
** Average of 120 fruits.
BDL = Below detectable limits
Figures in parentheses are average of bioaatsay and chemical assay . . • ' •
Temp,
. °C
A«T^,.»~ .».«.„ -tli^M n^.«y1*4.:^«n
RH
Rainfall
Max.
Min.
(mm)
1st spray 29-93
20-79
55-35
3-73
2nd spray 27-40
18-89
61-90
3-32
52 N P Kashyap and S F Hameed
Table 7. Persistence of insecticide deposits; on peach fruits (second Season, 1978)
two sprays.
Insecticide
Method of
estimation
No. ,:
Spray
>f Deposits (/Ltg/cm2) following treatment at
£ , . — - —
0-day 1 -day
3-day
5-day
7~day
14-day
Fenitrothion
Bioassay
7-42
2
•86
1-85
1
•10
0
•83
0*30
Chem. assay
I
7-39
2'
•97
1-83
1
•04
0
•84
0-30
r= 0-9998
(7-40)
(2
•91)
(1-84)
(1
•07)
(0
•84)
(0-30)
Bioassay
7-67
2'
88
1-76
1
•08
0
•85
0-28
Chem. assay
II
7-83
3-
02
1-91
1
•05
0
•77
0*30
r= 0-9995
(7-75)
(2-95)
(1-83)
(1
•06)
(0
•81)
(0-29)
Fentition
Bioassay
7-17
2-
•67
2-00
1
-23
0
•85
o-io
Chem. assay
I
7-24
2
•79
2-15
1
•26
0
•79
0-12
r =0-9998
(7-20)
(2'
73)
(2-08)
(1
•25)
(0
•82)
(0-11)
Bioassay
7-27
2
•92
1-96
1
•03
0
•70
0-10
Chem. assay
II
7-44
2'
•79
1-39
1
•04
0
•51
0-14
r =0-9993
(7*36)
(2-85)
(1-39)
(1
•03)
(0
•60)
(0'12)
Malathion
Bioassay
7-15
1-
•73
0-87
0
•42
0
•15
BDL
Chem. assay
I
7-10
1-
56
0-85
0
•43
0-14
BDL
r« 0-9997
(7'12)
(1-
64)
(0-86)
(0
•42)
(0
•15)
Bioassay
7-20
1-
40
0-85
Q
•36
0
•19
BDL
Chem. assay
II
7-1.3
1-
44
0-85
0
•37
o-
22
BDL
r = 0-9999
(7-16)
(I'
42)
(0-85)
(0
•36)
(0
•20)
Methyl parathion
Bioassay
9-13
3-
62
2-71
1
•26
0
•96
0^12
Chem. assay
I
8-89
3-
71
2-59
1
•41
0-93
0-12
r =0-9994
(9-01)
P-
67)
(2-65)
(1
•32)
(0
•95)
(0'12)
Bioassay
9-06
3-
25
2-32
1
•15
0
69
0-12
Chem. assay
II
8*81
3-
17
2%25
1
•08
o-
65
BDL
r =-0-9999
(8-94)
(3-
21)
(2*24)
0
•12)
(0
•67)
(0-.12)
Trichlorphon
Bioassay
7*22
2'
•81
1-97
1
•24
0
•43
0-07
Chem. assay
I
7-17
2-
87
2-07
r
•30
o-
47
0-06
r= 0-9998
(7-20)
(2*
84)
(2*02)
(i
•27)
(0-45)
(0-07)
Bioassay
7-20
2-
65
1-91
i
•06
o-
35
0-06
Chem. assay
II
7-14
2-
87
1-88
i
•13
o-
33
0-05
r = 9992
(7-17)
(2*
76)
(1-89)
(i
•10)
(0
•34)
(0-06)
Surface area (sq.
cm.)
I
25-1,3
21'
21
27-88
28
•69
29-
96
31-21
of one peach fruit**
II
31-41
32-
00
32-66
33
•06
33-
^73
36-60
Percentage increase in
I
0
8-
28
10-94
14'
•17
19-
22
24-19
fruit size over zero
II
24-99
27-
34
29-96
31-
56
34-
22
45-64
day sample
r = Coefficient of correlation Sigtiificant at P ==0*01
* Average of three replications
** Average of 120 fruits
BDL = Below detectable limits
Figures in parentheses? are average of bioassay and chemical assay
Temp. °C
Average weather conditions
- RH
Rainfall
Max.
Min.
(mm)
1st spray
33-54
20-96
27-17
.1-44
2nd spray
32-02
22-35
46-41
2-14
Evaluation of insecticides against Coquillett
53
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d
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8
^*"^*C*-ONC> t^t^c^oot^-
1?
•a
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£
*cS -"ti
.2
^o g
«
§ ^
«
l|
4
*§ "o
•§
*»+
N
<* sSrf
£
Insecticides
(at 0-05% cone.
- i j .1 .8 •
1 Bil j B|i § f§ § fg
• t||:|t f gjff la-.l&f |elM|
• Mffl Hilt
§ 11
54 N P Kashyap and S F Hameed
by fenthion, malathion and fenitrothion. Trichlorphon gave the lowest deposit
(i.e. 6-06/jg/cm2) for the 1st spray and 6-09/jg/cm2 for the 2nd spray (table 6).
The deposits of all these insecticides dissipated quickly up to the 1st day and
thereafter, the degradation was gradual. In the case of malathion, fast dissipation
of the dapo'its occurred and no residue could be estimated after the 7th day
following the treatment. The results were in agreement with the findings of Desh-
mukh and Singh (1975) and Singh (1977). The figures of average weather condi-
tion during this period are given in table 6. The deposits decreased as the ambient
temperature and the size of the fruits increased.
In the second season (table 7) maximum initial deposit of 9*0//g/cm2 (1st spray)
and 8-94/^g/cm2, (2nd spray) were obtained with methyl parathion, which was
followed by fenitrothion, fenthion, txichlor/phon and malathion. The deposits
were slightly more because of the absence of rain.
The relative persistence of 5 OP insecticides on peach expressed as half-life
values, revealed that fenitrothion was a highly persistent insecticide. It also
provided maximum period of protection agaiast the peach fruit fly larvae (10^11
days) following either of the two sprays (table 8). Methyl parathion was found
to be the next highly peristent insecticide giving adequate initial deposits and
providing about a week's protection following each spray. This chemical there-
fore could be considered as the next best insecticide with a safety interval of 9 days.
Comparing the biological performance of trichlorphon and malathion, trichlorphon
was more persistent than malathion, but it was of little benefit owing to its low
intrinsic toxicity to the fruit fly levae. Safety interval of trichlorphon (12-13 days)
was more than fenthion, malathion and methyl parathion, because of its low
tolerance level fixed on peach fruits. Safety interval of 16-17 days was found for
fenitrothion (table 8). Of the 5 OP insecticides, tested in the present investigation,
fenitroihion (in two spray schedule) outclassed the rest cf tb.s insecticides, provided
protection against the neonate larvae of fruit fly for about 12 days and the peach
fruits were safe for consumption after 14-16 days following each spray. Insecti-
cide residues at the time of harvest of peach in both the seasons were much below
the acceptable tolerance limits.
References
Anonymous 1969 Official FDA tolerance complete through 1969 ; NatL Agric. Chem. Ass, News
28(3) 10
Anonymous 1970 Malathion. Pesticide residues in food. Report of the 1970 joint FAO/WHO
meeting. FAO agricultural series No. 474, p. 33
Anonymous 1972 Fenthion, Pesticide residues in food. Report of the 1971 joint FAO/WHO
meeting. FAO agricultural series No. 88, p. 45
Anonymous 1975 Package of practices for fruit crops of Himachal Pradesh Puhln. of Directorate
of Extn. Edit. Agri. Compl. p. 22-27
Anonymous 1976a Fenitrothion. Recommended international maximum limits for pesticide
residues. Secretariat of joint FAO/WHO food standards programme, Codex alimeatarius
commission, Rome 5th series p. 13
Anonymous 1976b Trichlorphon. Recommended international maximum limits for pesticide
residues. Secretariat of joint FAO/WHO food standards programme, Codex alimentariu*
commission, Rome 5th series p. 502
Evaluation of some insecticides against Coquillett 55
Averell P R and Norris M V 1948 Estimation of small amounts of 0, 0-diethyl 0-p-n itrophenyl
thiophosphate ; Anal. Chem. 20 753-6
Deshmukh S N and Singh. J 1975 Dissipation of carbaryl and malathion from okra fruits ;
Indian J. Entomol 37 64-7
Gratwick M, Sillibourne J M and Tew R P 1965 The toxicity of insecticides to larvae of the codling
moth, Cydia pomonella (1) ; Bull. Entomol. Res. 56 367-88
Gratwick M and Tew R P 1966 A comparison of the toxicity of various carbamate, organo-
phosphorus and organo-chlorine compounds to the codling moth; Proc. 3rd British Insecticide
and Fungicide Conf. (1965), Brighton, pp. 276-85
Hameed S F and Rattan Lai 1971 Residues of malathion on eabbage, cauliflower and knol khol
estimated by bioassay and chemical assay methods ; Indian J. Entomol. 33 326-7
Hameed S F, Suri S M and Kahsyap N P 1980 Toxicity and persistence of residue of some
organophosphorus insecticides in cucumber fruits for the control of fruit fly Dacus cucurbitae ;
Indian J. Agric. Sci. 50 73-7
Hoskins W M 1961 Mathematical treatment of loss of pesticide residues ; PL Prot Bull. FAO
9 163-8
Jain H K, Pandey S Y, Agnihotri N P and Dewan R S 1974a Rapid estimation of organophos-
phorus insecticides ; Indian J. Entomol. 36 145-8
Jain H K, Pandey S Y, Agnihotri N P , Dewan R S, Saxena A N and Peshwam K M 1974b.
Dissipation of phorate and disulfoton in rape seed crop (Brassica campestris) ; Indian J. PL
Prot. 1 37-42
Myburgh A C 1961 Lebayoid as a cover spray for fruit fly control ; S. Afr. J. Agric. ScL 4
615-21
Nagappan K and others 1970 Insecticidal trial? for the control of the melon fruit fly Dacus
cucurbitae infesting snake gourd ; Madras Agric. J . 57 24
Peretz I R, Gavrielith E, Gurewitch and Frankel H 1966 Trials in the control of the Mediterranean
fruit fly Ceratitis capitata with organophosphorus insecticides ; Israel /. Entomol. 1 155-65
Pruthi H S 1969 p. 977. Text hook of agricultural entomology Indian Council of Agricultural
Research, New Delhi
S.impato A S, Rigitane O, Suplicyfilho N and Orlando A 1966 Tests on the control of fruit
flies on peach by the application of new insecticides ; Biologica 32 213-6
Sharma P L, Srivastava S and Dhaliwal HS 1973 Chemical control of peach fruit flies ; Pesticides
1 20-1
Singh G 1977 Persistence of malathion and Leptoplios (phosvel) residues in/on okra Abelmoschus
esculentus Moench fruits ; Thesis Abst. (Hau, Hissar) 3 157-8
Sutherland G L 1964 Milathion in Analytical method for pesticides, Plant Growth Regulators and
Food Additives (ed. G Zweig) (New York and London : Academic press) 2 283-93
Thakur A K and Hameed S F 1980 Biological performance of some organophosphorus insecticides
against Qiiadraspidiotus perniciosus Comstock on apple ; Proc. Indian Acad. ScL (Anim.
Sci.) 89 587-601
Thornburg W W 1963 Extraction and clean-up procedures. In analytical methods of pesticides -,
plant growth regulators and food additives (ed. G Zweig) (New York and London : Academic
Pt-ss) 1 87-108
Turrell F M 1946 Tables of surface and volumes of spheres of prolate and oblate spheroids
and spheroidal coefficients (Univ. Calif : Berkeley) p. 153
Proc. Indian Acad. Sci. (Anim. ScL), Vol. 91, Number 1, January 1982, pp. 57-66.
© Printed in India.
Structure and chemical composition of the cuticle of Cirolana
fluviatilisy Sphaeroma walkeri and Sphaeroma terebrans
D LEELA VALLABHAN
Wood Preservation Centre (Marine), Forest Research Institute, C/o. Zoological
Research Laboratory, University of Madras, Madras 600 005, India
MS received 2 July 1981 ; revised 2S October 1981
Abstract. A comparative study has been made of the cuticular organisation of
isopod wood borer Sphaeroma terebranjf, a fouler Sphaeroma walkeri and a free
living isopod Cirolana fluviatilis. The cuticle of S. tcrebrans s hows both structural
and chemical peculiarities. In S. walkeri, the cpicuticle contains fuchsinophilic
protein and gives evidence of primary tanning. In C. fluviatilis the epicuticlo is.
similar to that of other isopods.
Keywords. Cuticle ; structure and chemical composition ; Sphaeroma terebrans ;
• histochemistry.
1. Introduction
Although the cuticle of arthropods conforms to a basic pattern comprising of an
inner procuticle formed of chitin-protein complex and an outer lipo-protein epi-
cuticle, it shows a wide range of modifications in structure and chemical compo*
sition in different groups. Dennell (1947) observed that the abbreviation of
Banning, occurrence of a two layered epicuticle and calcification of the cuticle
of crusta ceans may be related to their aquatic habitat and to the ready
availability of calcium in their natural environment.
Earlier work on cuticle of isopods is more limited than on decapod cuticle. The
structure and chemical composition of the cuticle of Porcellio scaber, Ligia exotica,
Armadillidium vulgare and Oniscus asellus have been studied by George and Sheard
(1954), Mary (1968), Lagarrigue (1970) and Mary and Krishnan (1974). It is
known that there is a general conformity in structure and chemical composition to
that of the cuticle of decapod crustaceans. A point of interest is that isopods
unlike decapod crustaceans, have a number of adaptive devices for terrestrial life
It is of interest to investigate the nature of modifications in the cuticle structure
and chemical composition relevant to their adaptation to semiterrestrial and
terrestrial mode of life.
The Sphaerornatidae, which include wood borers and epifoulers, are presum-
ably adapted for their mode of life as borers or as foulera. The nature of the
adaptation of the cuticle structure and chemical composition is investigated by a
comparative study of a typical borer like Sphaeroma terebrans with a closely
57
5& D teela Valldbhan
allied species Sphaeroma walkeri which is not a "borer but shows a substratum
affinity to sfb merged wood. The results were compared with the ctitieular struc-
ture of a free living type, Cirolana fluviatilis.
2. Material and methods
Specimens of S. walkeri and S. terebrans were collected from Madras harbour by
immersing timber panels in the sea. Specimens of C. fluviatills were also collec-
ted from Madras harbour. Tne animals were maintained under laboratory condi-
tions by changing the sea water every day.
For histological preparations of the cuticle, the material was fixed in 5%
formaldehyde, decalcified in 3% glacial acetic acid or 3% EDTA and embedded in
paraffin or celloidin. The stains used were Maliory's triple stain, Masson's tri-
chrome stain and Heidenhain's haematoxylin (Mallory 1938; Pan tin 1948; Lillie
1954). Histochemical tests were performed on frozen sections of the cuticle which
were prepared by impregnating the specimens with 12£% and 25% gelatin solution
and the blocks were hardened in 5% formaldehyde (Carleton and Leach 1938).
For detection of chitin, the tests used were Chitosan test (Campbell 1929) and
Schulze test (Clark and Smith 1936). For sulphydryl and disulphide groups,
tetrazolium test (Barnett and Saligman 1952), nitroprusside test and ferric ferri-
cyanide test (Lillie 1954; Pearse 1968; were performed. To detect protein consti-
tuents the tests included xanthoproteic test, Millon's test (Pearse, 1968), Hg/nitrite
test (Lison 1936) and biuret test (Fearon 1946). The presence of lipids was tested
by treatment with dyes such as Sudan black B (Baker 1946; Lillie 1954). For
detecting calcium, alkaline pyrogallon test (Lison 1936), alizarine red-5 and
Vonkossa's test (Lillie 1954) were employed.
3. Results
The quticle of Cirolana fluviatilis varies in thickness in different regions from 10
to 30 #. Sections passing through the tergite reveal two well defined regions in
the cuticle corresponding to epicuticle and procuticle. An outer, thin homogeneous
layer, 7 to 10 /z thick is different in appearance and colour from a thicker lamellated
region which may be subdivided into three distinct layers in the intermoult stage.
The epicuticular nature of the outer thin part is confirmed by treatment with
chlorafed nitric acid which separates the epicuticle from the procuticle by the
differential solubility of the two layers in this reagent. At this stage the epicuticle
is not light yellow coloured ; the procuticle is not distinguishable into sub -divisions.
When stained with Mallory, the epicuticle may be divisible into two regions, an
outer thin blue staining membrane and below it, a fuchsinopb.il region (figure 1).
The two parts correspond to outer epicuticle and inner epicuticle of other arthro-
pods, The procuticle stains uniformly blue in Mallory and green in Masson's
stain. Tests for protein show that the inner epicuticle contains a protein contain-
ing phenyl groups (table 1). The protein in the procuticle on the other hand is
negative to these tests but reacts positively to biuret test. In this respect the protein
constituents of the cuticle conform to those reported in the cuticle of decapod
crustaceans and insects (Deniiell 1947; Wigglesworth 1948). A feature of the
Structure and chemical composition of the Cuticle
g i r to 1 s n a f 1 u v 1 a tills
Outer eplcutlcle
inner eplcutlcla
Exocutteie
Undocuticle
Dpidermi*
Figure 1. Transverse section through the interinoiilt cuticle, stained in Mallory's
triple stain.
Table 1. Results of Staining reactions and histochemioal tests obtained with the
late freahmoult cuticle of Cirolanafluviatilis.
No.
Stain£ and tests
Epicutklc
K-olcrenccS
Procutiolc
Outer Inner
layer layer
1.
Mallory's triple stain
Mallory 1938 Blue Red
Blue
2.
Ma$$or»'s? trichrome stain
Trim 1.941 Green Red
Green
3,
Heidenhain's liaematoxylin
Lillie 1954 Blue Grey
—
black
4.
ChitoSan test
Campbell 1929 — —
' -f-
5.
Schultz modified test
Clark and Smith — —
-4-
1936
6.
Sudan Black B
Baker 1946 +•
, —
7.
Lieberaann-Burchardt test
LiSon 1953 . -f -
—
8.
Biuret test
Fearon 1946 — —
4-
9.
Xanthoproteic test
Lillie 1954 - +
10.
Millon'S test
Pear§e 1968 — +
11.
Eg/nitrite test
Baker 1946 — +
•
12.
Argentaffioi test
LiSon 1936 — +
13.
Ferric chloride test
Liston 1936 — +.
— •
14.
Blue tctrazolium test
Barnett and — —
Seligman 1952
15.
Ferric ferrycyanide test
PearSe 1968 - -
16.
Alkaline pyrogallol test
LiSon 1936 — -
17.
Alizarin ied-5
Lillie 1954 - _
.
18.
VonkoSSa's test
Lillie 1954 - _ '
—
4- positive reaction ; — negative reaction.
60 D Leela Vallabhan
protein of the cuticle of the isopod studied above, is the negative reaction to biuret
test in the cpicuticle which is positive to the Million and xanthbprotcie tests.
The outer epicuticle reacts to tests for lipids and sterok. The inner cpicuticle
is only feebly reactive to these tests. It shows a positive reaction to argentaffin
test which may be indicative of the presence of reducing substances which in the
present context, considering this reaction together wilh the positive reaction
obtained in the region with ferric chloride, may suggest that the reacting materials
may be diphenols or polyphenols.
The structural features and staining reactions as well as the chemical composi-
tion of the cuticle differ in intermoult stage (figure 1). The cpicuticular region in
a section shows, an amber colouration and is unrceactive to stains. The outer lipid
cpicuticle is less prominently seen in the sections. The procuticle is now distin-
guishable into an outer region which is amber colour and an inner region in which
the lamellations are still clearly seen and still below is another region in which the
lamellations are closely set. The results of histochemical tests are given in table 2.
It is seen that the chemical composition of the epicuticle conforms to that in a
number of decapod crustaceans in undergoing tanning resulting in acquisition oi
rigidity and resistance to chemical reagents.
In the procuticle prominent changes are brought about by the formation of an
outer amber region giving rise to exocuticle and the part of the procuticle under
it appears to be calcified and this region reacts to tests for calcium, like Vcnkossa's
test, alkaline pyrogallol and alizarin red-S. A region immediately below the
calcified procuticle is free from calcium and is designated as the non-calcified layer,
Results of tests applied for protein in the procuticle show that at this stage in
addition to biuret positive protein and a protein involved in tanning, there is
evidence of another protein which reacts positively to the blue tetrazolium and
ferric ferricyanide tests. The presence of such a protein containing organic
sulphur associated with calcified region has been earlier re ported in decapod
crustaceans like Orconectes virilis (Travis 1965). This author suggested thai
in the absence of tanning in this region the protein containing the SH group
may play a role in facilitating calcification.
To examine how far the cuticular organisation of a closely allied fouler associated
with wood differs from a free living form (described above) a detailed study of the
cuticle of S. walkeri was made. Examinations of the stained and unstained sec-
tions of the cuticle of S. walkeri in the fresh moult condition showed epicuticle as
in Cirolana fluviatilis distinguished by its homogeneity and in being formed of aji
outer thin membrane of the outer epicuticle (figure 2). The procuticle conform*
in all respects to the condition reported in the corresponding stages of moult cycle
of Cirolana fluviatilis (table 3). But in the intermoult stage there are seen markcc
differences in chemical features of the cuticle compared to those of intermoul
cuticle of Cirolana fluviatilis (table 4). Unlike in C. fluviatilis the inner epicuticl<
does not undergo tanning. It however stains red in Mallory's and reacts positively
to tests for protein like xanthoproteic and Millon's. Similarly in the procuticle
the outer part is not differentiated into an exocuticle but the middle region of th<
procuticle undergoes calcification and reacts to tests for calcium like VonkOssa'i
alizarin red-S and alkaline pyrogallol tests (table 4). From a comparative stud:
of the intensity of the reaction to tests for calcium it may appear that calciun
content is more than what was noted in the allied type. The region of the pro
Structure and chemical composition of the Cuticle
61
Outer eplcutlclo
• Inner epieirticle
- Poro canal
" Procirbicle
Figure 2. .transverse section through the freslunoult cuticle, stained in Mallory's
triple stain.
Table 2. Results of Staining reactions and histochemical tests obtained with the
intermoult cuticle of Cirolana fluviatillis.
No.
Stains and tests
Reference
Epicuticle Procuticlc
E\ocu •
Outer inner tide Calcified Uacalci-
layer layer layer fied
layer
1.
Mallory's triple stain
Mallory 1938
Blue Amber Amber Blue
Light blue
2.
MasSon'S trichrome
Trim 1941
Green Amber Amber Green
Light blue
Stain
3.
Heidenhain's
Litlie 1954
Blue Grey — —
~—
.haematoxylin
baclk
4.
ChitoSan test
Campbell 1929
_ - -f, -|.,
j-.
5.
Schultz modified test
Clark and
+• -]„
H-
Smith 1936
6.
Sudan Black B
Baker 1946
-H" -1- - -
7.
Liebermann Burchardt
Lison 1953
_L JL, JL, ___
,
8.
Biuret test
Fearon 1946
~ - - . 4-
-h
9.
Xanthoproteic test
Lillie 1954
- H- 4- -
.
10.
Millon's test
Pearse 1968
~ -f. -i- _
11.
Hg/nitritc test
Baker 1946
— 4- -j- —
12.
Argentaffin test
LiSon 1936
- -f- H- -
13.
Ferric chloride test
Lison 1936
— — — . __.
—
14.
Blue tetrazolium test
Barnett and
- - 4- -h
Seligman 1952
15.
Ferric ferrycyanide
Pearse 1968
— — 4. ^.
—
test
16.
Alkaline pyrogallol
Lison 1936
_i t
— — -j~, _j-
»_
test
17.
Alizarin red-£
Lillie 1954
— — — JL J—
-j- -p
_
18.
Vonkossa's test
Lillie 1954
- . - + -f
—
4- positive reaction ; •+•+ intensely positive ; — negative reaction.
62 & Leela Vallabhaii
cuticle reacting to calcium test is also abbreviated. In other respects it recalls the
condition noted in the cuticle of C. fluviatilis. In the cuticle of S. terebrans which
is a borer, the epicuticle shows a further deviation from the condition reported in
C fluviatilis (figure 3). These differences refer to the protein compound of the
epicuticle which unlike in S. walkeri is not the fuchsinophil tyrosine containing
protein. There is evidence of only the basal protein which is biuret positive, stains
blue with Mallory's and green in Masson's stain. Complete absence of tanning
is a feature of the epicuticle of this animal in all the stages of moult cycle
(tables 5, 6).
Table 3. Results of staining reactions and histochemical tests obtained with the
late freshmoult cuticle of Sphaeroma walkeri.
No.
Stains and tests
Epicuticle
• Procuticle
Outer Inner
layer layer
3.
Mallory's triple stain
Mallory 1938 Blue Red
Blue
2.
MasfSon's trichrome stain
Trim 1941 Green Red
Green
3.
Heidenhain'S haematoxylm
Lillie 1954 Blue black Grey
—
4.
Chitosan test
Campbell 1929 — -
-h
5.
Schultz modified test
Clark and — —
Sinith 1936
. 4-
6.
Sudan black B
Baker 1946 +• -
—
7,
Liebermann Burcliardt
Lison 1953 + -
—
8.
Biuret test
Fearon 1946 — —
4-
9.
Xanthoprotoic test
Liliie 1954 - +•
—
10.
Milton's test
Pearse 1968 - H-
—
11.
Kg/nitrite test
Baker 1946 - 4-
—
12.
Argentaffin test
Lison 1936 - —
—
13.
Ferric chloride test
Lison 1936 - -
_
14.
Blue tetrazolium tost
Barnett and — —
Seligman 1952
••*"
15.
Ferric ferrycyanido test
Pearse 1,968 — —
_
16.
Alkaline pyrogallol test
fcfcon 1936 - -
_
17.
Alizarin red-S
Lillie 1954 -
_
18,
VonkoSSa's test
Liliie 1954 - -
— •
+• positive reaction ; — nogative reaction.
Structure and chemical composition of the Cuticle
63
Spbaeroma *t 6 r e ."b._r a us
2,5^
Epidermis
Figure 3. Transverse section through the freshraoult cuticle, stained in Mallory's
triple stain.
Table 4. Results of staining reactions and histochetnical tests obtained with the
in tei moult cuticle of Sphaerome walker i.
No.
Stains and tests
References
Epicuticle
Procuticle
Outer Inner
layer layer
Calci-
fied
layer
Uncalci's
fied
layer
1.
Mallory'S triple Stain
Mallory 1938
Blue Red
Blue
Light
blue
2.
Masson's trichrome stain
Trim 1941
Green Red
Green
Light
blue®
3.
Heidenhain's haematoxyUn
Lillie 1954-
Blue Grey
black
—
~
4.
Chitosan test
Campbell 1929
— —
4,
4.
5.
Scbultz modified test
Clark and
Smith 1936
— —
4,
•f-
6.
Sudan black B
Baker 1946
4- ~
_«
_
7.
Liebennann-Burchardt test
Li&on 1953
+ _
—
—
8.
Biuret test
Fearon 1946
~~ ~_
. • -~ .
•f-
9.
Xanthoprr.'teic test
Lillie 1954
i
— ~p
—
-.
10.
Milloit's test
Pearse 1968
_ 4.
—
—
11.
Hg/nitrite test
Baker 1946
- 4.
—
—
12.
Argentaffia test
Lison 1936
— _
-.
— -
13.
Ferric chloride test
Lison 1936
_ —
—
—
14.
Blue tetrazolium test
Barnett and
Seligtnan 1952
— —
4-
—
15.
Ferric ferricyanide test
Pearse 1968
_ —
-h
-_ .
16.
Alkaline pyrogallol
LiSon 1936
— _
4.
—
17,
Alizaiin red-5
Lillie 1954
— —
4"
— .
18.
Vonkoj>sa's test
Lillie 1954
— _
4*
—
64 D Leela Vallabhan
Table 5. Results of staining reactions and histocfomical tests; obtaiued with the
freshmoult cuitcle of Sphaeroma terebraiis.
No.
Stain and tests
References
Epicuticle Procuticle
1,
, Mallory's triple stain
Mallory 1938
Blue Light blue
2.
Massoii's trichrome stain
Trim 1941
Green Light green
3.
Heidenhain's hasmotaoxylin
Liliie *954
Light blue —
4.
Chitosan test
Campbsll 1929
+•
5.
Schultz modified test
Clark and Smith
_ 4-
1936
6.
Sudan Black B
Baker 1946
— —
7.
Liebermann-Burchadrt test
Lison 1953
— —
8.
Biuret test
Fearon 1946
4- +
9.
Xanthoproteic test
Liliie 1954
^ _
10.
Millon'S test
Pearse 1968
__ _
11.
Hg/mtrite test
Baker 1946
«_ -.
12.
Argentaffin test
Lison 1936
— —
13.
Ferric chloride test
Lison 1936
— . —
14.
Blue tetrazolium test
Barrnett an 2
Seligmaa 1952
15.
Ferric ferrycyanide test
Pearse 1968
_ __
16.
Alkaline pyrogallel
Lison 1936
17.
Alizarin red-S
LiUie 1954
18.
Vonkossa's test
Liliie 1954
— —
•i- positive reaction ; — negative reaction.
In the absence of a tyrosine containing protein which is the precursor of tanning
of the cuticle, S. terebrans may be said to lack the essential mechanism for tanning.
In the procuticle also there are seen marked deviations from the condition noted
in C. fluviatilis. This refers to the non-differentiation of outer part of the pro-
cuticle into an exocuticle or a mcsocuticle. Although there is evidence of calci-
fixation, compared to the other two types studied, it is much abbreviated. The
results of tests are recorded in table 6.
4. Discussion •
It deserves to be noted here that the cuticle of the wood borer S. terebrans is
devoid of the outer epicuticie while those of S. walkeri which is a fouler and of
the free living C. fluviatilis, have this layer. The outer epicuticie is formed of lipid
and is believed to check evaporation of water from the surface of the body
(Beament 1961, 1964). Recent work has shown that the cuticle lining the gut in
decapod crustacean like Ocypoda platytarsis lacks an outer lipid epicuticie which
accounts for the increased permeability to water through the layer (Marv and
Krishnan 1974). The significance of the absence of an outer epicuticie ^ the wodo
Structure and chemical composition of the Cuticle
65
Table 6. Results of Staining reactions and histochemical tests obtained with the
mtermoult cuticle of Sphaeroma terebrcms.
No.
Stains and tests
References
Epicuticle
Proculide
1.
Mallory's triple stain
MaUory 1938
Blue Light blue
2.
Masson's trichrome stain
Trim 1941
Green Light green
3.
Heidenhain'S haematoxylin
Lillie 1954
Light blue —
4.
ChitoSan test
Campbell 1929
— -4~
5.
Schultz modified test-
Clark and Smith
— .1.
1936
6.
Sudan Black B
Baker 1946
_U
7.
Liebermann Burchardt test
JbiSon 1953
8.
Biuret test
Fearon 1946
4~. ..:.
9.
Xanthoproteic tcsi
Lillie 1954
_
10.
Millon's test
PearSe 1968
.
11.
Hg/Bitrite test
Baker 1946
12.
Argentaffin test
Lison 1936
13.
Ferric chloride test
Li$on 1936
—
14.
Blue tetrazoliurn test
Barnett and
— _:.
Seligman 1952
35.
Ferric ferry cyanide test
Pearse 1968
_ ..;,
16.
Alkaline pyrogallol
Lison 1936
_.!„
17.
Alizarin ed-S
Lillie 1954
-j-
18.
Vonko$$a*$ test
Lillie 1954
— -\'
-h positive reaction ; — negative reaction.
borer Sphaeroma terebrans may be that the cuticle in it is more permeac.le than
that of the closely allied species Sphaeroma walker i. This species in its natural
habitat within the wood may not be exposed to fluctuations in ambient tempera-
tures, to need protective devices against water loss, similarly the absence of an
outer epicuiicle which would restrict the permeability to water and possibly ions,
may not be an important and a necessary factor as the borer imlike the free living
forms living within a restricted environment.
The wood boring species 5. terebrans is characterized by the occurrence of spines
on the cuticular surface. Spines are absent m S. walken. It thus appears that
the presence of cuticular spines is somehow related vith boring habit. The precise
functional role and the spikes in boring is not known.
The inner epicuticle which may undergo tanning is important in bringing about
a restraint on water loss. In the wood borer £ ttrebrans the protein compo-
sition of the cuticle is very different from the allied species S. walken and C.fluvia-
tilis in the absence of the fuchsinophil protein and the presence of only a biuret
positive protein in the epicuticle. This is an unusual feature in an intennoult
cuticle. Immediately i fter moulting or in preecdysial cuticle it has been reported
that the epicuticle may contain only a biuret positive protein which is seen over-
66 D Leela Vallabhan
laid by a fuchsinophil protein which is a precursor of tanning (Demnell and Malek
1955). Tanned protein and precursors of tanning are known to prevent water
loss (Sundararajulu and Krishnan 1968; Mary 1968).
Acknowledgements
Thanks are due to the authorities of the Forest Research Institute forgiving
permission and encouragement to carry out the investigation. Thanks are also
due to Dr G Krishnan for valuable comments on the manuscript.
References
Baker J R 1946 The histochemical recognition of lipine ; Q. J. Micr. Sci. 87 441-471
Barnett R J and Seligmen A M 1952 Demonstration of protein bound sulphydryl and disulphtde
groups by two new histochernical methods ; /. Natl. Cancer Inst. 13 215-216
Beament J W L 1961 The water relations of insect cuticle ; JBiol. Rev. 36 281-320
Beament J W L 1964 Advances in insect physiology Vol. II (London and New York : Academic
Press)
Campbell F L 1929 The detection and estimation of chitin and the relations of chitinization to
hardness and pigmentation of the American cockroach Periplaneta americana ; Ann. Ant.
Soc. America 22 401-426
Clark G L and Smith A F 1936 X-ray diffraction studies on chitin, chitosan and derivatives ;
/. Phys. Chem. 40 863-879
Carleton H M and Leach E H 1938 Histological technique (London : Oxford University Press)
p. 383
Charniaux-Legrand J 1951 Le cycle d'intermue des amphipodes et ses particularite's chez less
formes terrestres (Talotridae) ; Arch. Zool Exp. 88 178-204
Dennell R 1947 The occurrence and significance of phenolic hardening in the newly formed
cuticle of Crustacea Decapoda ; Proc. R. Soc. B134 485-503
Dennell R and Malek S R A 1955 The cuticle of cockroach Periplaneta americana III. The
hardening of the cuticle impregnation preparatory to phenolic tanning ; Proc. R. Soc. B143
414-426
Fearon M R 1946 An introduction to biochemistry (London : William Heinmann)
George R W and Sheard K 1954 Ecdysis in the isopod Porcellio scaber (Latereille) ; Aust. J.
Zool 2 75-85
Lagarrigue J Q 1970 Recherches ecophysiologiques sur les oniscoides (Isopodes Terrestres) ;
(Universite de Mont pellier)
Lillie R D 1954 Histopathologic Technique and practical histochemistry (New York : Blackiston)
p. 501
Lison L 1936 Histochemice animate (Paris : Gauthier-Villar) p. 534
Lison L 1953 Histochemie et Cytochemice animales, Gauthier Villars, Paris.
Mullory F B 1938 Pathological technique (Philadelphia : Saunders)
Mary F 1968 Studies on the integument of some invertebrates in relation to regulation of body
fluid concentration Ph.D. Thesis, Madras University.
Mary F and Krishnan G 1974 On the nature and role of protein constituents of the cuticle of
crustaceans in relation to permeability of the cuticle ; Mar. Biol (Berlin) 125 229-309
Pantin G F A 1948 Microscopic technique for zoologists (London : Cambridge University Press)
Pearse AGE 1968 Histo chemistry , theoretical and applied (London : Churchill) p. 759
Sundararajulu G and Krishnan G 1968 The epicuticle of millipedes belonging to the genera
Cingalobolus and Aulacobolus with special references to seasonal variations ; 2. Maturg
23 845-851
Travis D F 1965 The deposition of skeletal structures in the Crustacea. 5. The histomorpho-
logical and histochemical changes associated with the development and calcification of the
branchial exoskeleton in the crayfish, Orconectes virilis ; Hagen Acta Histochem. 20 193-222
Trim ARH 1941 Studies on the chemistry of the insect cuticle. 1. Some general observa-
tions on certain arthropod cuticles with special reference to the characterization of the
proteins ; Blochem. J. 35 10884098
V B 194$ Tfce insect cutipje ; £fo/. fyv, «3 408-431
f*roc. Indian Acad. Sci. (Anim. Sci.), Volume £>J, No. 1, January 1082, pp. $7-77.
© Printed in India.
Effect of some antibiotic compounds in cotton on post-embryonic
development of spotted bollworm (Earias vittella F.) and the
mechanism of resistance in Gossypium arboreum
H G SHARMA*, R A AGARWAL and MUNSHl SINGH
Indian Agricultural Research Institute, New Delhi 110 012, India
* Present address : Sorghum Entomologist, ICRISAT, ICRISAT Patancheru
P.O., 502324, India
MS received 6 March 1981 ; revised 31 August 1981
Abstract. Larval survival and post-embryonic development of the spotted bollworm,
Earias vittella was studied on 23 genotypes belonging to three cultivated species of
cotton (Gossypium arboreum, G. barbadense and G. hirsutum). There were significant
differences in larval survival and post-embryonic development on different genotypes.
The larval survival varied from 27-1 to 93-3%, developmental period from 16-6
to 20-3 days, pupation from 60 to 100% and adult emergence from 78 to 94%.
Gossypol increased the post-embryonic developmental period. Majority of the
larvae entered the bolls through the thallic region, possibly, to avoid higher concen-
trations of gossypol in the pericarpic region. Tannin content of bolls was signifi-
cantly and negatively correlated with adult emergence.
Crosses between resistant and susceptible genotypes of G. arboreum segregated
into pigmented (red) and non-pigmented (green) plant types. The former were
rich in gossypol and tannins compared to the latter. Gossypol and tannia content
of bolls showed negative correlation with spotted bollworm incidence.
Keywords. Gossypium ; gossypol ; tannins ; spotted bollworm ; Earias ;
antibiotic ; post-embryonic ; resistance.
1. Introduction
Cotton is an important commercial crop in Asia, Africa, America and Australia.
It is damaged by over 130 different species of insect pests. Spotted bollworms
(Earias spp.) cause serious losses to cotton in India, China, Southeast Asia, Iraq,
Israel and Africa (Sohi 1964; Chang et al 1963 ; Walker 1952; Avidov and Harpaz
1969 ; Reed 1974). Pigment glands characterizing genus Gossypium (Gillham
1965) had been identified as a source of resistance against the insects feeding on
cotton plant (Bottger et al 1964). Antibiosis, as one of the mechanisms of resis-
tance in cotton was. first demonstrated by Brazzel and Martin (1956, 1959) in
G. tomentosum against Pectinophora gossypiella. Later, antibiosis was reported
against Heliothis zea and H. virescens (Lukefahr et al 1966; Lukefahr and
Houghtaling 1969; Oliver et al 1970, 1971;. Lukefahr et al 1974, 1975; Meisner
et al 1977); Anthonomus grandis (Douglas 1966, Bailey et al 1967) and Amrasca
67
68 8 C Sharma, k A Agarwat and Munshi Siitgh
biguttula biguttula (Chakravarty and Sahni 1972). Gossypol (Lukefahr and
Houghtaling 1966; Lukefahr et al 1966, 1975; Meisner et al 1977), p-hem/gossy-
polone (Gray et al 1976), heliocides (Stipa&ovic et al 1976, 1977) and a condensed
tannin (Chan and Waiss Jr. 1978) have been reported to confer resistance to insects
feeding on cotton. Considering the colossal losses caused by this pest vis-a-vis
the limitations of insecticides to control it, the host plant resistance can be used
as one of the mechanisms to keep its populations at a low level. Asiatic diploid
species (Gossypium arboreum L.) have been reported to be comparatively less
damaged (Hussain and Khan 1940; Butani 1974). Singh et al (1972, 1976) found
that within G. arboreum, damage by bollworms varied among different varieties
and that resistance was genetically inherited. The present studies report the
extent of antibiosis and the antibiotic factors affecting development of E. vittella
in different cotton genotypes, and the mechanism of resistance in G. arboreum.
2. Materials and methods
The insect culture was raised in the laboratory on green cotton bolls of Bikaneri
Nerma. Genotypes tested included 3 lines from G. arboreum (Sanguineum, Virnar
and G-^27), one from G. barbadense (Line 199-^5) and 19 from G. hirsutum. The
effect of antibiotic factors was studied on the survival of first instar larvae and the
post-embryonic development. Larval survival on bolls of different genotypes was
studied by releasing newly hatched larvae on green bolls (7-10 days old) in plastic
boxes (15 x 15 x 5 cm). Five larvae were released on each boll. The plastic
petridishes were kept inside B.O.D. incubator at 30 ± 1°C. Three days after
inoculation, the bolls were dissected and the number of survivors recorded. The
observations were made on 20 bolls of each genotype arranged in four sets and also
on the number of larvae entering the boll through the thallic and pericarpic regions
of the boll on a few genotypes.
The post-embryonic development was studied on 7-10 days old bolls of different
genotypes. Food was changed every third day. The rearing was carried out at
30 ± 1° C. Observations were recorded on pupation, adult emergence, pupal
weight, and larval and pupal developmental periods. Growth indices on different
genotypes were calculated by the following formulae:
Larval growth index (LGI) = ,Percf * P^**'
Larval period (days)
Total developmental growth index (TGI) = ^j^ emergence
Total developmental
period (days)
Spotted bollworm incidence was recorded on 100 green bolls during the peak
activity period (August) in 1978 on five varieties of G. arboreum and three F2
populations of intra-arboreum crosses involving resistant and susceptible types.
The different genotypes were grown in 2 row-4 m plots. For chemical analysis,
10-15 days old bolls were collected. The bolls were dried at 40° C and powdered
finely in a grinder. The gossypol content was determined by the method of Yang
and Davis (1976) and tannins were estimated by indigocarmine volumetric method
Antibiotic compounds in cotton 69
(AOAC, 1975). The gossypol and tannins were expressed as per cent of dry
vjeight of the sample taken. The data were analysed and simple correlations
between the different parameters were worked out.
3. Results and discussion
The larval survival varied from 27-1 to 93-3% in different genotypes (table 1).
Larval survival was minimal on SH-269, SS-265, Aeala, Cocker^ 100A, Sangu-'ncum,
XG-15 and South Carolina. Only 27- 1 to 40-7 per cent larvae survived on these
lines. Comparatively, more larvae survived on PS- 10, Virnar, RS-89 and 320-F
(68-8 to 93-3%). This difference in survival values in different genotypes is indi-
cative of the resistar.ce offered by bolls of some genotypes. Larvae showed a
tendency to enter the bolls through the thellic region (table 2), possibly, to avoid
higher concentrations of antibiotic factors in the pericarpic region.
Table 1. Survival of first instfar larvae and pupal weights of s;x.ttcd bollworm
(E. vittella) on the bolls, and amounts of gossypol and tannins in different varieties.
Variety Larval survival
<%)
Gossypol
<%)
Tannins
<%>
Pupal Weight
(mg.)
PS-1G
93-3
0-96
1-42
48-2
Virnar
76-0
0-76
1-60
66-9
32GF
70'0
, 1-10
1*40
K.S-89
68*7
; 1-04
1-40
52-4
I>33
60-4
1*12
1-35
55-3
JR-81
5S-1
0*79
1-06
52*3
Stoit-73IN
51-6
0*79
1-63
. .
Bikaneri ncrma
48-8
0-92
1-22
, .
Frego bract
48-3
1-00
1-49
M-495
47-3
1-04
1-58
50*3
H-14
47-0
1-08
1-87
43-7
G. bwbadense
46-0
1-31
, .
. 65-6
HR-26# 8X H.HCf-6
1M
44-8
0-95
1-88
56-*0
Hindiweed
43-3
1-13
1-6)
46-3
BJR
42-0
0-98
1-57 '
. .
XG-15
40-6
1-33
1-87
45-1
South Carolina
40-6
1-11
1-15
58*5
SH-269
40-3
1-02
1-60
42-3
SS-265
40*2
1-02
1-34
' 57-4
Acala
39-3
0-58
1-30
56-0
Cocker-lOOA
34-6
0-61
1-61
45*2
Saitguineum
27-1
1-46
1-96
57-2
CJD. at 5% t 13-22 0*13 0-04 1Q-1
72 H C Sharma, R A Aganval and Munshi Singh
(r = - 0-7638). High concentrations of gossypol in the pcricarpic pov^iy
deterred the larvae entering the boil through this rcg'on.
Larval period was prolonged by gossypol and tannins. Total pcriod^ required
for completing post-embryonic development showed a positive mid
correlation with gossypol content of bolls (r = 0-4974) (table 5), Tnc shorter
developmental period on Virnar and Empire was possibly da* to lower amounts <*!'
gossypol in these genotypes, while the longT developmental period-* on
SS-265, XG-15, GH27 and G.barbademc could bi attributed to concen-
trations of gossypol in these varieties. Sinvlar antibiot'c factor* m cotton have
b:en reported against P. gossypidla (Brazil and Marlin 1(>56; 19S1*);
spp. (Oliver et al 1970, 1971); H. vlrcswtis (Lukcfahr a nl 1966; Mctswr ri «/
1977) and Anthonimits grandis (Bailey ct al 1967; l>oug»as Il>66).
Gassypol seemed to affect pupation and adult emergence advcricly. f tic-
correlation coefficients w<,rc very low (tables 6? 7). Tannins showed
negative correlations with adult emergence (r ~ - 0*7H13)and growth (r **••
— 0-7432) (table 7). Tnc regression coefficients were also Mgniftcwtt and negative.
Gossypol incorporated in artifhial d'et had b:tn found to affect the
and survival of pinkbollwoim (Siavcr and Parrot 1970). Tnc larval foul
b'ien found to bs negatively correlated with gossypol content (Wihon
1973).
Somi biochemical components other than gossypol and taniuit\ al\it «iccoui-itcd
for the antibiosis expressed by different genotypes. Eagle et n/ ( 19511) m*
correlation between cotton seed pigmont glands toxicity and cxtnictablc gottypoi.
S^m^ growth inhibiting factors have recently b:en reported in the nice Mtvks <*f
G. hirsutum, which contained medium amounts of gossypol but were anti-
biotic against P. gossypidla and Heliothix spp, (Lukcfahr «•/ nl I*>74), The^c
additional growth inhibiting factors were later idtauiihd as /Hiciiitgfi\-%>'f%iliiiic
(Gray et al 1976) and Heliocides Ht and H2 (Stipanovic ct at 1976, In the
present studies, soms factors other than gossypoi also accounted for the
effect against E. vittella. Major role among these factors was thai of
Elligar et al (1978) investigated the toxicity and relative importance of
terpenoid; in the pigment gland* and .suggested that these
the toxicity of gossypol but themselves are of minor importance.
redi :es the nutritional quality of boll contents (Carter and Lymun, 1969 ;
et al 1959). It also inhibits the activity of enzymes protease, amytttsc anil
g;n (Mcisner et al 1978 ; Tanksley et al 1970). Antibiotic effects of arc
possiblydin to reduced nutritional ualit or non-ava
^ nutritional quality or non-availability of «ir
enzyme inhibition, which lead to the prolong 'd development and reduced
of the insect,
Bollworm incidence on different varieties and Pt populations of inter»VArietai
crosses have been presented in table 8. It was observed that Dttutat CJf-71 and
non-pigmented F2 segregates had > 75% spotted boliworm incidence
to Lohit, G^27 and pigtnonted P2 segregates which manifested < 55%
The red pigmented segregates showed 32-42-46-87% incidence mnl
pigmented (green) segregates had 62-50-80-06% boliworm incidence
There vrere signiflcant differences among different genotypes k aosnypol and
tannm content (table 9). The less susceptible genotype, viz., G-27 md L6bii
as well as P1gmented F2 segregates had comparative!), higher
Antibiotic compounds in cotton
73
Table 5. Correlations between gossypol and tannin content in bolls with post
embryonic development of E. vittella.
Larval
Pupal
Total deve-
Gossypol
Tannins
Period
period
lopmental
period
Gossypol
1-000
Tannins
0-2397
1-000
Larval period
0-3398
0-3480
1-000
Pupal period
0-1724
-0-0725
0-051.9
1-000
Total developmental period
0-4974*
0-1491
0-7364*
0-5242*
1-000
/ value
Yi Larval period
X^ Gossypol
0-7239
1-1745
Xg, Tannins
0-7743
1-2208
0-1908
Y2 Pupal period
Xi Gossypol
0-4473
0-7992
X% Tannins
-0-2760
0-4793
0-0434
y3 Total developmental
period
Xi Gossypol
1-9673
2-1937*
X2 Tannins
0-1311
0-1421
0-2483
* Significant at P = (
Table 6. Relationship between gossypol and tannin content of bolls with pupation
and growth index.
% Gessypol
% Tannin
% Pupation
Growth index
% Gossypol
% Tannin
% Pupation
Growth index
1-000
0-5095
-0-2485
-0-0359
1-000
-0-0148
-0-0131
1-000
0-9825*
1-000
/ value
Yi Pupation
Xi Gosaypol
X^ Tannins
Ta Growth index
Xi Gossypol
X4 Tannins
-19-1455
8-2009
- 3-8546
1-7104
0-8255
0-3830
1*2135
0-5835
0-786
0*1556
74 H C Sharma, R A Agarwal and Munshi Singh
Table 7. Relationship bttwoen gossypol and tannin content in bolls to per cent
emergence and growth index.
GoslSypol Tannins Emergence Growth index
Gossypcl
1-000
Tannins
0-4605
1-000
Emergence
-0 3510
-0*7313*
1-000
Growth index
-0-3411
-0-7432*
0-9198*
1-000
bi t value
YI Emergence
Xi Go&ypol
0-3281
0-0446
X« Tannins
- 17-1281
3-1635*
0-6104
7a Growth index
Xl Gossypol
0-0027
0-0052
X4 Tannins
- 1-0993
2-791,3*
0-5523
* Significant at p = 0-05
Table 8. Incidence of shotted bollworm (E. vittella) on arboreum genotypes.
SI.
No,
Genotype
Incidence/
100 b-ill*
].
Daulat
90-00
2.
CJ 73
75-00
3.
Cernuum
69-20
4.
Lohit
55-00
5.
G-27
53-00
6.
Ccntuum x Lohit
(a) Non-pigmented F8
80-00
(b) Pigmorttcd Fa
46-67
7.
IXiulat x Lohit
Non-pigmented F%
80-00
Pigmor^tod Fa
45-45
8.
Cernuum x G-27
Non-pigmented Fz
62-50
Pigmertted F2
32*43
Average for F2 segregates
Non-pigmented
74-17
Pigmented
41-52
Antibiotic compounds in cotton
Table 9. Gossypol and tannin content in some G. arboreum genotypes.
SI. Genotype
No.
Gossypol (%>
Taaaiii (%)
1.
G 27
1-
58
d
0
•74
d
2.
Lohit
1-
40
c
0
•84
d
3.
* Pigmented F3
1-
20
b
0
•47
c
4.
Cernuum
1-
24
b
0
•41
be
5.
CJ 73
1-
02
a
0
•31
b
6.
* Nonpigmeitted F3
0-
99
a
0
•17
a
CD at 5% (0
o-
10
0
•12
* Samples from the Fa Segregates of the cross Daulat x Lohit.
(1-20-1 -58%) and were also rich in tannin content (0-41-0-84%). The spotted
bollworm incidence was found to be negatively correlated with gossypol (r =
— 0-7133) and tannin content (r = — 0-6420). Gossypol and tannins were also
significantly associated between themselves (r = 0-9040). Gossypol is the principal
antibiotic compound in the cotton plant, and is also genetically inherited (Lee et al
1968; Rhyne and Smith 1965; Wilson and Smith, 1976).
Tne spotted bollworm incidence in arboreum varieties seems to be largely influ-
enced by the gossypol and tannin content. On the basis of these results, it is
sug*)sted that while selecting plants resistant to spotted bollworms in F2 popu-
lations, the plant pigmentation (red-pigmented) may be used as an important
character along with the gossypol and tannin content of the genotypes.
Acknowledgement
The authors are thankful to Aspee Agricultural Research and Development
Foundation for awarding the fellowship to the Senior author. Our thanks are
also due to Dr W Reed for going through the manuscript and to Mr Krishna
Murthy for typing the manuscript.
References
A.O.A.C. 1975 Official methods of analysis. A.O.A.C., P.O.B. 540, Benjamin Franklin Station,
Washington, B.C. 20044. pp. 1094
Avidov Z and Harpaz J 1969 Plant pests of Israel (Jerusalem : Israel University Press)
Bailey J C, Maxwell F G and Jenkins J N 1967 Mortality of boll weevils in squares of geno-
typically different lines of cotton ; /. Econ. Entomol. 6 1279-1280
Bottger G T, Sheehan E T and Lukefahr M J 1964 Relation to gossypol content of cotton plant
to insect resistance ; J. Econ. Entomol. 57 283-285
Brazzel J R and Martin D F 1956 Resistance of cotton to pink bollworm damage ; Bull, Tex.
Agric. Exp. Sta. No. 843, pp. 20
76 H C Sharma, R A Agdnvdl and Munshi Singh
Brazzel J R and Martin D F 1959 Pink bollworm resistance in cotton ; /. Econ. Entomol. 32
385-390
Butani D K 1974 Insect pests of cotton. XVII. Effects of cotton varieties, cultural practices and
fertilizer on infestation by bollworms ; Colon et Trop. 29 237-240
Carter C M and Lyman C M 1969 Reactions of gossypol with amino acids and other amino
compounds ; /. Am. Oil Chem. Soc. 46 649-653
Chakravarty S C and Sahni V M 1972 Biochemical basis of resistance to jassids (Empoasca
spp.) in G. hirsutum. Cotton ; Indian Agric. 16 45-4$
Chan B G and Waiss Jr, A C 1978 Condensed tannin, an antibiotic chemical from Gossypium
hirsutum ; /. Insect Physiol. 24 113-118
Chang G S, Meng H L and Bao J A 1963 The status of spotted bollworms, Earias fabia (Stoll)
and E. insulana (Boisd.) after the changes of cultural system in Lukang Poashan District,
Yunnan Province ; Acta Entomol. Sin. 12 28
Douglas A G 1966 Selection in cotton for antibiosis to boll weevil ; /. Econ. Entomol. 59 32-34
Eagle E, Hall C M, Castillan L E and Miller C B 1950 Effect of fractionation and treatment
on the acute oral toxicity and gossypol and gossypurin content of cotton seed pigment
glands ; /. Am. Oil Chem. Soc. 27 300-303
Elliger C A, Chan B G and Waiss Jr. A C 1978 Relative toxicity of minor cotton terpenoides
compared to gossypol ; /. Econ. Entomol. 71 161-164
Gillham F E M 1965 Evolutionary significance of glands and their importance in cultivated
cotton ; Emp. Cotton. Grow. Rev. Ill 101-103
Gray J R, Marby T J, Bell A A and Stipanovic R D 1976 P-hemigossypoione : a sesquiterpenoid
aldehyde quinone from Gossypium hirsutum ; /. Chem. Soc. Chem. Commurt. pp. 109-110
Hussain M A and Khan M H 1940 Studies on Platyedra gossypiella Saunders in Punjab. IX.
Relative incidence on exotic and indigenous varieties of cotton ; Indian J. Entomol. 2 45-57
Lee J A, Cockerham C A and Smith F H 1968 The inheritance of gossypol level in Gossypium.
I. Additive, dominance, epistatic and maternal effects associated with seed gossypol in tw«
varieties of Gossypium hirsutum ; Genetics 59 285-298
Lukefahr M J and Houtghtaling J E 1969 Resistance of cotton strains with high gossypol content
to Heliothis spp. ; /. Econ. Entomol. 62 588-597
Lukefahr M J, Houghtaling J E and Cruhm D G 1975 Suppression of Heliothis spp. with
cottons containing combinations of resistant characters ; /. Econ. Entomol. 68 743-746
Lukefahr M J, Noble C W and Houghtaling J E 1966 Growth and infestation of bollworms
and other insects on glanded and glandless strains cf cotton ; /. Econ. Entomol. 59 817-820
Lukefahr M J, Shaver T N, Cruhm D G and Houghtaling J E 1974 Location, transference
and recovery of a Heliothis growth inhibition factor present in three Gossypium hirsutum
race stocks ; Beltwide Cotton Production Research Conference Proceedings, 1974
Lyman C M, Balliga B P and Margaret S W 1959 Reactions of proteins with gossypol ; Arch.
Biochem. Biophys. 84 486-497
Meisner J, Kehat M, Zur M and Asner K R S 1977 The effect of gossypol on the larvae of
spiny bollworm, Earias insulana ; Entomol. Exp. Appl. 22 301-303
Meisner J, Ishaaya I, Asher K R S and Arom M Z 1978 Gossypol inhibits protease and amylase
activity of Spodoptera littoralis larvae ; Ann. Ent. Soc. Am. 71 5-8
Oliver B F, Maxwell F G and Jenkins J N 1970 A comparison of the damage done by the
bollworm to glanded and glandless cottons ; /. Econ. Entomol. 63 1328-1329
Oliver B F, Maxwell F G and Jenkins J N 1971 Growth of bollworm on glanded and glandless
cotton ; /. Econ. Entomol. 64 396-398
Reed W 1974 Populations and host plant preferences of Earias spp. (Lepidoptera : Noctuidae)
in East Africa ; Bull Entomol. Res. 64 33-44
Rhyne C L and Smith F H 1965 Genetic aspects of gossypol content of leaves and flower buds
of Gossypium ; /. Heredity 56 242-247
Shaver T N and Parrot W L 1970 Relationship of larval age to toxicity of gossypol to bollworm,
tobacco budworm and pinkbollworms ; /. Econ. Entomol. 63 1802-1804
Antibiotic compounds in cotton 77
Singh H G, Mathur R K and Yadava H M 1965 A study of the thickness of green mature bolls
in relation to the incidence of pink bollworm (Pectinophora gossypiella Saund.) ; Indian
Cotton J. 19 253-255
Singh M, Agarwal R A and Katiyar K N 1972 Preliminary studies on the inheritance to pink
bollworm (Pectinophora gossypiella Saunders) resistance in Gossypium arborewn ; Entomo-
logist's Newsletter 2 40-41
Singh M, Joshi A B and Agarwal R A 1976 Genetics of cotton bollworms (Pectinophora gossy-
pialla Saund.) resistance in an intervarietal cross of Gossypium arboreum L. ; Colon et Fibres
Trop. 31 369-372
Sohi G S 1964 Pests of cotton ; In : Entomology in India (ed.) N C Pant Entomological
Society of India pp. 111-148
Stipanovic R D, Bell A A and Lukefahr M J 1976 Natural insecticides of cotton. Structural
analysis and toxicity of heliocides. 172nd Nat. Mfg.Amer. Chem. Soc. 29 Sept. 1976,
San Francisco ; Pest. Div. Abs. No. 78
Stipanovic R D, Bell A A, O'Brein D H and Lukefahr M J 1977 Heliocide-Ha. An insecticidal
sesquiterpenoid from cotton (Gossypium) ; Tetrahedron Lett. 567-570
Tanksley T D, Neumann H, Lyman L M, Pace C H and Prescott J M 1970 Inhibition of pesino*
gen activation by gossypol ; /. Biol. Chem. 245 6456-6461
Walker R L 1952 Spiny bollworm of cotton in Iraq ; FAO Plant Prot. Bull. 1 42
Wilson F D and Shaver T N 1973 Glands, gossypol content and tobacco budworm develop-
ment in seedlings and floral parts of cotton ; Crop Sci. 13 107-110
Wilson F D and Smith J N 1976 Some gent tic relationships between gland density and gossypol
content in Gossypium hirsutum L. ; Crop Sci. 16 830-832
Yang H C and Davis D D 1976 Variations in gossypol concentrations of flower buds of cotton;
Curr. Sci. 16 485-488
ftroc. Indian Acad. ScL (Anim. Sea.), Vol. 9l, lumber 1, January 1982, pp, 79-98.
© Printed in India. , .
Some biometric studies of certain closely related species of the
genus Anus (Pisces : Siluriformes : Ariidae)
J R DHANZE and K C JAYARAM
Zoological Survey of India, 27 J L Nehru Road, Calcutta 700 016, India
MS received 6 March 1981 ; revised 17 August 1981
Abstract. The marine catfish genus Arius of the family Ariidae comprising 21
species have been divided into six complexes and three groups based on interspecific
relationships and morphometric affinities. In this paper the maculatus complex of
four species, v/z., Arius maculatus* Arius arius, Anus gagora and Arius jella has
been critically examined in respect of a selected list of 20 morphological characters
based on examination of a large series of examples collected first hand by the authors.
The samples have been statistically analysed, and the range of variation in respect
of each character as exhibited by each species has been delineated. The probability
significance test has been made to establish the interspecific relationship.
Keywords. Biometric study ; Arius species ; Ariidae.
1. Introduction
The genus Arius Valenciennes, 1840 forms a commercially important group of
marine catfishes comprising 21 species from India, Pakistan, Bangladesh, Burma
and Sri Lanka. Most of the species are marine often entering estuarine waters
and occasionally even in freshwaters such as A. acutirostris, A. burmanicus and
A. gagora etc. About 80% of the total catfish landing in our country is of Arius
species. Despite the economic value of these fishes, the taxonomic identity of
most of the species is in a state of confusion. The main reason for such ambi-
guity is because earlier workers depended mainly on one or two characters which
were highly variable interspecifically if not associated with the changes in growth
or sex.
Day (1877, 1889) gave a comprehensive account of 23 species by using the anal
fin counts, relative head length and eye diameter as diagnostic characters, besides
the shape and size of teeth bands on the palate. Weber ajid de Beaufort (1913)
also utilised the dentition pattern, besides the shape of the occipital process for
separating the species of this genus. Smith (1945) considered the dentition pattern
as one of the very important taxonomic character and stated, " the most important
character for separating the species are teeth. " Chandy (1954) framed a key
mainly based on the dentition pattern on the palate, for the identification of
Arius species present in the NZC of ZSI, Calcutta. Subsequent ichthyologists
also relied upon this character (Munro, 1955 ; Smith, 1962 ; Wongratana and
Bathia, 1974 ; Misra, 1976). Taylor (1978) adopted the length of the median
19
80
j R Dhatize and K C JayarcMi
longitudinal groove on the head, the shape of the bony shield for separating
Arius species of western central Atlantic (Fishing Area 31).
It any bs seen that for separating the various species of Arius the pattern of
teeth patches on the palate still remains to be an unavoidable necessity. How-
ever, it may be indicated that whereas the basic contour, the number and posi-
tion of the patches remain constant, the size, number and nature of the teeth
themselves vary highly and alter considerably with age and growth. Earlier
ichthyologists seem to be unaware of this fact and established species like
A. serratus Day, A. maldbaricus Day, A. satparanus Chaudhuri for such variants
which are invalid (Jayaram and Dhanze 1978a, 1981).
Based on the number and contour of the patches we have placed the 21 species
of Arius in six complexes under three groups. The constituent species of each
complex are closely interrelated and some may even prove later either to be
synonyms or subspecies. In this paper 'maculatus' complex which has four species
(A. maculatus, A. anus, A. gagora and A.jelld) (Text-figures 1A-H), have been
analysed to determine their interspecific affinities and systematic status.
2. Materials and methods
The material for this study is based on 430 specimens collected by the authors
during extensive survey tours of the entire eastern coast and a part of southwest
*l$^w*if'\tf,wW
w*
«$\' ..-v
Ot)(> >00
•' Oor> " q » °*o0 -.
% /
'%8S«S^
B
H
Text Figure 1 A.— -A. maculatus (dorsal view of head). B. — A. maculatus (dentition).
C.— A. gagora (dorsal view of head).. D.— A. gagora (dentition). E.— - A. arius
(dorsal view of head). F. — A. arius (dentition). G. — A. jella (dorsal view). H,
A. jela (dzntition).
(Figures A— E, G and H, after Chandy, 1953, Figure F— after Chaudhuri 1916).
Biometric studies of the genus Arius 81
coast of India. The specimens present in the National Zoological Collections of
the Zoological Survey of India, Calcutta, have also been examined. Fresh
material of species such as A.jella and A. maculatus were collected and studied
by the second author (KCJ) during the FAO consultation, Cochin in 1980 . A total
of 45 characters were mensurated and of which 20 alone are selected for the
statistical analysis. All the measurements were taken with dial calipers to the
nearesthalfof a millimeter for the size range up to 150 mm and by measuring tape
abcva this size.
Taxonomic characters are generally found to intergrade of overlap between
closely related species when a large series of specimens are studied. The reliability
or otherwise of such characters are to be evaluated. Different methods of
measuring intergradation or divergence have been proposed (Davenport and
Blan kinship, 1898 ; Pearl, 1930 ; Ginsburg, 1938 ; Simpson and Roe, 1939 ;
Amadon, 1949 ; Snedecor, 1956; and Simpson et al 1960). Methods deviced
tby Simpson et al (op. tit.) for the comparison of two populations irrespective of
heir taxonomic identity seems to be useful here. The "Student's Mest" to
determine the probability value at 95% confidence intervals have been applied.
Before deducing any numerical conclusion, a hypothesis was set forth that all the
specimens of different populations examined belong to a same species, and the
universally used rejection value of 5 per cent was chosen as a criteria for the
rejection of this hypothesis. However, the establishment of the significance of
a difference between two species by numerical derivation is not in itself a zoological
conclusion. Thus the numeiical expressions for each character were further
compared or rather standardized by employing geometrical expressions proposed
by Dice and Leraas (1936), and later on adopted with some modification by
Hubbs and Perlmutter (1942), Pillay (1951), Hubbs (1952), and Wmterbottom
(1980). In this method, for each character the range, mean, one standard deviation
and one standard error on each side of the mean were delineated on the graph.
The degree of overlap or separation of the standard deviations in respect cf the
arithmetic mean of each species was determined.
3. Results
Tables 1-6 and graphs 1-20 present the biometric comparison of the four species
with each other for ell the 20 characters selected.
3.1. A. maculatus vs. A. aritts
It is seen that excepting the head length, in respect of all oth?r characters the t\vo
species have a probability of less than 0 • 1% and are significantly different (table 1)
From the graphs XIV-XVI, XVIII, XX, Dice diagram A and B in each, it is seen
that the mean of each population as well as standard deviation (S) diverge to a
considerable degree, thereby justifying the separate specific status of A. maculatus
and A. arms. Both the species have a single large oval patch of teeth on each
side of the palate (text-figure 1 B, F). Further, the two species can be morpho-
logies lly distinguished by the size and position of the eye The eye diameter is
18-50% in A. maculatus vs. 21-40% in A. arius in the head length ; 33-30% \s
45-60% u the interorbital width and 57-35% vs. 63-30% in snout length,
82
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Biometric studies of the genus Arius 83
3.2. A. maevlatus vs. A. gagora
The two species differ significantly in respect of 14 characters having the probability
of less than 5% (table 2) and the standard deviation not overlapping with arith-
metic mean of the other in respect of 11 characters (Graphs I, II, V, XI-XIII, XV,
XVI, XVIII-XX, Dice diagram A and D in each).
A. gagora and A. maculatus have a single oval large patch of teeth on each side
of the palate (Text-figure 1 B, D), though the teeth may be set sparsely in the
former and densely packed in the latter. However, we have observed sparse
arrangement of teeth in a few adult male specimens of A. maculatus also. Further,
A. gagora is significantly different from A. maculatus in respect of eye size
and internostril distance. The eye diameter in snout length is 57-37% in A. macu-
latus vs. 39-03 in A. gagora ; the internostril width in snout length is 65-94% vs.
48 '17%. It may be mentioned here that in respect of the other so
called significant characters such as predorsal length, width of dorsal fin, height
of head etc., the difference is not very high. From the distributional pattern of
both the species it would seem that A. maculatus is replaced by A. gagora in the
Hooghly estuarine system.
3.3. A. maculatus vs. A. jella
From the data presented in table 2, it can be seen that only in respect of 10 charac-
ters the probability is less than 5Jtf and in respect of the remaining ten characters
it is more than 5%. Among the significant characters, the least depth of caudal
peduncle in its length is 53-89% in A. maculatus vs. 43-96% in A. jella ; inter-
nostril width in snout length 65-94% vs. 60-42% and the size and position of eyes
are noteworthy. Further, A. jella is clearly separable from A. maculatus by the
length of the pectoral spine which in A. jella is longer than the dorsal spine.
In most species of Arius the pectoral spine is equal or shorter than the dorsal
spine. We have examined specimens of all sizes in both the sexes in each species
and have not found any variation in respect of this character (Graphs XII, XVIII-
Dice diagram A and C in each).
3-4. A. anus vs. A. jella
These two species differ statistically in respect of ten characters in the fact that
their probability is less than 5%. Table 4 and Graphs XEWCIV, Dice diagram
B and C in each, indicate the degree of intergradation or divergence. Here again
the size and position of the eye appears to be an important character. The eye
in head length is 21-30% in A. arius vs. 15-95% in A. jella ; in snout length
63-30% vs. 50-35% j in interorbital width 45-60% vs. 33-90%. Further, as
stated already A. jella is separable by the character of pectoral spine being longer
than dorsal spine as compared to other species of Arius. A. jella is darker in colour
than A. arius.
3.5. A. arius vs. A. gagora
The systematic position of these two species is slightly vague. A. gagora is not
very well represented and apparently not collected extensively as A. arius. One
of us (JRD) was able to obtain four ftesh specimens (195-»245 mm SL) of A. gagorq
84
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Biometric studies of the genus Arius 87
from the river Hooghly at Serampore (W.B.) and eight specimens (12(M85 mm SL)
from Haldi estuary at Halida lart (W.B.). Prom a critical examination it is
seen that A. gagora is clearly separable from A. arius by its shallow median longi-
tudinal groove which extends up to the supraoccipital crest as compared to
A. arius which has the median longitudinal groove narrow extending only up to
the frontal bones -(text-figure .1 C, E). Besides, both the species differ in respect
of the size of the eye which is larger in A. arius than in A. gagora ; the eye diameter
in head length is 21-35% in A. arius vs. 14-95% in A. gagora ; in snout length
63-30% vs. 39-00% ;. in interorbital width 45-60% vs. 34-25% Further, 13 charac-
ters having less than 5% probability indicate the statistical differences between
the two species (Table 5 ; graphs, I, II, V, XKXIV, Dice diagram B and D in each.)
3-6. A. gagora vs. A. jetta
Table 6 presents the comparative data in respect of A. gagora and A.jella. Nine
characters showing the probability of less than 5% are delineated in the Dice
diagram C and D (graphs I, II, V, VII, XI, XIII, XV). Morphologically the two
species can be distinguished by the size and position of the eye and also the rela-
tive distance between the pairs of nostrils on each side. The nostrils in A.jella
are closer to each other on either side than in A. gagora. The eye diameter in
snout length is 39-00^ in A. gagora vs. 44-45% in A.jella ; the internostril width
in length of snout 48-20% vs. 55-60%.
4. Discussion
The above analyses of four species forming the maculatus complex of the genus
Arius indicate clearly their close inter-relationship. Morphologically also these
species resemble each other in one or other character and in juvenile stages they
are hard to separate, more particularly since all of them have a single oval patch
of teeth on the palate (text-figure 1 B, D, F, H). The 20 characters which appeared
helpful to differentiate these species were utilized for statistical interpretation ;ajad
the extent of range of variation of each character was computed. The probability
significance in respect of each such morphometric character as shown by each
species is summarised in table 7.
Of the 20 characters selected there is not a single character which can distin-
guish each species from the other. The size and position of the eye is most signi-
ficant followed by the internostril width, snout length and the least depth of the
caudal peduncle. The body depth, head width, head length etc., the conven-
tional characters used by the earlier ichthyologists do not appear to te of much
help, at least in respect of these four species. Considering the fact that the macu-
latus complex of species are inhabitants of clear oceanic and estuarine waters
feeding on carnivorous diet in midwater, the differences in structure and position
of the eye seems justified.
Of the four species it is seen that A. maculatus and A. arius are well established
separate populations, each occupying its own separate habitat. Thus A. macu*
latusis extensively' distributed in the Arabian sea with stray individuals occasio-
nally caught in Bay of Bengal. A. anus on the other hand is extensively found in
Bay of Bengal havng not been so far recorded south of Portamovo. Moreover,
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Mometric studies of the genus Arius
93
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Biometric studies of the genus Arms
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Graphs I- XX (See for Captions in p. 98)
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Biometric studies of the genus Arlrn 97
A. arius is an inhabitant of brackish water lakes such as Chilka, while A. macu-
atus seems to prefer deeper waters of the open seas. It would seem that A. macu-
atus is replaced by A. anus in the Bay of Bengal north of Coromandel coast.
A. gagora is found in the Hooghly esturaine system and is known from compa-
ratively lesser saline waters than the other species of this complex. It is most
closely related to A.jella which is also known from Orissa and Bengal coast. It
is clearly separable from A. maculatus and A. arius by the relative extension of
the median longitudinal groove, besides size and position of the eye (vide supra).
The data of probability distribution depicted in table 7 substantiated by Gins-
burg's method of analysis (table 8) would seem to indicate that A. jella is only
a subspecies of A. gagora. Pending further studies with the fond hope of obtaining
more material of these two species, we have kept A.jella as a separate species for
the present. It is concluded as such that A. maculatus complex comprises of
four species : A. maculatus (Thunberg, 1792), A. arius (Hamilton, 1822), A. gagoro
(Hamilton, 1822) and A. jella Day, 1877.
Acknowledgement
We are thankful to the Director, Zoological Survey of India, Calcutta, for
facilities.
References
Amadou D 1949 The seventy five per cent rule for subspecies ; Condor 51 250-258
Chandy M 1954 A key for the identification of the catfish.es of the genus Tachysums Lacapede
with a catalogue of the specimens in the collection of the Indian Museum (Zool. Surv. ) ;
Rec. Indian Mus. 51(1) 1-18, 3 pis. text figures.
Devenport C B and Blankiaship J W 1898 A precise criterion of species ; Science (N.S.) 7
684-695
Day F 1877 The fishes of India ; London, Wm. Dawson and Sons 778 pp. 198 pi-
Day F 1889 The fauna of British India including Ceylon and Buma, Fishes 1 169-192, Taylor
and Francis, London
Dhanze J R and Jayaram K C 1979 The family of catfishes of the genus " Arius " (Siluriformes) ;
Curr. Sci. 48 (22) 1008
Ginsburg 1 1938 Arithmetical definition of species, subspecies, and race concept with a proposal
for a modified nomenclature ; Zoologica 23 253-286
Dice L R and Leraas H J 1936 A graphic method for comparing several sets of measurements,
Contribs. Lab. Vertebr. Genetics, Univ. Mich. Ann. Arbor. 3 3
Hubbs C L and Perlmutter A 1942 Biometric comparison of several samples with particular
reference to racial investigations ; Amer. Nat. 76 582-592
Hubbs C 1952 A contribution to the classification of the blennoid fishes of the family Clinidae
with a partial revision of the eastern pacific forms ; Stanfard ichthyol. Bull. 4 (2) 41-165
Jayaram K C and Dhanze J R 1978a Siluroid fishes of India, Burma and Ceylon. 21. A note
on the systematic position of Tachysums serratus (Day) (Ariidae) ; Bull. zooL Surv. India
1(2)203-205
Jayaram K C and Dhanze J R 1978b Siluroid fishes of India, Burma and Ceylon 22. A
preliminary review of the genera of the family Ariidae (Pisces : Siluroidea) ; Matsya 4
42-51.
98 / R Dhanze and K C Jayaram
Jayaram K C and Dhanze J R 1981 Siluroid fishes of India, Burma and Ceylon. 23. The
specific status of Tachysums malabariucs (Day) (Ariidae) ; Bull zool Surv* India 4(1)
121-123
Misra K S 1976 The fauna of India and the adjacent countries, Pisces, 3 xxi -J- 349 pp, 2nd ed.
Munro I S R 1955 The marine and Freshwater fishes of Ceylon, Canberra, xvi -J- 349 pp.
Pearl R 1930 Introduction to Medical Biometry and Statistics., Philadelphia and London
Pillay T V R 1951 A morphometric and biometric study of the systematics of certain allied
species of the genus Barbus Cuv. and Val.; Proc. natn. Inst. Sci. Inida 17 (5) 331-348
Smith H M 1945 The freshwater fishes of Siam or Thailand ; Bull US. Natn. Mus.9 Washington
(188), xi -h 622 pp.
Smith J L B 1962 Fish from the cape described by Liethtenetein 1833 ; S. Afr. J. Sci. 58 39-40
Simpson G G and Roe A 1939 Quantitative zoology, New York
Simpson G G, Roe A and Lewontin R C 1960 Quantitative zoology New York, Rev. ed.
Snedecor G W 1956 Statistical methods applied to experiments in agriculture and biology, 5th
Ed. Anus Iowa, Iowa State Collge Press
Taylor W R 1978 FAO species identification sheets for fishery purposes Western Central Atlantic
(Fishing Area 31) Rome 1
Weber M and de Beaufort L F 1913 The fishes of the Indo-Australian Archipelago Leiden 2,
xx -f 404 pp.
Winter bottom R 1980 Systematics, osteology and phylogenetic relationships of fishes of tho
osteriophysan subfamily Anostominae (Characoidei, Anostomidae) ; Life Sciences Contri-
bution Royal Ontario Museum 123 Canada
Wongratana T and Bathia U 1974 FAO species identification sheets for fishery purposes Eastern
Indian Ocean (Fishing area 71) Rome 1
Graphs I-XX* Dice diagram showing the intergradation and divergence in respect
of 20 characters in the four species of the genus Arius. In each diagram, the hori-
zontal base line indicates the extreme range ; the vertical line in the middle represents
the arithmetic mean ; the solid area on either side of the mean is the extent of one
standard error ; the hollow area delimits: one stai dard deviatioi on either Side of
the mean ; the hatching lines represent the extent of standard deviation beyond the
extreme range.
Proc, Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 2, March 1982, pp. 9M12
© Printed in India.
Electron microscopic study of the sperm a the ca of Gesonula puttctifrons
(Aerididae : Orthoptera)
- ; & G PAL and D GHOSH
" ••; . ' , Department tof Zoology, University of Calcutta,'35, B. C. Road, Calcutta 700 019,
India
MS received 17 July 19-81 ; revised 1 January 1982
Abstract. The present transmission electron microscopic study of the spermatheca
of a common Indian grasshopper, Gesonula punctifrons, has highlighted the pre-
• sence of the glandular secretory cells (SGC) and ductule cell (DC) in the spermathe-
• .-. . cal epithelium and additionally the occurrence of muscle cells, tracheoles and haemo-
f ;•:..•< •.' i cytes. Both the former cell types are secretory in nature and probably their dis-
• i . . charges in the lumen of the cuticle-lined spermathecal duct or ductule vary in their
chemical nature. The ultrastructural evidence gives ample support to a concept
of a lysosomal control of the secretory materials prior to their liberation in the
lumen. The characteristic features of the plasma membranes of the secretory cells
clearly suggest their involvement in the transepithelial transport of ions and smaller
:, > , molecules across the basement membrane. A neuronal supply to the spermathecal
. -.:... >. ..wall is yet to be demonstrated to , explain the filling in and out of the male
..•....., gametes by this organ.
Keywords^ Transmission electron microscope ; spermathecal gland cell ; ductule
- cell ; rpugh endpplasmic reticula ; plasma membrane ; secretory granule ; micro-
yilli uk nucleus ; euchromatin ; heterochromatin ; ; male gamete; muscle cell;
s ,.>^j ., tracheole ; haemocyte.
1. Introduction
Pal and Ghosh (1981) have described earlier the cytological, histological and
his tochemical features of the spermatheca of Gesonula purtctifrons. The presence
of glandular cells in the spermatheca of several insects has. been documented (Imms
1957 ; Wigglesworth 1965 ; Adiyodi and Adiyodi 1975). According to Adiyodi
and Adiyodi (1975), 'spermathecal cells have the capacity to resorb excess germ
cells'. In recent years, conventional transmission electron microscopy (TEM)
$n4 sga^ning electron microscopy (SEM) have been employed to study the sperma-
th^ca^ .of insects like, Aedes aegypti (Clements and Potter 1967 ; Jones and Fisch-
nian 197())9 Sitophiltis granarins (Tombes and Roppel 1971), Dytiscus marginahs
al 1972), Apis mellifera (Dallai 1975),- Drosophila melanogaster (Filosi
1975), Tenebrio m&litot (Happ.and, Kfe.pp.1975), etc. These studies
issues with regard to the homology; .ar;d analogy of the spermathecae
insects. (Huebner 1980), the n^ture,,pf{ secretion (Copland and King
99
100 S G Pal and D Ghosh
1972 ; Filosi and Perotti 1975), the possible role in oogenesis (Dumser 1969;
Bouletreau -Merle 1977), etc. The present paper would provide a comprehensive
description of the spermathecal epithelium of G. punctifrons as seen under the
TEM.
A detailed account on the fine structural morphology of the spermathecal epi-
thelium, musculature, tracheoles and innervation is necessary before forwarding
an explanation for the mechanism(s) of controlled movement of spermatozoa
into and out of the spermathecae. The mode or style of functioning of the sper-
mathecae either in the yellow fever mosquito, or in the Chalcids or in cockroaches
is not free from confusion (Jones and Wheeler 1965a, b; Dent 1970; Jones and
Fischman 1970 i Copland and King 1972).
According to Pal and Ghosh (1981) the spermathecal epithelial cells of G. punc-
tifrorts are highly active and secrete copious, amount of mucoprotein. Usually
osmiophilic and PAS-positive materials appear juxta-nuclearly and these coalesce
apically to be subsequently transported to the lumen of the spermatheca by means
of cuticle lined ductules (Clements and Potter 1967; De Camargo and Mello 1970;
Poole 1970). It has been claimed by Lensfcy and Ailimot (1969) that some frac-
tions of the haemolyrnph proteins migrate to the spermathecal fluid in honey-bee.
An alternative route of release of some unknown spermathecal contents or factors
responsible for the growth of the female gonad in insects has been suggested by
Dumser (1969) and Boggs and Gilbert (1979).
According to Huebner (1980) the ultrastructural features of the spermathecae
of Rhodniits protixus differ remarkably from those of other insects studied so far.
The characteristic presence of apically situated secretion-loaded tubular inpocke-
ting or invagination in the glandular cells is a novelty and is not represented widely.
Furthermore, this apical cone lacks a cuticular lining. Therefore, it is suggested
that a systematic inventory on the similarities and dissimilarities of the sperma-
thecal epithelium in insects may indicate its true nature in reproduction, its rela-
tion to other ectodermal invaginations/glan ds etc . Gupta and Smith (1969) showed
in Perlplaneta americana the presence of nerves and my oneural junctions in the
striated muscles of spermathecae which could be analogous to the myoepithelium
of newt spermathecae (Dent 1970). A future publication would include the map-
ping of the fine structural details of the muscle cells, haemocytes and the nerve-
supply (?) of the spermathecae of Gesonula punctifrons.
2. Materials and methods
Spermathecae from the adult female grasshoppers were dissected in insect Ringer
solution and small pieces were fixed in 1% ice-cold glutaraldehyde in 0- 1 M phos-
phate buffer (pH 7-2-7-4) for one hour (Sabatini et al 1962) at 4°C. Subse-
quently these were washed twice in the buffer and post-fixed in 1% OsCMndis tilled
water at room temperature for two hours. After double fixation, tissue pieces
were dehydrated in ethanol with or without uranyl acetate and embedded in plastic
capsules with araldite mixture (Luft 1961). Ultrathin sections were cut on a LKB*
ultrotome with glass-knives and stained with uranyl acetate and lead citrate (Rey-
nolds 1963) and viewed under a transmission electron microscope (Siemens Ehni-
sfcop* I) with nafcgd copper grids.
Study of the spertnatheca of G> punctifrons
101
3. Observations
Figure 1 gives the essential features of the spcrmuthccal glandular cells (SGC) and
the peri-luminal diactule cells (DC). The glandular epithelial cells (SGC) of
the spermatheca of Gesonula punctifrons rest on a thick basement membrane (BM)
which. is supported by an underlying layer of deep striated muscles (MC) and
supplied by fine branches of the tracheoles (T) (figure 2). The tracheoles have a
diameter 0-3 /*. The basal plasma membranes of these cells make extensive and
characteristic infoldings, while the lateral plasma membranes run slightly unevenly
leaving a minimum of intercellular space. The entire thickness of two lateral
plasma membranes and the space between them is 340 A. Apically the cell
membrane forms numerous brush-border like processes around the cuticle-lined
lumen of the spermathecae. The nuclei (N) are large (10 ju in diameter), covered
by a double-layered nuclear envelope (NE). The outer leaflet of the nuclear
envelope is studded with ribonucleo-protein particles. Frequently, a medium-
sized nucleolus (NCL) is observed within the nucleus. The en tire nucleoplasm
shows uniform but moderate electron density. However, small but regularly-
sized dense-particles are observed at the boundary between the nucleolus and the
MC
Figure 1. Semi-diagrammatic representation of the iiltrastructural features asso-
ciated with the spermathecal glandular cell (SGC) and the ductule cell (DC) of
Gesonula punctifrons. Both muscle cells (MC) and. tracheoles (T) surround -the
spermathecal epithelium ,
102 S G Pcd and D Ghosh
nucleoplasm. These dense mtranucleolar RNt>' (^particles measure about 300 A
in diameter (figure 3). .. - «««\ " 'i
The cytoplasm is full of rough surfaced endoplas'mic reticula (RER), ; several
of which appear in the form of Ovojd or spherical bodies containing electron
dense materials. These measure about 0-3 /* in /diametef. Associated with
these small bodies there occur many medium-sized membralne^ound dense bodies
with slightly granular peripheral zones! These measure ab'out 0-9^ in diameter.
The rest of the contents, of these bodies has similar electron opacity as those
of the RER vesicles. Supranuclearly the cytoplasm contains many membrane-
delimited pleomorphic entities (figure 2)*. These are filled' with 'a grammar matrix,
corpuscular dense microstmctures, membranous profiles and ffiicrovesicles. Some
of these bodies also occur in the apical cytoplasm of the glandular cells of the
spermatheca. There are several round or ^void mitochondria (M) containing
relatively fewer cristae.
Topographically the ductule cells (DC) appear in groups surrounding the lumen
(L) of the spermatheca (figures 1 and 4). Further, the ductule cells (DC) are mono-
nucleated cells which are characterized by the dense nuclei with irregular
outlines. Both densely-staining heterochromatin and lightly staining euchro-
matin are present within the nuclei of these cells. The nuclear envelope has a thick>
ness of 300 A. Nucleolus may be present or absent.' Occasionally pores, on the
nuclear envelope are observed. The cytoplasm 'of these cells is characterized by
the presence of numerous large rounded mitqcKondria and hiyelin bodies (MB)
having a diameter 0-5 /*. "Myelin bodies consist' of whotls 6f fine membranes-
Near the lumen the cell apices are thrownlnto numerous; microvilli which surround
the ductule. A single microvilli tfus process has '& diameter pf 0- 1 //. Laterally
the plasma membranes show extensive ifttetdigitatfons and folds to increase the
surface area, though the intercellular space is exceedingly delimited. The total
width of two lateral plasma membranes "and the intercellular space varies from
300 to 350 A (figure 4). Besides^ the cytoplasm has fewer ribosomes, RER and
SER. A few vesicles with varying contents are usually observed in these cells.
The paragonadial haemocoele is filled with numerous haemocytes, muscle cells
and the tracheoles. The haemocytes" possess prominent rounded nuclei with
numerous cytoplasmic granules (figure 5). There is only a particular haemocyte
which is very common around the spermatheca of Gesonula piinctifrons. These are
rounded or rarely irregularly outlined cells occurring between the muscle cells and
the tracheoles reaching the spermathecal wall. Infrequently binucleate haemocytes
are observed (figure 6). Usually the 'rfueleus is 7 n in diameter. Both euchrcr
matin and heterochromatin are ^istingiushe^ in these nuclei. The cytoplasm is
populated by three different types of gtaiitiles r (a) small, dense bodies, measuring
approximately 500 A in diameter, (6) intermediate type of dense granules mea*
suring about 1-2 n in dia;meter and (c) a larger variety of 2-5 /* in diameter,
These three classes of intracytoplasmic granules are unifofniay ''distributed within
the haemocyte. The larger type' of granules have characteristic electron-lucent
zone of separation between their membranes and the moderately granular con-
tents. The rest of the cytoplasm is occupied by mitochondria and rough-surfaced
endoplasmic reticula. A clear-cut Golgi apparatus has not been seen in these
preparations.
Study of the spermatheca of G. punctifrons
103
Figure 2. A low magnified electron microphotograph of the spermathecal glandular
cells (SGC) detailing the different cellular components. Presence of numerous RER
vesicles and a large nucleus is a constant feature, x 6,500.
104
G Pat and D Ghosh
Figure 3. A large electron micrograph, of the nucleus (AO and the perinuclear
cytoplasm of the SGC showing nucleolar peculiarities and the cytoplasmic large
dense pleomorphic bodies (arrows), x 16,000.
Study of the spermatheca of G. punctifrons
105
1
A
Figure 4. Electron micrograph detailing the nucleated ductule cells surrounding
the lumen (DTL), the interdigitating lateral plasma membranes (LPM) and myelin
bodies (MB). X 6,000.
S G Pal and D Ghosh
Figure 5. Haemocytes (H) and muscle cells (MC) populate the para-spermathecal
coelome of grasshoppers. Fine structures of haemocytes and in particular the
cytoplasmic granules (CG) are apparent in this micrograph, x 3,200.
Study of the spermatheca of G. punctifrons
!07
Figure 6. Electron micrograph detailing the ultrastructural features of a binucleate
haemocyte (H). X 3,200.
Study of the spermatheca of G, punctifrons
4. Discussion
According to Adiyodi and A&yodi (1975) and Huebner (1980) the glandiular cells
of the spermatheca are characterized -by lateral interdigitations between the adja-
cent cells and the basally situated large ovoid nuclei. -Besides, ribosome rich cyto-
plasm in Aedes aegypti (Clements imd Potter 1967) or basophilic cytoplasm in
Apis meltifica (De Camargo and Mello 1970 •; Poole 1970) is an essential feature
for the protein synthesizing glandular cell in the spermatheca of insects. The im-
portance of a large nucleus with prominent nuc-leolus, numerous rough endoplasmic
reticula, Golgi apparatus, etc. are well-known: for cellular protein synthesis
(Palade et al 1962). We have not observed a Golgi apparatus, in the spermathecal
gland cells of G. punctifrons but this does not mean that it is definitely absent.
Huebner (1980) has described the presence of Golgi apparatus in the sperma-
thecal epithelium of Kkodnius prolixus.
This paper records the occurrence of two distinct cell types in the epithelium of
the spermatheca of an Indian grasshopper. Moreover, the fine structural diffe-
rences between these cells and their topographic distributions clearly suggest their
long morphogenetic separation and functional specialization. Both these cells
are partly or entirely secretory and glandular but they sharply differ in several
features and probably also in their secretory products. The mechanism of release
or drainage of the secretory products -by these cell types presumably differ due to
the presence of a cuticle-free cluctule in one case and the presence of extensive
apical plasmalemmal infoldings in the large glandular cell. Apparently there is
no comparable organelle as that of the apical imagination as described by Huebner
(1980) for Rhodnius prolixus. '* However, it is hot clear whether the intracellular
canaliculi of Apis mellifica ideally correspond with the cuticle-lined due tule observed
by several workers (Copland and King -1972). The due tule cells" of 'the sperma-
theca of G. punctifrons are somewhat low with prominent lobate nuclei containing
dense chromatin masses. There are swarms of mitochondria around the micro-
villi of these cells. The lumen is cuticle-free. The cytoplasm possesses' several
small-sized, membrane-bound dense mierostructures containing; secretory materials.
Apart from these, there also occur a? few myelin bodies, cells release their products
through the smooth-surfaced microvesicles to the ductule^ the lumen of which
demonstrate the presence of "moderately electron dense granular substances. It
is likely that these cells liberate chemically different substances from the true sper-
mathecal glandular cells "(SGG). There are, however, claims that spermathecae
in different insects liberate dissimilar chemical moieties to their lumen (Clements
and Potter 1967 ; Bhatnagar and Musgrave 1971 ; Pilosi and Perotti 1^975). The
secretory products may be mucoprotein or lipoprotein. It is still unsettled Whether
a similar situation exists in a species of insect. The discharge of a sperm-activa-
ting factor from the spermatheca of Eurytomidae has been reported by Copland
and King (1972).
On the contrary, the sperinathecal glandular cells of grasshoppers are tall epi-
thelial cells with narrow width and a large ovoid basal nucleus . TThe basal plasma
membrane of these cells show deep and elaborate infolding. These may be in-
volved in active transport of ions and probable absorption of" some protein frac-
tions of the haemolymph. Similar claims have been separately reported in Peri-
"plarteta americana (Gupta and Smith 1969) and in Apis mattifera (Lensky arid
110 S G Pal and D Ghosh
Allimot 1969). A minimum of intercellular space over the lateral plasma mem-
branes is suggestive of a low diffusion of ions and small molecules. However*
this cell gives an unmistakable evidence for the synthesis of proteins, likely to be
transported to the lumen of the spermatheca. Numerous RER vesicles gradually
enlarge and store granular materials. These slowly loose the surface ribosomes
from their membranes and attain peripheral condensations as they enlarge to form
medium and large sized secretory spheres or droplets. Supramiclearly the cyto,
plasm shows the presence of a different class, of pleomorphic organelles. It remains
to be resolved in future whether these entities are the later developmental stages
of the secretory spheres or an entirely new class of sub-cellular bodies or vesicular
organelles as described by Copland and King (1972). However, both these, classes
of the secretory bodies move apically and ultimately release their contents by means
of exocytosis (?) near the apical foldings to the spermathecal lumen. It is hardly
known how the intracellular transport and the direction of the secretory products,
etc., are regulated by means of the microtubules as reported by Huebner (1980)
or by the intervention of a lysosome system to control the overproduction of the
secretory materials (Smith and Farquhar 1966).
• Jones and Fischman (1970) have given the ultrastructural details of the plasmato-
cytes occurring in the vicinity of the spermathecal complex of Aedes aegypti. Our
description of the haemocytes from, the para-spermathecal haemocoele of the
grasshoppers is suggestive of their granular nature; and additionally the presence
of a large ovoid nucleus and the absence of the pseudopodial extensions justify
that these belong not the plasmotocytic cell type. Again the spherule cells and
the cytocytes both possess granules which enclose several microtubular profiles
(Ratcliffe and Price 1974). These workers have further suggested that it is difficult
to clearly identify the various haemocytes of the insects under.both the light and
the electron microscope. However, it is extremely premature at this stage to indi-
cate the active participation of these haemocytes in the adult reproductive struc-
tures of insects.
From the data presented here and also from those reported earlier (Jones and
Fischman 1970 ; Huebner 1980 ; Pal and Ghosh 1981) it is abundantly clear that
the spermathecal epithelium varies quite strikingly from the distal portion to the
duct. But in Gesonula pimctifrons the spermathecal gland cells (SGC) have unique
distribution both in the proximal and distal regions as well as in the duct zone.
An extensive examination of the sectioned materials both with light and transmis-
sion electron microscope (TEM) may provide information on the histological and
subcellular transitions in the spermatheca of grasshoppers. This could be extended
to include the cuticular intima, muscles, haemocytes; tracheoles and nerves so that
a comprehensive account on the insect spermatheca may be established.
AckBowlsdgsmqnts
The authors are grateful to Prof. K C Ghose for providing the laboratory faci*
lities and to late Prof. t> N Raychoudhuri for encouragement, One of them
(DG) is indebted to the authorities, of this University for the grant of a junior
research fellowship to him in collaboration with the UGC, New Delhi. They
Study of the spermatheca of G. punctifrorts ill
are particularly thankful to the Saha Institute of Nuclear Physics, Calcutta for
valuable help and cooperation during this study.
References
Adiyodi K G and Adiyodi R G 1975 Morphology and cytology of the accessory sex glands of
invertebrates ; Int. Rev. Cytol. 43 353-398
Bhatnagar R D S and Musgrave A J 1971 Aspects of hi stophysiology of the spermathecal gland
of Sitophilus granarius (L.) (Coleoptera) ; Can. J. Zool. 49 275-277
Boggs C and Gilbert L 1979 Male contribution? to egg production in butterflies: Evidence for
transfer of nutrients of mating ; Science 206 83-84
Bouletreau-Merle J 1977 Role des spermatheques dans 1'utilisation du sperme et la stimulation
de I'ovogenese Chez Drosophila melanogaster ; /. Insect Physiol. 23 1099-1104
Clements A N and Potter S A 1967 The fine structure of the spermathecae and their ducts
in the mosquito Aedes aegypti ; /. Insect Physiol. 13 1825-1836
Conti L, Cioft-Luzzatto A and Autori F 1972 Ultrastructural and htstochemical observations
on the spermathecal gland of Dytiscus marginalis L. (Coleoptera) ; Z. Zellforsch. 134 85-96
Copland M J W and King P E 1972 The structure of the female reproductive system in the
Eurytomidae (Chatcidoidea : Hymenoptera) ; /. Zool. 166 185-212
Dallai R 1975 Fine structure of the spermatheca of Apis mellifera ; /. Insect Physiol. 21 89-109
De Camargo IMF and Mello MLS 1970 Anatomy and histology of the genital tract, sper-
matheca, spermathecal duct and glands of Apis mellifica queens (Hymenoptera : Apidae) ;
Apidologie 1 351-373
Dent J N 1970 The ultrastructure of the spermatheca in the red spotted Newt. ; /. Morphol.
132 397-424
Dumser J B 1969 Evidence for a spermathecal hormone in Rhodnius prolixus (Stal) ; M.Sc.
Thesis, Biology Department, McGill University, Montreal, Quebec, Canada
Filosi M and Perotti M E 1975 Fine structure of the spermathecae of Drosophila melanogaster
Meig ; /. Submicrosc. CytoL 7 259-270
Gupta B L and Smith D S 1969 Fine structural organization of the spermatheca in the
cockroach Periplaneta americana ; Tissue Cell 1 295-324
Happ G M and Happ C M 1975 Fine structure of the spermatheca of the milkworm beetle
(Tenebrio molitor L.) ; Cell Tissue Res. 162 253-269
Huebner E 1980 Spermathecal ultrastructure of the insect Rhodnius prolixus ; J. Morphol. 166
1-25
Imms A D 1957 A general text-book of entomology (London : Methuen) p. 325
Jones J C and Fischman D A 1970 An electron microscopic study of the spermathecal com-
plex of virgin Aedes aegypti mosquitoes ; /. Morphol. 132 293-312
Jones J C and Wheeler R E 1965a Studies on the spermathecal filling in Aedes aegypti (Linneaus).
I. Description ; Biol Bull. 129 134-150
Jones J C and Wheeler R E 1965b Studies on the spermathecal filling in Aedes aegypti (Linneaus).
II. Experimental ; Biol Bull. 129 532-545
Lensky Y and Alumot E 1969 Proteins in the spermathecae and haemolymph of the queen
bee (Apis mellifica L. var. lingustica Spin) ; Comp. Biochem. Physiol. 30 569-575
Luft J H 1961 Improvements in epoxy resin embedding methods ; /. Biophys. Biochem. CytoL
2 409-414
Palade G E, Siekevitz P and Caro L G 1962 Structure , chemistry and function of the pancreatic
exocrine cell; Ciba Foundation Symposium on the Exocrine Pancreas (London;
J and A Churchill Ltd.) pp. 23-49
Pal S G and Ghosh D 1981 Functional histomorphology of the spermatheca of an Indian
grasshopper, Gesonula punctifrons (Acrididae : Orthoptera) ; Proc. Indian Acad. Sci. (Anim.
Sci.) 90 161-171
112 S 6 Pat and i> Ghosh
Poole H K 1970 The wall structure of the Honey Bee Spermatbeca with comments about its
function ;, Ann. Em. Soc. Am. 63 1625-1628
Ratclifle N A and Price C D 1974 Correlation of light ancj ..electron microscopic hemocyte
structure in the Dietyoptera ; J. Morph. 144 484-498
Reynolds E S 1963 The use of lead citrate at high pH as an electron opaque stain in electron
microscopy ; /. Cell Biol. 17 208-212
Sabatini D D, Bensch K G and Barrnett R J 1962 New Fixatives for Cytological and Cyto-
chemical Studies. Proc. 5th Intern. EM. Cong. Vol. 2, L-3, Philadelphia
Smith R E and Farquhar M G 1966 Lysosome function in the regulation of the secretory
process in cells of the anterior pituitary gland ; J. Cell Biol 31 319-347
Tombes A S and Roppel R M 1971 Scanning electron microscopy of the sperrnatheca io
Sitophilus granarius (L.) ; Tissue Cell. 3 551-556
Wigglesworth V B 1965 Tlie principles, of insect physiology (London : Methuen)
Abbreviations
BBP w Brush border processes
BM „. Basement membrane
BPM .„ Basal plasma membrane
C .. Cuticle
CO .. Cytoplasmic granule
DC .. Ductule cell
DTL . . Ductule lumen
H . . Haemocy te
L .. Lumen of the spermathec'
LPM . . Lateral plasma membrane
M .. Mitochondrion
MB . . Myelin body
MC . . Muscle cell
N .« Nucleus
.NCL .. Nucleolus
NM . * Nuclear membrane
RER . . Rpugh endoplasmic reticvlum
SGC * . Spermathecal glandular cell
SV . . Secretory vesicle
SSV .. Small smooth surfaced inicrovesiclc
T . . Fine tracheole.
Proc. Indian Acad. Sci. (Anim. ScL), .Vol.. 91* No. 2. Match 1982, pp. 113-120.
© Printed in India.
Histology and histochemistry of adrenal glands of Indian mongoose
Herpes tes edwardsii edwardsii (Geoffrey)*
P VARADA RAJUt and K HANUMANTHA RAO
Department of Zoology, Andhra University, Waltair 530 003, India
t Present Address : Department of Zoology, M.G. College,
Atroyapurarn 533 235, India
MS received 3 October 1980 ; revised 15 October 1981
Abstract. The histology of the adrenals of the mongoose Herpestes edwardsii
edwardsii has been studied. Three layers in the cortex, namely zona glomerulosa,
zona fasciculata and zona reticalaris and central medulla surrounded by the cortex
have been observed.
Employing histochemical techniques it was revealed that the cortex is rich in
glycoproteins, lipids and protein bound Amino groups. It has moderate amounts
of proteins containing sulphydrit and disulphide radicals and tyrosine. Tryptophan
has not been detected.
Negligible amounts of mucopolysaccharides were detected in the medulla.
Aspects dealing with the occurrence of carbohydrates, pioteins and lipids in various
regions of the cortex are discussed.
Keywords. Histology ; histochemistry ; adrenal glands ; Herpestes edwardsii
edwardsii.
I. Introduction
Studies on the smaller terrestrial mammals have been generally confined to rodents.
Carnivores seem to have been neglected probably due to the difficulties encountered
in their collection and rearing. Histology of adrenal glands have been studied by
Meyers and Charipper (1956), Pauly (1957), Holmes (1961), Houser et al (1962) and
McKeever and Tomich (1963). Hunt and Hunt (1959) studied the glycogen con-
ten tin the adrenal glands of rats at different ages and a detailed account of glyco-
gen in adrenals was furnished by Girod (I960). Sinha and Ghosh (1961) gave
information on the adrenal cortical cytochemistry in the pigeon. Prasad and
Yadav (1974) made observations on the histologtcal and histochemical details of
the adrenal glands of the Indian buffalo. Recently Carole et al (1979) studied
the histologtcal details of adrenals in newborn alpacus. Our knowledge of the
adrenal glands of carnivorous wild mammals is meagre. In this paper an attempt
* This paper was "presented in Second All India Symposium on Comparative Endocrinology
held at Manasagangotri, Mysore in 1976.
113
P. (B)-2
114 P Varada Raju and K Hanumantha Rao
has been made to bring out histological and histochemical aspects of the adrenal
glands of the Indian mongoose Herpestes edwardsii edwardsii.
2. Materials and methods
Mongooses were obtained from villages nearby Visakhapatnam town and were
acclimatized to laboratory conditions. Adrenal glands were removed from the
animal and were fixed in Zenker or Bouin's or Susa or formol-calcium. After
routine procedures of dehydration and embedding, 5 to 7// thick sections were
cut. Heidenhain's Azan, Mallory's triple stains were employed to study the histo-
logical details. The histological and histochemical techniques were adopted from
Gornori (1952), Lillie (1954), Carleton and Drury (1957), McManus and Mowry
(1960), Gurr (1962), Barka and Anderson (1963), Humason (1965), Pearse (1968),
Culling (1974) and Bancroft (1975).
3. Observations
Anatomically the adrenals of mongoose appear quite regular in shape. The left
gland is relatively long and flattened whereas the right one is thick with latero-
ventral angular borders. Both left and right adrenals lie closely pressed to the
dorsal body wall anterior to the kidneys. The caudate lobe of the liver envelopes
the right gland whereas the left one is free and is lightly pressed by the pancreas
and stomach.
Two regions could be distinguished in the adrenals the outer cortex and central
inedulla. The gland is ensheathed by thin fibrous capsule. The cortex has 3
layers, the outer zona glomerulosa, middle zona fasciculata and an inner zona
reticularis.
The capsule is formed by a combination of collagen ous, elastic and reticular
fibres. Smooth muscle fibres are also associated with the connective tissue.
The zona glom^Fulosa has a cellular structure and is delineated from the capsule
on the outer side and the zona fasciculata from the inner side. The cytoplasm is
basophilic in nature. In this zone the cells are more columnar and arranged in
vertical single rows. The cells with a single nucleus which have usually one
nucleolus each but some with double nucleoli could be seen occasionally.
The zona fasciculata is the major portion of the cortex with cuboidal cells and
some columnar cells. The cells are polygonal in shape and arranged in radiating
columns. The cytoplasm is homogeneous and the nucleus is spherical and centrally
situated. The size of the nuclei increases progressively towards the medullary part.
The cells and their nuclei are larger than those of zona glomerulosa. The cells
usually display a single nucleus with a nucleolus, but double nucleated cells also
occur (figure 1).
The histological details of zona reticularis are in agreement with those described
for other mammals. This region is interspersed with sinusoids of various sizes
giving the appearance of a broken network.
To mafcs a clear-cut demarcation between the zona fasciculata and zona reti-
cularis is rather difficult (figure 2). Zona reticularis is well developed in adulte than
in young ones and a distinct demarcation between the cortex and medulla is notice-
Histology of adrenal glands of Indian mongoose
115
11
rS M
!i
j^j o
'00
«
§
.x o
Ri PH
C *
tD
W N/ C/5
.2P to ctf
o ^*
0 §
•a N
c "d
|g
^»
"3 .5
On CO
8
i o ;>
PlS
i Cl c/5
116
P Varada Raj'i and K Hanumantha Rao
I
8|
3 *O
&fi v
5-2
Histology of adrenal glands of Indian mongoose
117
lable in adult animals. There is a mixing up of medullary cells with reticularis
cells, but the extension of these cells is limited to zona fasciculata only (figure 3).
The medullary cells are arranged in irregular rows and the cells are smaller
than those of cortical cells. The cells are arranged in irregular groups of 2 to 10
and mainly surrounded by thick strands of interlocking connective tissues and are
lightly stained with histological stains when compared to cortex (figure 4).
4. Histochemical observations
The medulla in general gives a moderate reaction with bromophenol blue and
Millon's reactions but tryptophan and arginine are absent as evidenced by negative
response to p-dimethylaminobenzaldehyde nitrite and Sakaguchi reactions respec-
tively (table 1). Medulla is moderately positive to ninhydrin/Schiff and chlora-
mine-T/Schiff when compared to other protein reactions such as KMnO4/AB,
ferric ferricyanide indicating that large amounts of protein bound amino groups
rather than disulphides and sulphydrils, are present. The medulla is positive to
lipids (Sudan black B) and phospholipids (copper phthalocyanin). With Congo
red it stains moderately indicating the presence of glycoproteinsi
Table 1. Higtochemical reactions of the adrenal glands.
Test applied
Medulla
Zona
Zona
Zona
Capsule
glomorulosa
fasciculata
reticularis
PAS
4-
+ 4-
+ -h
4-4-
4-4-1-
PAS/Acetylation
4~
-f-h
4-4-
4-4-
4-4-h
PAS/Deacetylation
4~
-h-f
•f-f
4-4--
4-4-4-
PAS/Methylation
4-
-H
4-
• 4-4-
+,+,
Schiff '$ alone
4-
+
-H
-H
4-
Alcian blue-1 pH
d-
±
±
±
±
Mcian blue^2*5 pH
-h
+
+
4-
+•
Congo red
4~4~
. +•
4-4-
4-
4-4-4-
Bromophenol blue
4-4-
+4-
+•4-
+•4-
4-4-
BPB/Vanslykes
-
•—
—
—
_
Millon's reaction
• 4-4-
4-4-
4-4-
+•4-
4-4-4-
DMAB/Nitnte
-
—
—
__
—
Sakaguchi
-
• —
—
__
—
KMnOJalcian blue
4-
• 4-4-
4-
4-4-
4-4-
Ninhydrin-Schiff
-f-f
+4-
+4-4-
4-4-
4-4-4-
Chloraraine-T/Schiff
+4-
4-f
4-4-4-
4-4-
+ 4-4-
Ferric ferrycyanide
4-
++•
4-4-
4-4- •
+•+•
Sudan black B
++•
4-4-4-
4-4-
4-4-
+ + +
Copper phthalocyanin
+4-
4-4-
+ + +
+ 4-
+ + +
+ 4- + -Strongly
positive ; + +
= IVloderately
positive ;
+ = Faintly
positive ;
*- =* Negative.
118 P Varada Raju and K Hanumantha Rao
Zona glomenilosa is moderately positive to all protein tests showing their pre-
sence in small quantities. Protein bound amino groups and basic proteins like
tyrosine are present in little amounts, but tryptophan and argiriine are absent.
This is the lipid rich part of the cortex. Its intense staining with Congo red indi-
cates that it is rich in glycoproteins. But negligible amounts of Mucopolysaccha-
rides are noticed. The zona fasciculata is rich in protein bound amino groups
but tyrosine is present only in moderate amounts. This part of the cortex like
other parts is devoid of tryptophan and arginine. Moderate amounts of sulphy-
drils and disulphides have been localised. Phospholipids are abundant in this
area. No mucopolysaccharides have been detected, but glycoprotein is present
in abundance.
The reticular zone is rich in tyrosine but is devoid of mucopolysaccharides or
bzsic proteins containing arginine and tryptophan. The protein bound amino
groups, sulphydrils and disulphides are present in moderate amounts. As in the
case of adrenals of other mammals, this region displays mild amounts of lipids.
5. Discussion
In the zona glom^rulosa the presence of double nucleolated cells was also observed
in the Indian buffalo by Prasad and Yadav (1974). In the ferret (Holmes 1961)
and in the Indian buffalo (Prasad and Yadav 1974) it was observed that zona
glomsrulosa took lighter stain with histological stains than the cortical layers, an
observation which is in agreement with the present findings. This may be due
to glucocorticoids that are secreted by the zona glomerulosa which take lighter
stain.
The presence of faintly stained cytoplasm in this zone also agrees with the condi-
tion reported by Meyers and Charipper (1956) for the golden hamster, by Hewer
and Foster (1966) for man. Holmes (1961) for ferret and Houser et al (1962) for
Panama monkeys.
McKeever and Tomich (1963) observed an arc of cells at the capsular end in
Herpestes auropwtctatus in mature females but this condition could not be seen
in the present study. The zona fasciculata occupies the major portion of the
cortex in mongoose as is the case in bulls (Cupps et al 1954; Das et al 1965) and
in Indian buffalo (Prasad and Yadav 1974). This is attributed to the fact that
this may be synthesizing and secretory zone for steroidal hormones. McKeever
and Tomich (1963) reported that in Herpestes auropuitctatus there is a clear
demarcation between inner and outer fasciculata in sexually active female, which
could not be corroborated in our study on Herpestes edwafdsii edwardsii, as our
observations were made on females in captivity. Estrogen secreting activity may
augment the bulk and reactivity of zona fasciculata which actually forms a band
of cells and could be considered the estrogen secreting zone. tHiring the course
of development this estrogen secreting zone may extend into zona glomerulosa
side constituting distinct zona intermedia— a condition which occurs in Indian
buffalo. .
Progressive increase in size of the cells in deeper parts of zona fasciculata has
been reported by Copenhaver (1964) in man and by Prasad and Yadav (1*74) in
Indian buffalo. The large* sizes of the nuclei and cells in zona fasciculata- are fa
Histology of adrenal glands of Indian mongoose 119
agreement with the -findings in bull and bullock (Gupps et al 1954; Sohal and
Chaturvedi 1962). However, Hartman (1959) found that the cells of this zone were
smaller than those of zona glomerulosa in the adrenal glands of the sloth.
The zona reticularis is comparatively better developed in adult animals than in
young ones, thus agreeing with the observations of Prasad and Yadav (1974) in
Indian buffalo. As far as. mixing up of cells in the medulla is concerned Holmes
(1968) also found this condition in Macaca mulatta but Prasad and Yadav (1974)
state that medullary cells migrate up to the level of zona glomerulosa.
The medulla in general is rich in lipids obviously because of the principal secre-
tions of medulla, adrenaline and noradrenaline. A positive ninhydrin/Schiff reao
tion is due to free amino group in adrenaline and noradrenaline. As a whole
the cortex is strongly positive to lipid stains, since gluco and adrenocorticoids
are lipids in nature. The cortex displays a comparatively more intense reaction
for proteins. After deamination with van Slykes reagent cortex as well as medulla
became negative to bromophenol blue and other basic protein tests,
Acknowledgements
The authors thank Dr K Shyamasundari, Department of Zoology, for her help
in histochemical investigations. One of us (PVR) thanks University (Grants
Commission for award of Junior Research Fellowship.
References
Bancroft J D 1975 Histochemical techniques (London and Boston : Butterworths)
Barka T and Anderson P J 1963 Histo chemistry theory , practical and bibliography. Hoeber
Medical Division (New York, Evanston and London : Harper and Row)
Carleton H M and Drury R A B 1957 Histochemical techniques for normal and pathological
tissues and the identification of parasites (London : Oxford University Press) pp. 1-343
Carole A, Samuel J, Sumer and Nathanielz P W 1979 Histological observations of the adrenal
glands of newborn alpacas (Lama pacos) ; Comp. Biochem. Physiol. A62 387-396
Copenhaver W M 1964 Bailey's text-book of histology, 15th edn. (Baltimore : Williams and
Wilkins) pp. 582-583
Culling C F A 1974 Hand-book of histopathological and histochemical techniques (London and
Boston : Butterworths)
Cupps P T, Laben R C and Mead S W 1954 Histology of the pituitary, testis and adrenal in
relation to reproduction in the btill ; /. Dairy Sci. 37 1074-1087
Das L N, Mishra D B and Brswal G 1965 Comparative histological study of adrenal and thyroil
glands of the bull and the bullock ; Indian Vet. J. 42 824-830
Girod C 1960 Adrenal glycogen ; C.R. Soc. Biol 154 2049-2051 (Vide Excerpta Med. 16 229 ;
1962)
Gomori G 1952 Microscopic histo chemistry, Chicago University Press
Gurr H 1962 Staining : Animal tissues, practical and theoretical (London : Leonard Hill)
Hartman F A 1959 Notes on the adrenal of the sloth ; Anat. Rec. 133 105-114
Hewer E E and Foster C L 1966 Hewer's text-book of histology for medical students (London :
William Heinmann Medical Books Ltd.) pp. 204-210
Holmes R L 1961 The adrenal glands of the ferret, Mustela putorius ; /. Anat. 95 325-336
Holmes R L 1968 The adrenal glands of Macaca mulatta, with special reference to the cortico-
medullary zone ; /. Anat. 103 471-477
120 P Varada Raju and K Hanumantha Rao
Housfr R G, Hartman F A, Knouff R A and McCoy F W 1962 Adrenals in some Panama
monkeys ; Anat. Rec. 142 41-52
Humason G L 1965 Animal tissue techniques (San Fransisco and London : W H Freeman)
Hunt T E and Hunt E A 1959 Glycogen in the adrenal gland of rats at different ages ; Awt*
Rec. 133 537-551
Lillie R D 1965 Histopathological technique and practical histochemistry (New York : Blakiston
Div., McGraw-Hill)
McKeever S and Tomich P Q 1963 Observations on the adrenal glands of the mongoose ; Anat.
Rec. 147 163-170
McManus J F A and Mowry R W 1960 S taming methods : Histological and histochemical
(New York : Paul B. Hoeber)
Meyers M W and Charipper H A 1956 A histological and cytological study of the adrenal
gland of the golden hamster (Cricetus auratus) in relation to age ; Anat. Rec. 124 1-25
Pauly J E 1957 Morphological observation on the adrenal cortex of the laboratory rat ;
Endocrinology 60 247-264
Pearse AGE 1968 Histochemistry : Theoretical and applied 3rd edn. (Londor. : J A Churchill)
Prasad G and Yadava R C P 1974 Histological and histochemical studies on the adrenal
glands of the Indian buffalo ; Indian J. Anim. Sci. 44 243-248
Sinha D and Ghosh A 1961 Some aspects of adrenocornc?! cytochemistry in the donks*ic
pigeon; Enfokrinologie 16 270-280 (Vide Excerpta Mea. 16 742-743, 1962)
Sohal H S and Chaturvedi R P 1962 Adrenal glands of Indian buffalo • /. Anat. Soc. India
w 46
roc. Indian Acad. Sci. (Anim. Sci), Vol. 91, Number 2, March 1982, pp. 121-133.
J) Printed in India.
Effect of x-rays on the somatic chromosomes of the exotic fish,
rilapia mossambica
G K MANNA and R C SOM
Department of Zoology, Kalyani University, Kalyani 741 235, India
MS received 10 July 1981
Abstract. Male and female T. mossambica were x-raycd with 100 r and the meta-
phase chromosome aberrations in their gill epithelia were studied at 13 different
intervals against suitable control. The chromosomes of males appeared more
radio-sensitive than those of females. Among the diploid complement of 44
chromosomes, the individual type aberrations were non-random in both sexes. The
longest pair of chromosomes, taken as the marker pair, was found very highly
radio-sensitive, while the remaining 21 pairs as non-markers were somewhat
resistant to x-radiation when the observed and the expected numbers were subjected
to statistical analysis. The break in the marker chromosome was also non-randomly
distributed as the distal half had a significantly large number of breaks.
Keywords. Fish ; Tilapia mossambica ; x-irradiated chromosome aberrations ;
differential radio-sensitivity.
[. Introduction
n comparison to some insect and mammalian models, very limited studies on the
'adiation induced chromosome aberrations in fish have so far been carried out.
5uch studies have, however, dual importance because fish in general serve as an
mportant biological monitor in aquatic environment for the study of radiation
)ollution and secondly their stock could be improved through radiation induced
nutation and selection. Schroder (1973) reviewed the works on radiation induced
nutations in fish while Hickling (1962) reported the genetids and hybridization
effect of some fish including Tilapia. At the chromosomal level Kama et al
[1976) studied the chromosome aberrations in gill epithelia of the mosquito fish,
Oryzias latipes from 2 to 10 days, after radiation, while Pechkurenkov (1976)
itudied the chromosome aberrations in embryonic fish induced by chronic radia-
;ions. The do^dependent effects of x-rays on the frequency of mitosis in regene-
rating tail fin of O. latipes was studied by Hama and Egami (1977). Mong and
Bena (1979) also studied the effect of x-rays on chromosomes of mud minnow
using different doses. The present paper deals with the x-ray induced chromo-
some aberration in the fish T. mossambica with special reference to the study of
the differential radio-sensitivity of chromosomes between males and females and
between and within the chromosomes in each sex which were not studied before.
T. mossambica has been chosen not only for its easy rearing and handling, but
121
122 G K Manna and R C Som
also its nrtotic m^taphase complements containing a pair of conspicuously
large chromosomes which, as markers, served better to study the problem of the
intraand interchromosomal radio-sensitivity.
2. Material and methods
The herbivorous freshwater higher group of teleostean fish, Tilapia mossambica
Peters (Family Cichlidae, Order Perciformes) domestic to the rivers of East coast
of Africa was introduced to the Indian inland waters for its exotic habit of breeding.
They breed throughout the year almost every 2 months except in winter (see
Jhingram 1974). Specimens used in the present investigation were from the 4th
inbred generation raised by us. Before irradiation living male and female
specimens were acclimatized in the aquarium for a day or two. Immediately
after taking them out of the aquarium, their body was gently rubbed once with a
piece of dry cloth to remove surface water. They were then irradiated with the
dose of 100 r from the x-ray machine operated at 1 10 fcV, 4 mA with 1 mm aluminium
filter emitting 2-5r per second. After irradiation the specimens were stocked
into the aquarium for fixing their gills at different intervals. As controls unirra-
diated specimens of the same brood were kept in to another aquarium under similar
laboratory conditions. An hour before the fixation time each specimen was
intramuscularly injected with 0-1% colchicine solution at the rate of 2 ml per
100 gm body weight. No colchicine was injected if the fixation of the tissue
was to be done within an hour after irradiation. The gills, of each specimen
immediately after removal were minced in 1% sodium citrate solution and the
minced tissue was left into citrate solution for an hour at room temperature. The
tissue was then fixed in acetic -alcohol (1 :3) mixture for a brief period after
removing the citrate solution by centrifugation. The fixed tissue suspension
was taken on a slide and after air-drying the slide was stained with Giemsa stain
at pH 7-2 . The observations were made from the stained air-dried slides. .
3. Observations
3-1. Control series
The diploid number of chromosomes in both the sexes of T. mossambica was 44,
the sex chromosomes being undifferentiable cytologtcally (figure 1). With regard
to the morphology of the chromosomes different workers (Natarajan and Subra-
manium 1968 ; Hideo and Muramoto 1975 ; Prasad and Manna 1976 ; Manna
and Som, unpublished) were not incomplete agreement with one another excepting,
of course on the first pair of the longest subtelocentric chromosomes, referred to
here as the marker pair. The controversy was on the exact morphology of the
remaining 21 pairs of non-marker chromosomes. Their relatively small size and
variable length and disposition of the shorter arm caused confusion. Anyhow
none of the chromosomes was of the tnie metacentric type which helped us to
determine the cases of centric fusion leading to form metacentric chromosome in
the treated material (vide infra). Thus, without entering into any controversy
for our present analysis we put the first longest pair into the marker group and
Effect of x-rays on somatic chromosomes of T. mossambica 1 23
il It f « Vt Wf
tf
m
ft *± -& .'* ' • 4$
«*' '.f***tfa: :' * ' *''.&
Figures 1-13. Photomicro.graphs, part and full metaphases. 1. A normal comple-
ment in male (2n = 44), 2. Male karyotype, 3. Polyploidy, 4. Stickiness, 5. A
marker chromosome with a subchromatid break, 6-8. Each with a chromatid
break in a marker chromosome, 9. Two isochromatid and one chromatid fragment
of unknown origin, 10, 11. Each with a small metacentric chromosome formed
by the centromeric fusion of two non-marker chromosomes, 12, 13. Terminal
association and/or chromatid exchange between two chromosomes.
Effect of x-rays on somatic chromosomes of T. mossambica 125
the remaining 21 smaller pairs into the non-marker group (figure 2). Since the
first marker pair was about double the size of the second pair (figure 2), there
was not the least difficulty in identifying the first marker pair in any plate. This
marker pair formed 1/22 part in the haploid number and approximately measured
1/10 (average 15 •()//) of the total genome length (149 -6^). The second pair also
considered as marker chromosome (Hideo and Muramoto 1975) is, however, not
considered as its size difference from the 3rd pair is not very conspicuous (figure. 2).
In the control series out of 150 metaphas.es examined in each sex at each of the
13 intervals corresponding to the treated series (table 1), only 2 constrictions were
encountered at 96 hr in females. Thus these two were the individual type solely
found in a total of 3,900 metaphases examined in two sexes while there were meta*
phases with gross, effect found at all intervals. The frequency in the combined
data of two sexes was 5-0% in 5 min, 8-7% in 1 hr, 4-3% in 6hr, 6-0% in 12 hr,
8-0% in 24 hr, 8-7% in 48 hr, 3-3% in 72 hr, 7-0% in 97 hr, 5-0% in 120 hr,
6-0% in 144 hr, 5-0% on 7th day, 7-7% on 10th day, 3-7% on 15th day and
6-0% as average (table 1). The gross effect was mainly due to the stickiness of
chromosomes and the frequency fluctuated erratically.
3.2. SH-rayed series
In comparison to the control series, the gill epithelia of the x-irradiated specimens
contained various types of aberrations (figures 3-23). For the sake of convenience
**
*
V
18
19
22
23
Figures 14-23. Camera lucida drawings x ca. 3,000 showing some rearranged
metaphase chromosomes mostly with aberrations induced by x-rays. 14. One
marker chromosome with a terminal chromptid break 15. Each marker chromo-
some with a chromatid break, and three non-markers with terminal association
or chromatid exchange, 16. A marker chromosome with a proximal chromatid
break and a non-marker chromosome with a constriction, 17. A marker chromo-
some with two breaks in the same chromatid, 18. A marker chromosome with
isochromatid breaks, 19. A marker chromosome with a chromatid constriction,
20. A marker chromosome with beaded constrictions in one chromatid and the
other with a break while a non-marker chromosome with a chromatid break,
21. A chromatid gap in a marker, a chromatid break in the first non-marker
(2nd marker) chromosome and a fragment of unknown origin, 22, 23. Each with
a fragment of unknown origin while one (No. 22) also contained a chromatid
break in a non-marker chromosome and the other (No. 23) a centric fusion.
126 G K Manna and R C Som
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Effect of x-rays on somatic chromosomes of T. mossambica 127
polyploidy (figure 3), stickiness (figure 4), c-mitosis etc. were put under gross effect
in which the entire chromosome complement was affected while subchromated
(figures 5, 14), chromatid (figures 6^8, 14-47, 20, 21) and isochromatid (figure 18)
breaks, fragment of unknown origin (figures 9, 22, 23), translocation and fusion
(figures 10-13, 15), constriction (figures 16, 19, 20), gap (figure 21) etc. were put
under individual effect in which one (figures 5-8) or more (figures 14^21) chromo-
somes of the whole complement were involved. It appeared that the individual
type aberrations were mostly of the chromatid type. If the marker chromosome
was arbitrarily divided into 3 equal regions as proximal, middle and .distal from
the centromeric end, the chromatid breaks were somewhat localized in the distal
region because out of 106 breaks in the marker pair, 15 were in the proximal,
33 in the middle and 58 in the distal region against the expected number of 35- 3
breaks per region with random occurrence. The difference was statistically signi-
ficant at 1% level (%* = 26-40, d,f. 2). Thus, broadly speaking the distal half
was more radio-sensitive than the proximal half of the marker chromosomes.
However, in the present material such an analysis in the non-marker chromosomes
was not possible for the inherent difficulties with the morphology and size of
chromosomes. Definite translocation between the marker and non-marker
chromosomes except for som^ terminal chromatid association or exchange (figure 12)
was not encountered but centric fusion between two non-marker chromosomes
(figures 10, 11) was common. While scoring the data some individual type of
aberrations in the non-marker group might have escaped che to the inherent
observational difficulty for the small size and morphology of the chromosomes.
But the frequency of such an omission, if it occurred at all, would not exceed more
than 2%.
In presenting the data the different individual type aberrations were put into
one category e.g., siAchromatid, chromatid and isochromatid breaks as breaks
etc. while for the gross types all of them were put together (table 1). An analysis
of the data (table 1) indicated that chromosomes in irradiated males were affected
more than those in females because in the same number of 150 metaphases, the
number of aberrations was higher in males at all intervals and in their total except
at 5 min, and from 24 hr to 96 hr for breaks, except at 48 hr for fragment of
unknown origin and except at 12 hr, 96 hr and 144 hr for gaps and constrictions.
As the translocation data were limited, we made no comment. The statistical
analysis of the total data showed that the difference was below the significant
level because 3?2 =0-20, d.f. 1 for breaks, %* = 1-47, d.f. 1 for fragment of
unknown origin and Jif2 =0-74 for gaps and constrictions. In the combined
data of all individual type aberrations, it was also higher in males at each interval
except at 48 hr and 96 hr (table 1). In the total of all intervals, the males had
255 aberrations against 225 in females. The difference was also a little below
the significant level because the X2 value was 1 • 87 with 1 d.f. Therefore, on the
whole the higher frequency of individual type aberrations in males was somewhat
indicative that the sex factor might have some differential radio-sensitivity", but
the data needed be extended for further confirmation. That the sex factor
could have differential radio-sensitivity was supported by the fact that when the
number of affected metaphases which contained individual type aberrations was
Compared between the two sexes, it was found higher in males in 9 intervals while
128 G K Manna and R C Som
it was at par with females at 120 hr, 7th day and 10th day (table 1). In the total
1950 metaphases 177 were affected in males against 142 in females. The X2 test
gave a value of 3- 84 with 1 d.f., indicating that the difference was significant at 5%
level.
The number of affected metaphases with gross effects like polyploidy, stickiness
etc. was not significantly different in the two sexes. It was a little higher in males
at 6 out of 13 intervals and in the total (table 1). The 3T2 value was 0-08 with
1 d.f. which indicated that the difference was highly insignificant This was expected
because gross effect was mostly physiological in origin. The number of affected
metaphases with individual and gross type aberrations if combined, would be
higher in males in 8 out of 13 intervals and in the total (table 1). The JP2 test
showed that the value 2-17 with 1 d.f. was a little below the significant level.
Thus, though the analysis of the data of aberrations and the affected metaphases
did not conclusively prove, that the males and females responded differentially,
there were reasonable indications beyond doubt for the same.
The individual type aberrations did not show a regular mode of incidence in
both the sexes. The maximum number of the different types was mostly found
in 5 min (breaks, fragments in male, total) and 1 hr (gaps and constriction) which
reduced to nil on the 1,5th day or earlier (breaks) but the mode of decrease was
very erratic as number fluctuated oddly at different intervals (table 1). The
frequency of affected metaphases in male showed the same trend but in female the
maximum number was found at 48 hr. The occurrence of the affected metaphases
with gross effects was still more erratic as the maximum number of 27 was found
at 48 hr in males and 29 in females at 5 min and the effect continued in a lower
frequency in both sexes even on the 15th day (table 1). On the whole, the present
data showed that the individual type of aberrations did not continue up to the
15th day while the gross type continued longer and in both cases the frequency
fluctuated at different intervals (table 1). That the x-radiation induced a higher
frequency of chromosome aberrations and affected more metaphases was beyond
any doubt. The net increase in the individual type aberrations when the data
of two sexes were combined was 22-7% at 5 min, 21-0% at 1 hr, 12-7% at 6 hr,
17- 3% at .12hr, U-3%at24 hr, 19-3%at48hr, 12-0%at72hr, 15-7%- (2 constric-
tions in control) at 96 hr, 8-7% at 120 hr and 144 hr, 6-7% on 7th day, 3-3% on
10th day and nil on 15th day. The net increase in an average was 12-4%. On
the other hand, the net increase in the frequency of total affected metaphases over
the control was 25-3%, 15-6%, 10-0%, 24-3%, 11-3%, 22-3%, 20-4%, 18-0%,
15-3%, 7-0%, 0-3%, 7-3% and 2-6% respectively in U intervals and 13-8% in
the average (table 1).
3-3. Non-random distribution
To find out if the aberrations were non-randomly distributed between the marker
and non-marker chromosomes, some individual type aberrations like breaks, gaps
and constrictions were quantitatively assessed at each interval from 150 metaphases
examined in each sex (table 2). The other individual types like fragment of
unknown origin and translocation were not considered as the chromosome involved
was not known in the former type.
Effect of x-rays on somatic chromosomes of T. mossambica
129
Table 2, Frequency distribution of some individual type aberrations between 1st
* Marker* pair and 21 pairs of non-marker chromosomes in X-irradiated
and female Tilapia mossambica. Data of females are in brackets ( ).
Fixa
time
No of
metaphase
Marker chromcSoms
Non-marker chromosome
Grand
total
Break
Gap and
Cons
Total
Break
Gap and
Con.
Total
5 min
150(150)
7(7)
4
(2)
11(9)
6
(7)
*(7)
14
(14)
25 (23)
1 hr
150 (150)
5(1)
8
(5)
13 ( 6)
8
(6)
6(6)
14
(12)
27(18)
6 hr
150 (150)
5(3)
7
(8)
1-2(11)
1
(1)
3H
4
(D
16(12)
12 hr
150 (150)
7(7)
6
(*)
13 (15)
4
(3)
-d)
4
(4)
17 (19)
24 hr
150(150)
4(7)
4
(4)
8(11)
1
(1)
3B
4
(D
12 (12)
48 hr
150 (15C)
9(11)
8
(2)
17 (13)
1
(1)
1(5)
2
(6)
19 (19)
72 hr
150(150)
5(3)
6
(3)
11(6)
1
(6)
!(-)
2
(6)
13 (12)
96 hr
150 (150)
4(5)
6
(6)
10(11)
2
(4)
2(4)
4
(8)
14 (19)
120 hr
150 (150)
4(5)
4
(4)
8(9)
1
H
2(1)
3
(1)
11 (10)
144 hr
150 (150)
3(2)
~
(2)
3(4)
6(2)
-(-)
6
(2)
9(6)
7 day
150 (150)
-(2)
2
(3)
2(5)
6
<~)
3(2)
9
(2)
11(7)
10 day
150(150)
~ ( ~)
3
(1)
3(1)
-
(~)
-(2)
-
(2)
3(3)
15 day
150 (150)
~(-)
-
(-)
-<-)
-
H
-H
-
(-)
-<•->
Total
ob;-.
1950(1950)
53 (53)
58
(48)
111 (101)
37
(31)
29 (28)
66(59)
177 (160)
Expected per number
8(7)
169
(153)
177(160)
Expected per length
18 (16)
159
(144)
177 (160)
It was interesting to note that there was some difference in the data of the two
sexes. In the marker chromosome no difference was seen in the total number of
breaks, while it was higher by 10 in males for gaps and constrictions (table 2). In
the non-marker chromosomes it was. higher in males by 6 for breaks and meagrely
by 1 for gaps and constrictions. Therefore, no definite claim was made as to
the differential response of the two sexes, it was just to draw attention to the
trend.
That the marker chromosomes in each sex were highly sensitive to x-radiation
was clear when the observed and the expected values calculated according to the
number of chromosomes and according to the mean length were compared. Out
of the total 1,77 individual type aberrations in males, III were observed in the
marker pair-againstthe expected number of only 8 as calculated per proportionality
of number indicating thereby that the marker pair was about 14 times more
susceptible to x-ray damages. The expected number was 18 if the mean length
was considered. Even then the observed number was more than 6 times indi-
cating the higher susceptibility of the marker pair. On the other hand, in the
non-maker chromosomes of males, 66 aberrations were found against 169 expected,
calculated per number of chromosomes and 159 calculated per length of chromo-
130 G K Manna and R C Som
somes, indicating thereby that the susceptibility of the non-marker chromosomes
was 2-5 times and 2-4 times less. The chi-^square tests of the expected data per
number and per length against the observed number showed in each case that
the difference was highly significant (P < 0-001). Therefore, in males some indi-
vidual type aberrations mentioned early were non^randomly distributed between
the marker ajad non-marker chromosomes, the former group was highly susceptible
and the latter group was somewhat resistant to the x-ray damages.
In females like males the marker chromosomes were also found to be highly
radio-sensitive, while the non-marker ones were somewhat less susceptible. Out
of the total 160 individual type aberrations analysed, 101 were observed in the
marker pair against the expected number of 7 as calculated per proportionality
of number and 16 as calculated per length of chromosomes (table 2). Thus, like
in males, in females also the marker chromosomes were 14 times more susceptible
according to the number and over 6 times susceptible according to the length of
chromosomes. On the other hand, in the non-marker chromosomes the observed
number of individual type aberrations was 59 as against the expected number of
153 calculated per proportionality of number and 144 calculated per proportio-
nality of length of the non-marker chromosomes, indicating that they too, like
marker chromosomes, were 2-5 times less vulnerable according to the number
and 2-4 times less vulnerable according to the length of chromosomes. The chi-
square tests of the observed number and the expected number calculated per
number and the expected number calculated per length of chromosomes showed
that the difference in both the cases was very high (P < 0-001). Therefore, just
like males, the two groups of chromosomes showed the same type of response to
x-rays, the marker chromosomes were highly susceptible, while the non-markers
were somewhat less responsive.
Since the data of each sex showed differential radio-sensitivity, it was expected
naturally that in the combined data of the two sexes, the same manifestation
would be shown. Thus, out of the total 337 aberrations, 212 were observed in
the marker pair against the expected numbers of 15 and 34 calculated per number
and length of chromosomes respectively which also showed 14 times and 6 times
more susceptibility of the marker chromosomes (table 2). In the non-marker
chromosomes 125 aberrations were observed as against the expected number of
322 and 303 calculated per number and per length of chromosomes respectively
which also showed that the non-marker chromosomes were 2-5 times and 2-4
times less vulnerable to x-ray damages. The chi-square test of the observed
number and the expected numbers calculated in two different ways showed in
each case that the difference was highly significant (P < 0-001).
4. Discussion
Most of the effects of ionizing radiations on chromosomes of fish reported by
different investigators did not elaborate on the aberration types. The quali-
tative aspect of the present study revealed that the aberrations were of a similar
nature as found in the somatic chromosomes of some classic material induced
by radiations. But as the chromosomes of fish were not cytologically ideal, all
types could not be studied in every detail. The individual type aberrations
Effect of x-rays on somatic chromosomes of T. mossambica 131
could only be studied more elaborately in the marker chromosomes. The quanti-
tative study of the chromosome aberrations in T. mossambica at different intervals
showed that the effect lingered for a long time. There was not much indication
of the cell lethality caused by the dose of lOOr. The individual types continued
mostly up to the 10th day, while the gross type did so till the, end of the fixation
intervals. Anyhow the persistence of mainly the chromatid type aberration as
long as. 10 days after irradiation deserved some consideration. Though the timing
of the cell cycle in T. mossambica has not been worked out, yet within 10 days
some cells must have completed the cycle unless their further division was inhi-
bited. The prevalent occurrence of the chromatid type aberrations at late intervals
indicated the possibility. Further, the chromatid type break has been supposed
to be induced by the radiation acting on the post-synthetic period of DNA or else
after the replication of chromosome, the reason for which could not be suggested.
It has been a matter of common experience that the chromosome aberrations
induced by odd chemical (Kihlman 1966 ;. Manna 1971, 1975, 1978) and living
mutagens (Manna 1980) were mainly of chromatid type. The same type of
chromosome response of having mainly chromatid type aberrations to ionizing
radiations like other chemically induced ones might lead us to think that the post-
synthetic period, in general, was most sensitive for mutagenic damage to chromo-»
somes. Since the aberrations were found within 5 m!n after radiation it was all
the more suggestive that the chromosome nearing metaphase was more vulnerable
to x-ray lesion. The occurrence of more or less the same chromatid type aberra-
tions from the beginning to almost the end of fixation interval would further lead
us to suspect if the chromosomes approaching metaphase were the vulnerable
stage. This was suggested to explain the chromosome aberrations induced by
odd mutagens. in mice (Manna 1971, 1975). The present study indicated the
differential radio-sensitivity of chromosomes and metaphase nuclei of males and
females irradiated with x-ray. In the past various parameters were used to test
the differential radio-sensitivity in different materials (Evans 1962; Sparrow 1962;
Manna and Mazumder 1968) while the testing of the differential radio-sensitivity
of chromosomes in the two sexes in fish has not been carried out. The present
data need be extended to confirm because there were somp> lecunae in the data.
The analysis of the data of the region-wise distribution of chromatid breaks
in the marker chromosomes of T. mossambica revealed that the distal region or
more broadly the distal half was more vulnerable to x-ray damages. More or
less the same trend was shown by the chromosomes of mice treated with physical^
chemical and living mntagens (Manna 1971, 1975, 1978, 1980) for which it was
suggested by Manna (1975, 1978) that there could be some inherent weaker region
in chromosomes. The same might be the reason for the somewhat localized
break found in the marker chromosome of T. mossambica. The other possi-
bility of having localized breaks by radiation was the differential restitution as
suggested to explain the localized breaks in the i? chromosome of irradiated grass-
hopper (Manna and Mazumder 1962).
Interchromosomal radiation damages by x-ray have been studied in different
animals. The differential radio-sensitivity between chromosomes, of the same
species was seen in the Syrian hamster (Manna and Dey 1981), grasshopper
(Manna and Mazumder 1962* 1968) and Heteroptera (Manna and Dey 1978,
132 G K Manna and R C Sorri
1980). In the above cases the differential radio-sensitivity was shown between
the sex chromosome and autosome of the species concerned, while in the present
study on Tilapia, it was found between two groups of autosome. It was claimed
that the radiation injury was directly proportional to the chromosome volume
(Marshals 1937), length etc. but it was not found in other material (Manna and
Mazumder 1968). It was also not supported from the present data because the
marker chromosomes had more breaks than the expected number calculated
proportional to the length of the chromosomes. The present study, therefore,
revealed some interesting results on the x-ray induced chromosome aberrations
in T. mossambica. Further studies are in progress.
Acknowledgement
One of the authors (GKM) is grateful to the University Grants Commission, New
Delhi, for financing the major project.
References
Evans H J 1962 Chromosome aberrations induced by ionizing radiations. In : Int. Rev. Cytol.
(eds.) G H Bourne and J F Danielli (New York : Academic Press) 13 221-308
Hama A and Egami N 1977 The dose-rate effect of gamma radiation on the initiation of mitosis
in the regenerating tail fin of the fish Oryzias latipes, J. Fac. Sci. Univ. Tokyo, Sec. IV,
14 47-60
Hama A, Egami N, Aral R and Shiotsuki K 1976 Note on chromosome abnormalities found
in irradiated Oryzias latipes, J. Fac. Sci., Univ. Tokyo, Sec. IV, 13 405-408
Hickling C F 1962 In Fish culture (London : Faber and Faber) pp. 245-259
Hideo F and Muramoto J I 1975 Somatic and meiotic chromosomes of Tilapia mossambica
Peters. Chromosome Information Service, Japan 18 4-6
Jhingram V J 1974 Fish and fisheries of India (Delhi : Hindusthan Publishing Corporation)
Kihlman B A 1966 Actions of chemicals on dividing cells (New Jersey : Prentice Hall)
Manna G K 1971 Bone marrow chromosome aberrations in mice induced by physical, chemical
and living mutagen?, Proc. 1st All India Cong. Cytol. Genet. (Chandigarh), /. Cytol.
Genet. (Cong. Suppl.) pp. 144-150
Manna G K 1975 Induced breakage in chromosomes— mechanical and /or chemical. Proc.
Symposium on structural and functional aspects of chromosomes (Bombay, BARC) March
25-26, pp. 194-204
Manna G K 1978 Nonrandom aberrations and their implications on the possible existence of
weaker regions in chromosomes. Proc. 3rd All India Cong. CytoL and Genet. (Hissar) In :
Perspective of Cytology and Genetics (eds.) G K Manna and U Sinha (Delhi : Hindasia
Publisher) 3 221-233
Manna G K 1980 The living mutagens. In : Golden Jubilee Commemoration Volume of National
Academy of Sciences, India (Allahabad) (ed.) U S Srivastava (Calcutta : Naya Prakash)
pp. 573-606
Manna G K and Dey S K 1978 X-ray induced chiasma-like configuration between sex chromo-
somes of the pyrrhocorid bug, Physopelta schlanbuschi. Proc. 3rd All India Cong. CytoL
Genet. (Hissar), In : Perspective of Cytology and Genetics (eds,) G K Manna and U Sinha
(Delhi : Hindasia Publisher) 3 367-672
Manna G K and Dey S K 1980 Preferential sex chromosomal aberrations induced by X-rays and
their alterations by penicillin in male hoteropteran bug, Physopelta schlanbuschi. Symp.
on Chromosome Methodological approaches in cytogevetics (In Cytogenetics in India (eds.)
R P Roy and U Sinha (Delhi : Hindasia Publisher) In press
Effect of x-rays on somatic chromosomes of T. mossambica 133
Manna G K and Dey S K 19S1 Differential X-ray sensitivity of Syrian hamster chromosome,
Nucleus 24 48-56
Manna G K and Mizumder S C 1962 The grasshopper X-chromsome as an indicator for X-ray
induced chromosome breakage, Proc. Zool. Sac. (Calcutta) 15 103-110
Manna G K and Mazumder S C 1968 Induced X-chronmsomal aberrations in the study of intra-
. and interspecific radioscnsitivity of grasshoppers, in : Seminar on chromosomes, Nucleus
(cd.) A K Sharma (Calcutta) pp. 197-207
Manna' G K and Som R C Unpublished
MaishakA 1937 The effect of X-rays on chromosomes in mitosis, Proc. Natl. Acacl Set. U.S.A.
23 362-369
Mong S J and Bena'T M 1979 The effects of increasing dosages of X-radiation on tho chromo-
somes of cen.tr?! mud minnow ; /. Fish BioL 14 523-527
Natarajan R and Subramanyam K 196& A preliminary study on the chromosomes of Tilapia
mossambica (Peters) ; Curr. Scl. 37 362-363
Pechkurenkov V L 1976 Formation of chromosomal aberrations in fish embryos induced by
chronic radiation ; Radiobiologiya 16 842-846
Prrsad R and Manna G K 1976 Chromosomes of fishes, T. mossambica and N. notopterus ;
Chromosome Information Service Japan No. 21 11-13
Schroder J H 1973 Tclccsts as a tool in mutation research. In Genetics and Mtitagenesis in
Fishes (ed.) J H Schroder (New Yoik : Springei-Verlag) pp. 91-98
Sharma A K and Chaticrjee A 1962 Chromosome size as a factor on radio-sensitivity ; Nucleus
5 67-74
Sparrow A H 1962 The role of nucleus in determining radio-sensitivity ; Brookhaven Lecture Series
No. 17.
P.(B)~4
Proc. Indian Acad. Sci. (Anim. ScL), Vol. 91, No. 2, March 1982, pp. 135-141.
® Printed in India.
Histochemical changes in Setaria eervi caused by certain
anthelmintics
ABDUL BAQUf and HUMAIRA KHATOON
Department of Zoology, Section of Paiasitology, Aligarh Muslim University,
Aligarh 202 001, India
MS received 11 March 1980
Abstract. The present study deals with the preliminary in vivo screening of suramin
and levamisole in ra.t-Setaria cervi system with special reference to the histochemical
changes in the adult worms caused by the drugs. Levamisole proved to be highly
effective as a micro- and macro- filaricidal agent. It also appears to be interfering
with the normal activity of alkaline phosphatase and glycogen of the adult worms
with no apparent effect on its protein content. The drug also causes irreversible
paralysis in adult worms. Suramin, though an active pharmacological agent, proved
to be completely ineffective on microfilariae as well as on adult worms of Setaria
cervi. Consequently, no notable alterations in the histochemistry of the parasite
following suramin treatment were observed.
Keywords. White rats ; Setaria cervi ; histochemical observations.
I. Introduction
Numerous anthelmintics have been tried on nematode parasites in experimental
studies and their efficacy has been established; but their mode of action on the
worms and the consequent biochemical or histochemical alterations brought about
by the drugs are least understood. Levamisole and suramin are known potent
anthelmintics. Levamisole is the newly-discovered highly potent broad spectrum
anthelmintic effective on a variety of nematodes. But the mode of action of these
drugs on the biochemistry or histochemistry of the parasite is not fully known.
The present study deals with the preliminary screening of suramn and levamisole
in T&t-Setaria cervi system with special reference to the histochemical alterations
in the adult worms, caused by the drugs.
2. Materials and method?
About 20 laboratory bred white rats almost of the same age group and weight were
used in the present experiment. Adult worms (Setaria cervi), collected from the
peritoneal cavity of freshly slaughtered buffaloes, were implanted surgically into
the peritoneal cavity of white rats according to the method described by Baqui
135
136 Abdul Baqui and Humaira Khatoon
and Ansari (1975). Each rat received five adult worms of both sexes. Infected
rats were divided into two groups : one for the suramin and the other for levami-
sole treatment. The drugs were given to the microfilaria-positive rats after a week
of initial infection at the higher tolerant dose determined earlier. Levamisole
and suramin were administered orally and subcutaneously at 20 mg/kg/day and
9 mg/kg/day respectively. Administration of the drugs and microfilarial count
were made for 5-^10 consecutive days, thereafter the treated rats of both groups.
were aiytopsied to observe the condition of the worms and the apparent effect of
the drugs on the worms.
Untreated normal wonm (control) and those recovered from treated autopsied
rats were fixed in Carnoy's fluid and cold acetone for histochemical observations
of protein, gtycogen and alkaline phosphatase activities. Fixed materials were
cleared in benzene and paraffin blocks were made. Protein and glycogen were
localized by Mercury-^bromophenol blue and carmine stain methods respectively
$s suggested by Pearse (I960). Alkaline phosphatase was estimated by calcium
cobalt technique as described by Oomori (1952).
3. Results
It wd; observed that all the rats treated with levamisole for 5 consecutive days
cleared of mlcrofilariae (response 100%) from peripheral blood circulation (table IX
Microfilarial density continued to drop after the administration of the very first
dose of the drug. Further, rats autopsied after the disappearance of microfilariae
on the 15th day of infection showed only 20% recovery of live active adult worm8
(table 1). The remaining worms were either completely exhausted or degenerate.
Some of the worms were completely well organized in their architecture but
remained immobile and inactive even after transfer to the normal saline showing
the sign of doubtful viability. Such worms were also counted as dead. Posterior
i part of some live adult worms (male and female both) was found to be com-
pletely shrunk and contracted which remained unchanged even after transferring
into the normal saline indicating the paralysing action of the drug.
Histochemical observations of the levamisole-treated worms revealed that protein
content of cuticle, body muscles, boundary walls of ovary, uterus, microfilariae
and developing embryos remained unchanged as compared to that of normal con-
trol. However, a heavy concentration of alkaline phosphatase found in subcutiele
body muscles, lateral cords, embryos and microfilariae in control worms (figure 1)
was noted to have considerably decreased in treated worms (figure 2). Similarly,
glycogen content appreciably localized in muscles, boundary walls of uterus and
developing embryos of control (figure 3) was also found to have relatively decreased
in treated worms (figure 4).
Another drug, suramin,, was found to be completely ineffective on microfilariae
as well as adult worms of S. cervL Some of the rats (50%) treated for 10 conse-
cutive days did not show any sign of effectiveness on circulating microfilariae, con-
sequently microfilarial density continued to increase in the peripheral blood circu"
lation (table 1). Treated rats autopsied at 5 and 10 days intervals did not show
any apparent macrofiiaricidal effect either. Live worms recovered on autopsy
ranged from 40-60%. Further, no notable changes, in all the three biochemical
Histo chemistry of S. cervi
137
Figure 1. Alkaline phosphatase activity in the control worm.
Figure 2. Alkaline phosphatase activity in the levamisole-treated worm.
138 Abdul Baqui and Humaira Khatoon
Figure 3. Glycogen localization in the control worm.
Figure 4. Glycogen localization in the levamisole-treated worm.
Histochemistry of S. cervi
139
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140 Abdul Baqui and Humaira Khatoon
constituents, i.e., protein, glycogen and alkaline phosphatase, were recorded
histochemically as compared to the control.
4. Discussion
Levamisole, a broad spectrum anthelmintic, has been found to be highly effective
on microfilariae as well as adult S. cer'vi worms like its dextrous omer, tetramisole
as earlier reported by Baqui and Ansari (1976). Complete disappearance of
circulating microfilariae following a 5-day treatment with levamisole and low per-
centage of recovery of live adult worms on autopsy are indicative of the fact that
the drug con tains micro — as well as macrofilaricidal property against S. cervL As
earlier observed, the transplanted worms normally survive in the peritoneal cavity
of white rats for 4-.6 weeks (Baqui and Ansari 1975), Hence, disintegration of
the worms at this early stage of infection could be solely attributed to the effects
of the drug.
Studies regarding the histochemical changes in the nematodes following anthel-
raintic treatment are scanty. However, there are a few reports on the biochemical
changes of the worms brought about by certain drugs. Van den Bossche aitd
Janssen (1969), Van den Bossche (1972), Malkin and Camacho (1972) and Prichard
(1973) have reported that fumarate reductase activity is considerably inhibited in
Haemonchus contortus and Ascaridia gatti following treatment with tetramisole,
levamisole and thiabendazole. Tetramisole also inhibits the cholinesterase, aldo-
lase and acid phosphatase of Ascaridia galli (Vertinskaya et al 1972;. Chakraborty
et al 1976). Piperazine has been reported to decrease glycogen value in Ascaris
lumbricoides tissues (Abdulazizov 1975; Bogoyavlenski et al 1975) and histaminc
content in Ascaris suum (Phillips et al 1976).
The present study supports the above observations. Levamisole has shown
pronounced effects on adult worms which are characterized by death or irreversible
paralysis of the worms. Suramin, though an effective drug in other fllarial nema-
todes such as Ottchocerca and Dipetalonema (Burch 1955 ; Gayral and Pommies
1976) proved to be completely ineffective on S. cervL Hence no notable altera-
tions in the histochemistry of the worms were observed. However, it has been
reported that suramin inhibits strongly, in vitro, a variety of enzyme system of
trypanosomes (Von Brand 1966).
Levamisole appears to be interfering with the carbohydrate metabolism especi-
ally with the absorption of carbohydrates and their intracellular utilization. As a
result glycogen value is diminished in different organs. According to Von Brand
(1966) inhibition of glucose absorption results in decrease in the concentration of
energy-rich phosphate bond; finally the energy required for survival becomes
inadequate and the parasite dies.
The drug also in some manner, inhibits the normal alkaline phosphatase activity
of the worms as a result of which considerable decrease in its concentration in
various organs is observed. The protein value remains unchanged in treated
worms. There is very little information available, concerningnematode parasites
as to whether anthelmintics attack the parasite proteins or interfere with some
phase of its nitrogen metabolism. Levamisole appears to have a paralysing action
on adult worms and probably acts as a neuromuscular blocking agent like its
Histochemistry of S. cent 141
dextro-isomer, tetramis.ole (Gaitonde 1971). The sustained contracture of the
somatic muscles of S. cervi results in the irreversible paralysis of the worm — a
condition similarly reported in another filarial worm, Breintia sergenti and Ascaris
following in vitro treatment with levaiuisole (Natarajan et al 1974; Van den
Bossche 1972).
Acknowledgement
The authors are grateful to Prof. S M Alam for providing laboratory facilities.
References
Abdulazizov A J 1975 Histological changes in Ascaris lumbricoides caused by some anthdmmtics ;
Tr. Mask. Med. Inst. 84 14-17
Baqui A and Ansari J A 1975 Electrophorctic patterns of white rat sera infected with Setaria
cervi ; Indian J. Helminthol 28 106-115
Baqui A and Ansari J A 1976 Compar?tive studies on the chemotherapy of expeiimental
Setaria cervi infection ; Jpn. J. Parasitol. 25 409-414
Bogoyavlenski Yu K, Abdulazizov A I and Onushko N V 1975 Changes in the fine structure
of the female reproductive system of A. lumbricoides caused by some anthelmintics ; Tr.
Mask. Med. Inst. 84 45-48
Burch T A 1955 Treatment of .wucheriasis and ouchocerciasis with suramin sodium ; Am. J.
Trop. Med. Hyg. 4 332-333
Chakraborty AK,MehtaRKand Datte I C 1976 Effect of tetramisole upon certain biochemical
constituents of Ascaridia gdli ; Indian /. Exp. BioL 14 585-587
Gaitonde B B 1971 Pharmacological review of anthelmintics ; In Soil transmitted nematodes and
tetramisole, Ethnor Ltd. (Proceeding of the symposium) April 9 and 10
Gayral P and Pommies M 1976 Preliminary results on the use of a new rodent filaria, Dipctah
nema disselae in the evaluation of filaricides ; C.R. Hebd. Seances. Acad. Set. Paris. 283
861-864
Gomori G 1952 Microscopic hi sto chemistry: Principles and practice (Chicago: University Chicago
Press)
Malkin M F and Camacho R M 1972 The effect of thiabendazole on fumarate reductase from
thiabendazole— sensitive and resistant Haemonchits contortus ; J. Parasitol. 58 845-846
Natarajan P N, Zaman V and Yeoh T S 1974 In vitro activity of levamisolc oa the infective
larvae, microfilariae and adult worms of Breinlia sergenti ; Int. /. Parasitol. 4 207-210
Pearse A G E 1960 Histochemistry : Tlieoretical and applied, 2nd edition (London : J and A
Churchill)
Phillips J L, Sturman G and West G B 1976 The interaction between anthelmintic drug and
histamine in A. suum ; Br. J. Pharmacol 57 417-420
Prichard R K 1973 The fumarate reductase reaction of Haemonchus contortus and the mode
of action of some anthelmintics ; Int. J. Parasitol. 3 409-417
Van den Bossche H and Janssen P A J 1969 The biochemical mechanism of action of the
antineinatodal drug tetramisole ; Biochem. Pharmacol. 18 35-42
Van den Bossche H (ed.) 1972 Biochemical effects of tetramiscle ; In Companttive Biochemistry
of Parasites (New York and London : Academic Press)
Vertinskaya M K, Govorova S V and Polyakova O I 1972 The inhibition of Ascaridia galli
enzymes by tetramisole ; ByulL Vses. Inst. GelmintoL K I Skryabina 7 9-12
Von Brand T 1966 Biochemistry of parasites (London ; Academic Press)
Proc. Indian Acad. Sci. (A»im. Sci.), Vol. 91. Number 2, March 1982, pp. 143-152.
© Printed in India. .
Effect of salinity on the survival and growth of Chanda (^
gymnocephalus (Lac.) fry (Pisces; Centropomidae)
J RAJASEKHARAN NAIR, N K BALASUBRAMANIAN and
N BALAKRISHNAN NAIR
Department of Aquatic Biology and Fisheries, University of Kerala,
Trivandrum 695 007, India
MS received 12 May 1981
Abstract. The survival and growth of Chanda (= Ambassis) gynmocephalus (Lac.)
fry (8 -8 ±0-2 mm) collected from Murukumpuzha Lake (9-34%0) for a ninety
day period in different salinity grades were studied. A faster rate of growth is
exhibited by the fish in the highest salinity grades(22-41 and28-51%0), even though
during the first month, growth and health was apparently better in the lower
salinity grades (4-11, 10-21 and 16-31%0). Assimilation efficiency also showed
a similar gross picture. Thus in C. gymnocephalus, an euryhaline species, the fry
show preferred salinity gradients for optimum growth within the fluctuating salinity
regime at a stable temperature (26 ± 2° C) and hence may make salinity bound
emigrations with growth.
Keywords. Salinity ; growth efficiency ; assimilation efficiency ; satiation ;
Chanda gymnocephalus.
1. Introduction
Chanda gymnocephalus i$ an euryhaline glassy perchlet inhabiting the coastal waters
and the estuarine and brackish water tracts of Kerala. A shoal of fry (8-8 ± 0-2mm)
was collected from the shallow protected region of the Murukumpuzha lake
(9'34%0) about 2 to 3 km away from the Perumathura bar-mouth (pozhi) which
was open. According to Nair (1957) " Ambassis gymnocephalus spawns in coastal
waters near the bar-mouth. Large quantities of the pelagic eggs spawned in the
vicinity of the bar-mouth are passively carried into the lake by the strong tidal
currents. Reaching the main body of the lake, these eggs and larvae drift into
the shallow protected regions. " In order to understand the salinity "preferences
for growth and the salinity bound movements of the fry and early juveniles, the
salinity tolerance and its effect on the growth pattern of the fry for a three month
period under laboratory conditions were studied. Salinity preferences in emigratory
movements of the presmolt coho salmon have been studied by Garrison (1965)
in natural waters and by Otto and Mclnerney (1970) and Otto (1971) under labora-
tory conditions. There is also considerable field information (Canagaratnam 1959
1966 ; Gunther 1961 ; Holliday 1971) and experimental evidence (Gibson arid*
143
144 / Rajasekharan Nair et at
Hirst 1955 ; Kinne 1960 J Holliday 1971 ; Weatherly 1972) suggesting that
growth and size of euryhaline fish are influenced by salinity.
2. Materials and methods
In about four to five weeks after hatching, the larvae reach a length of 8 mm
(tsjair 1957). Thus the fry were abou 1 1- 1 • 5 months old at the start of the experi-
ment. Immediately on arrival at the laboratory, the fry were transferred into a
large cement tank (4' x 3' x 3') containing water of 10-21%0 at 26 ± 2°C for
two days for acclimation. The salinity tolerance of the fry for a 48 hr period was
studied in seven different grades (0-96, 4-11, 10-21, 16-31, 22-41, 28-51 and
31*56%0). At 31-56%0 (seawater) and 0-96%o (wellwater) there was 70% and
30% mortality in 48 hr while there was no mortality in other grades. Therefore
only the five salinity ranges (4*11 to 28-51%0) were chosen for the growth studies.
After the tolerance tests the shoal in the cement tank were divided into batches
of 100 each and reared in five aquarium tanks (60cm x 30cm x 30cm) in five
different grades, while about 200 specimens were kept in the cement tank as control
at 10*21%». Once in the OKperimental tanks they were allowed a 12-48 hr period
to acclimate. There was no mortality during this period. Feeding began the
next morning and the fish were fed to satiation twice a day. For the first twenty
days they were fed on Anemia nauplii and thereafter, till the end of the experiment
on chopped tubifex worms (Tubifex tubifex).
At the end of every ten days (11, 21, 31,. . ., 91) five fish were collected at ran-
dom (retarded individuals were discarded) and sacrificed for length and weight
measurements and the average taken for each grade. Length measurements were
made using a micrometer (up to l/100th of a mm) and weight using an electric
monopan balance (up to l/100th of a mg).
At the end of every ten days (15, 25, 35,....., 85) the satiation amount, the
amount of faecal matter excreted (in 24 hr) and the total weight offish in each
salinity grade were also measured for the rough estimation of digestibility
( = digestion coefficient) as given by Kapoor et al (1975) and dealt with in the
present study as 'assimilation efficiency', and for the estimation of growth effici-
ency for each ten-day period.
Assimilation efficiency = Afa * 10° ,
(Digestibility)
where AH « Af6 — Afm.
Growth efficiency 10 d»y,= /d ? 10
Where -I/, = amount of food eaten, Afm = amount of faecal matter excreted,
Aft * amount of food assimilated, (?io = growth in weight during the corres-
ponding ten-day period.
The different salinity ranges were prepared by diluting seawater with wellwatet
making 10, 30, 50, 70 and 90% seawater and were maintained throughout t&e
Effect of salinity on C. gymnocephalus fry
145
experimental period at ± 1%0. Salinities were determined using a salinometer. All
experimental tanks were maintained at a water temperature of 26 ± 2° C and
the oxygen kept at air saturation level using aerators every alternate day. The
excess of food was removed within two hours of feeding. To inhibit the accumu-
lation of metabolites and bacterial growth, water was changed every fifth day.
The salinity tolerance (48 hr) of a new stock of early jiveniles (22-27 mm)
collected from near the bar^mouth (poxhi) was also studied.
3* Results
The cumulative growth in length and weight are plotted against time (days) for the
different salinities and the corresponding regression equations are given in figures 1
and 2. The regression equations are calculated using the least square method of
analysis. The correlation coefficient values for cumulative growth in length and
weight for the different salinities are given in table 1. All the values are significant
at the 1% level. A faster rate of growth is exhibited by the fish in the highest salini-
ties (22-41 and 28-51%0), even though during the first month growth and health
were apparently better in lower salinities (4-11, 10-21 and 16-31%0).
Estimates of assimilation efficiency and growth efficiency are given in tables 2
and 3. Changes in growth pattern are clearly reflected in the growth efficiency
changes with time (figure 3) and the corresponding regression equations are given.
20
16
12
o
2
Q) y- 0-1577X * 8-4895
b) y= 0-1115X^9-6323
16-31 %0
Y=0-1128X + 9.3739
10-21%0
Y= 0-1102 X+9-32U
31 51 71 91 11 3V 51 71
. . DAYS
Figure 1. Regression lines and equations for growth in length in different salinities.
146 / Rajasekharan Nair et al
90r
70
SO'
ZE
o
70
30
10
a)Y=0-6747X-1-27l5
b) Y* 0-8924 X- 5-9455
16-31%,
Y * 0-6661 X- 2-4705
10-21%o
Y=0-6465X- 1-7092
22-41%)
Y =0-7610 X-3-8UO
21 41 61 ' 81
21 41
61
DAYS
Figure 2, Regression lines and equations for growth in weight in different salinities.
Table 1. The correlation coefficient values for cumulative growth in length .and
weight.
Correlation coefficient values
Salinity (%0)
Growth in length
Growth in weight
1.
4-11
0-9925
0-9716
2.
10-21
0-9955
0-9725
3.
16-31
0-9931
0-9695
4.
22-41
0-9957
0-9735
5.
28-51
0-9974
0-9765
In the initial stages better assimilation efficiency is shown by the lower salinities
but with growth higher salinity grades show greater efficiency as may be seen in
figure 4. The estimates of the satiation amount, in percentage offish wet weieht
are presented in table 4. ^ '
In the control (10-21%o) the fish weight and length were 67-4 mg and 20- 1 mm
at the end of the experiment (66-2 mg and 19-44 mm for the experiment 10-2ir *
showing that their growth was in no way inhibited.
Effect of salinity on C. gymnocephaJus fry
147
g 6
ts
<
u.
UJ
o
1,0
a) Y=
b)Y=0-U65X*1.0A83
Ys0.1113X*1-7916
16-31%o
Y=0-1009X*2-1S90
Y*0-1325X*0.7732
15 35 55 75 ' OAYS 15 35 55 75
Figure 3* Changes in growth efficiency factor with time in different salinities.
The salinity tolerance range of the early juveniles was between 5- 5%0 and
33*60%0 (figure 5).
4. Discussion
In order to analyse and understand growth phenomena, it is convenient to consider
short growth periods or stanzas for arbitrarily defined time periods (Webb 1978).
For the initial three ten-day periods (one month), growth was inhibited in the
two higher salinities of 22-41 and 28-51%5, whereas the three lower salinities had
little effect on the growth, the fish showing uniform growth. Of primary interest
in the series of experiments is the gradual maximisation of growth in the
tyro higher salinities (22-29%0) and the gradual decline in the rate of growth for
fish maintained at the two lower salinity grades (4-17%0). A precipitous decline
in the rate of growth and growth efficiency at all salinity grades occurred during
the 21-41 day period even though the assimilation efficiency was not. affected.
This may be due to the change in food from Artemia nauplii to chopped tubifex
worms, since after this period growth rates picked up fast and then showed steady
increase in all salinities especially in the two higher grades. •
Corresponding to changes in growth rates, the growth efficiency in different
salinities showed similar fluctuations (figure 3). In the initial stages, higher growth
efficiency was shown by fish in the thre* lower salinities. Even though: food
14S
/ Rajasekharan Nair et al
100-
92
Y= -0-0512X+ 2-0501
b)Y«-0-OU7X+V9990
i&s
75
65
35 55
DAYS
75
a)Y=0-2362X*1-5268
tjlYs 0-0353 X»t-8803'
15 35 55 75
DAYS
Figure 4. Changes in assimilation efficiency with, growth in different salinities.
consumption was high in the higher grades at this time, the assimilation efficiency
was as low as 62-S%(22-41%0) and 6l-3%(28-51%0) whereas it was as high as
97-61 %(4-ll%0); 94-66% (10-21%0) and 87-99% (16-3 1%0) during the 11-21
day period. But slowly the assimilation efficiency picked up in the higher salinities
while the lower grades showed marginal decrease with time as is clearly brought
out in figure 4. According to Patoheimo and Dickie (I966a,b) and Warren and
D5&vis (1967) salinity may affect growth through its influence on food conversion
efficiency and activity, which are important components of the Moenergetic budget
of fishes, as is seen in the present study also. Also according to Webb (1978)$
** fe general total foo<f intake is greatest and metabolism smallest under least
(s&ess so that growth is then maximal ", as is seen in the lower salinities during
the initial stages and in the higher gradfes with acclimation and passage of time*
Another interesting aspect was the schooling behaviour of the fry. In the higher
salinities during the initial stages of the experiment the fish were scattered as indi-
viduals but with passage of time formed loose-knit shoals and by the 41-51 day
period were shoaling well and behaving as a unit. In the lower salinity grades the
fry were shoaling well from the initial period ^introduction but tended to scatter
in the later stages. From the fluctuations in the assimilation efficiency it maybe
noted that schooling tend to reduce metabolism. Parker (1973) has made similar
ob^ervstions in the case of 21 species offish. According to Weihs (1973) schooling
to exert a 'calnrimg' effect and there niay be further hydrodynamic-energy
Effect of salinity on C. gyriinocephatus fry
Table 2. Changes in assimilation efficiency with growth in different salinities
Time (days) 4-ll%0 10-
21%0 1<5-31%0
22-41%0
28-51%0
15
25
35
45
65
75
97-61% 94-66
95-17 94-74
95-10 96-01
95-00 93-88
90-36 88-48
87-54 89-07
87-99
89-97
89-02
73-79
86-13
84-13
62-50
83-77
82-72
89-62
89-03
87-96
61-38
70-33
73-63
88-68
90-14
92-46
85
85-15 88-02
86-75
91-78
95-00
Table3. Changes
in growth
efficiency
with time in
different salinities.
Period
4-ll%0 10-21%0 16-31%0
22-41%0
28-51%0
11-21
6-74
6-44
5-65
5-25
4-94
21-31
2-68
3-79
4-50
2-22
3-69
31-41
2-95
3-41
5-60
3-43
3-39
41-51
8-55
7-09
4-02
8-33
9-44
61-71
6-82
6-72
7-45
8-59
11-99
71-81
9-10
9-53
9-97
9-61
10-17
81-91
12-68 12-94
13-75
13-70
14-25
Table 4. Changes
different salinities,
in satiation as percentage of wet
body weight with growth in
Time (days) 4-ll%0
10-21%0
*«x
D 22-41%0
28-51%0
15
26-48
31-57
29-05
29-58
38-41 '
25
' 32-43
32-29
29-88
30-00
34-88
35
31-90
34-80
35-54
37-98
46-52
45
31-17
34-95
35-57
34-64
45-18
65
33-36
27-61
36-05
43-98
56-55
75
35-16
26-00
48-48
52-55
57-39
85
34-42
26-53
48-85
53-16
58-05
130
3 Rajasekharan Nair et al
33-6
32-0
20
10 30 50 70 9°
MORTALITY (%)
Figure 5. 48 hr mortality rates of juveniles in different salinities.
economics. However, the 'size hierarchy9 of Brown (1957) or 'growth depensation
of Ricker (1958), i.e., dominant fish tend to monopolise food and show better
growth, even though apparent, was not taken into account in the present study
since the fish showing retarded growth were discarded from the growth studies.
The satiation amount as percentage of wet body weight (3-2-84-3 mg) ranged
from 26-48%-58-05% for the 91-day experiment period at 26 ± 2°C. Davis
and Warren (1968) found young chinook salmon Onchorhynchus tshawytscha(Q-6g)
would consume 20% body weight/day and Krivobok (1953) as cited by Winberg
(1956) obtained daily rations as high as 54% dry weight in very young carp
(0-016 g). Brett (1971) computed a daily intake of 30% dry weight at 15° C for
one gram sockeye fry.
By the time the fry reach a length of about 17-18 mm they move towards the
main body of the lake (Nair 1957). In the present study, by the time the fry reach
this length (61 days), the fish show much better growth in the two higher salinities
and better shoaling habits too and hence may start moving up the lake towards
the bar-mouth seeking the optimum salinity gradients in the niche. The capture
of early juveniles (22-27 mm) and their salinity tolerance level (5-5-33*6%0)
strengthen the above conclusion.
Using the regression formula for growth in length in the 28- 51%0 (Y = 0-1577 X
4- 8-4895), the fish may reach a length of about 6- 6 cm during the 0-1 year period
and about 4-8cm in 7-8 months time. Thus the 0-year class individuals of the
species mainly contribute to the annual fishery in the estuaries as also noted by
Nair (1957) and Raman et al (1975) and start breeding towards the end of the
0-1 year period. It is also quite possible that with their increased preferences to
higher salinities with growth, the adult fish may escape into the sea when the
bar-mouth is open contributing to the marine stock. Nair (1957) and Raman et al
(1975) have noted the seaward migration of this species and Raman et al report
that the adult fish migrate to the sea and grow to larger sizes there.
Effect of salinity on C. gymnocephdus fry 151
Thus in C. gymnocephalus an euryhalinc species; the fry show preferred salinity
gradients for optimum growth within the fluctuating salinity regime at a stable
temperature of 26 ± 2° C in the laboratory, while similar salinity bound emigra-
tions have been noted in the natural waters fcy other workers.
Acknowledgement
One of the authors (JRTN) is grateful to the Council of Scientific and Industrial
Research for the award of a Junior Research Fellowship during the tenure of
which this work was carried out.
References
Brelt J R 1971 Satiation time, appetite and maximum food intake of sockeye salmon (Oitchorhytt-
elms nerka) ; /. Fish. Res. Bd. Canada 28 409-415
Brown M E 1957 Experimental studies on growth ; In The Physiology of Fishes (cd.) M E
Brown (New Yai'k: Academic Press) 1 361-400
Canagaratnam P 1959 Growth of fishes in different salinities ; /. Fish. JR.es. Bd. Canada 16
121-130
Quiagaratnam P 1966 Growth of Tilapia mossambica Peters in different salinities ; Bull. Fish.
Res. Sta. Ceylon 9 47-50
Davis G E and Warren C E 196$ Estimation of food consumption rates ; in IBP Handbook
No. 3 Methods for assessment of fish production in freshwater s (cd.) W E Rickcr
(Oxford : BJackwell Scientific Publication) pp. 204-225
Garrison R L 1965 Coho salmon smalts in ninety .days ; Prog. Fish-Cult. 27 219-230
Gibson M B and Hirst B 1955 The effect of salinity and temperature on the prcadult growth
of guppies ; Copeia 3 241-243
G anther 1961 Salinity and size in marine fishes ; Copeia 2 234-235
Holliday F G T 1971 Salinity-Fishes ; In Marine ecology (ed.) O Kinne (New York : John
Wiley) Vol. 1 Part II pp. 997-1083
Kapoor B G, Smith H and Verighina I A 1975 The alimentary canal and digestion in Telcosts
in Advances in Marine Biology (eds.) F S Russel and Sir M Yonge (London: Academic
Press) 13 109-239
Kiniio O 1960 Growth, food intake and food conversion in a euryplastic fish exposed to 2 diffe-
rent temperatures and salinities ; Physiol. Zool. 33 288-317
Nair. G S 1957 On the breeding habits and development of Ambassis gymnocephalus (Lac.) ;
Bull. Cent. Res. List. Univ. Travancore 5 69-76
Otto R G 1971 Effects of salinity on the survival and growth of presmolt coho salmon
(Onchorhynchus kisutch) ; /. fish. Res. Bd. Canada 28 343-349
Otto R G and Mclnerney J E 1970 The development of salinity preference in presmolt coho
salmon (Onchorhynchus kisutch) ; /. Fish. Res. Bd. Canada 27 793-800
Paloheimo J E and Dickie C M 1966a Food and growth of fishes. II Effect of food and
temperature on the relation between metabolism and body weight ; /. Fish Res. Bd. Canada
23 869-908
Paloheimo J E and Dickie L M 1966b III. Relation among food, body size and growth
efficiency ; /. Fish. Res. Bd. Canada 23 1209-1248
Parker F R 1973 Reduced metabolic rates in fishes as a result of induced schooling : Trans.
Am. Fish Soc. 102 125-131
Raman K, Kaliyamurthy M and Rao G R M 1975 Studies on the biology of Ambassis gymno-
cephalus (Lac.) from Pulicat and Vembanad lakes ; Matsya I 49-52
152 / Rajasekharctn Nair et al
Ricker W E 195S Handbook of computations for biological statistica of fish populations ;
Fish. Res. Bd. Canada Bull. 119 1-300
Warren C E and Davis G E 1967 Laboratory studies on the feeding bioenergetics and growth
of fish ; In The biological basis of freshwater fish production (ed.) S D Gerking (London :
Blackwell Scientific Publications) pp. 175-214
Weatherly A H 1972 Growth and ecology offish populations (London and New York : Academic
Press) p. 293
Webb P W 197$ Partitioning of energy into metabolism and growth ; in Ecology of fresh'
water fish production (ed.) S D Gerking (London : Blackwell Scientific Publications)
pp. 184-214
Weihs D 1973 Hydrodynamics of fish schooling ; Nature (London) 241 290-291
Winberg G G 1956 Rate of metabolism and food requirements of fishes ; Belorussian State
University Minsk Fish Res. Bd. Canada Transl. Ser. No. 194 (1960) p. 251
Proc. Indian Acad. Sci. (Anira. ScL), Vol. 91, Number 2, March 1982, pp. 153-153.
© Printed in India.
A comparative study on the mineral composition of the poultry
cestode RailUetina tetmgona Molin, 1858 and certain tissues of its
host
A M NADAKAL and K V1JAYAKUMARAN NAIR
Dip.i,rtnimt o.f Zoology, Mar Ivaijios College, Triyandrura 695015, India
MS received 5 March 19.81 ; revised 26 December 1981
Abstract. The amounts of cations Ca, P, Na, K, Cu and Zn in RailUetina tetra-
gona (Cestoda) and in liver, intestinal tissues and blood serum of its host (Gallus
gallus dotmsticus) were determined using spectrophotometry, titrimetry, flame photo-
metry and atomic absorption spectrophotometry. Quantitative variations were
observed in the distribution of these minerals in the immature, mature and gravid
regions of the worm, on dry weight basis. There was a gradual decrease in Ca
content of worm along the antero-posterior axis. The Na content, on the other hand
showed a reverse trend with the greatest amount in the gravid proglottids. The
immature region contained the highest levels of P, K and Cu. The worms showed
significantly higher levels of Ca, P, Cu and Zn than the liver and intestinal tissues
o-n dry weight basis. R. tetragow, like host liver and intestinal tissues (but unlike
blood serum), had quantitative excess of K o.ver Na and other cations.
Keywords. Mineral composition ; poultry cestode ; RailUetina tetragona \ host
tissues.
1. Intraductian
Most of the earlier studies on the biochemistry of cestodes have dealt extensively
with their organic constituents, especially the carbohydrates, Hpids and proteins.
More recently several attempts have been made to identify and quantify the inor-
ganic contents of tapeworms (Salisbury and Anderson 1939 ; Wardle and
McLeod 1952 ; Goodchild et al 1962 ; Nadakal et al 1975 ; Singh et al 1978 ;
Jakutowicz and Korpaczewska 1979). The data available so far are largely con-
cerned with the larval cestodes and so little is known about the inorganic compost
lion of the adult cestodos. H^nce a study was designed to throw some light on
the m'.neral composition of a cosmopolitan poultry cestode, RailUetina tetragonu
and certain tissues of its host, by way of comparison.
2. Materials and methods
Day^old white leghorn chicks were procured and maintained in. the laboratory
on a basal diet adequate in all nutrients. Wnen three weeks old, 20 healthy birds
153
154 AM Nadakal and K Vijayakumaran Nair
of uniform weight were selected 25 cyaticercoids of Raillietina tetragana recovered
from naturally infected ant vectors (Nadakal et al 1971) were administered per
os to each of the 20 birdst. Tnree week* post-infection, blood was collected from
the wing veins for obtaining serum and then the birds were autopsied. The intes-
tines were split and the worms, carefully recovered. The liver, intestines and the
worm* were washed thoroughly in distilled water and blotted dry with low ash
filter paper. 100 worms, were pooled and each worm was cut into immature,
mature and gravid regions. 40 worms were set apart and sampled as whole worms*
The tissue samples were immediately processed for biochemical estimations of
ionic Na, K, Ca, P, Cu and Zn. For estimations of Ca and P, 5 samples of
each tissue were taken. Each sample was divided into 2 weighed portions. One
part was extracted with 10% trichloro-acetic acid for Ca and P determinations
and the other part was used for determining percentage of dry matter. For Na,
K, Cu and Zn estimations 5 samples from the pooled tissues were dried at 80-400° C
Measured quantities of these dried tissues and serum were ashed separately and
extracted with concentrated nitric acid and diluted with glass distilled water, the
diluted extracts being used for the estimation of Na, K, Cu and Zn.
Ionic Ca and P were determined following the methods of Clark and Collip (1925)
and Fiske and Subba Row (1925) respectively. Na and K were estimated using a
Him? photometer (Elico Pvt. Ltd., CL 22A), while Cu and Zn were determined
using an atomic absorption spectrophotometry (Unicam, SP 1900).
The data obtained for the different regions of the worm were statistically analysed
using student's t test for the probability of significance of difference between means.
The data for the whole worms were compared with those for the host liver and
in-testinal tissues and blood serum. P values at 5% level are considered to
represent significant differences.
3. Results
Quantitative findings for percentages of Ca, P, Na, K, Cu and Zn in the three
different regions of the worm are shown in table 1 and those in the whole worms
and in the host tissues and blood serum are presented in table 2.
There was a gradual decrease in the Ca content of the worm along the antero-
posterior axis. The Ca content of whole worms was 2-64 times a^d 7-9 times
greater than those in liver and intestine, respectively. The immature region con-
tained the greatest amount of phosphorus. The phosphorus content in whole
. worms was 1-27 timss and 2-53 timis greater than those in liver and intestine,
respectively. A gradation in the amount of Na was observed along the antero-'
posterior axis of the worm ; the peak value being noticed in the gravid region.
The whole worm; contained 2 • 53 times less Na than that in the liver. The contents
of K and Cu were highest in the immature region. The K content in the worms
was twice as much as that in the intestinal tissues and less than half as much as
that in liver. The amount of Cu in the worms was considerably less than the
amount of Zn. The worms contained significantly higher levels of Cu and Zn
than the liver and intestinal tissues. The worms had a quantitative excess of K
over the other cations studied. fi
Mineral composition of the poultry cestode R. tetragona
155
Table 1. Percentages of Ca, P, Ka, K, Cu and Zn in dry weight of Raillietina
tetragona.
Region
Immature
Mature
Gravid
Ca
mean±SE
0-099 i -006
0-082 ±'003
0/066 ±*005
P values
<Q-05*
<0-05**
<0-002***
P
meaiti SE
0-184 ± '013
0-110. ± -009
0/122 ± '013
P values
<0-002*
>o-i**
<o-oi***
Na
mcan± SE
0-091 i -009
0'120± -008
0-156 db '012
P values
<0,-05*
<0-05**
<Q-OI***
K
mean dr. SE
Ot-538± -018
0-472 ± -021
0-498 db "016
P values
<0-05*
>o-i**
<o-i***
Cu
mean ± SE
Q-OOSi -0008
0-005± -0005
0-Q04±-Oa05
P values
<Q-Q1*
>o-i**
<0-002***
Zit
mean ± SE
a-Q36i '004
0-021 ± -001
0-035 ± -003
P values;
<a-ai*
<0'01**
>o-i***
Probability of sigrtificance of difference between
gravid ; *** gravid and immature,
* immature and mature ; ** mature and
4. Discussion
The importance of inorganic substances to adult cestodes is often demonstrated
by experimental studies involving mineral deficiencies in the host's diet (Chand
1969 ; Deo and Srivastava 1962 ; von Brand 1966 ; Mathur and Pande 1969 ;
Nadakal et al 1975). Ca deficiency in the diet of the host birds, for instance,
leads to dwarfing of the tapeworm Ralllietina cesticillus (Mathur and Pande 1969)
and dwarfing and reduction in the ash and Ca contents of R. tetragona (Nadakal
et al 1975). These findings indicate that the amount of mineral components of
these worms depends on the nutritional condition of the host. In the present
study, since the host birds were maintained on a basal diet containing sufficient
amount of all the essential nutrients, the mineral levels shown by the worms may
be considered to be normal.
A sizeable quantity of mineral components of cestodes is known to be
incorporated in the calcareous corpuscles (Scott et al 1962 ; von Brand 1966). Large
numbers of calcareous corpuscles have been reported in JR. tetragona (Chowdhury
and Singh 1978). The variations observed in the quantitative distribution of the
minerals along the an tero-posterior axis of R. tetragona may reflect a metabolic
gradient that might exist in the strobila.
The pattern of distribution of calcium in the three different regions of R. tetra*
gonai& in conformity with that in Hymenofepis diminuta as reported by Goodchild
et al (1962). The decrease in Ca content in the gravid proglottids may be corre-
lated with the loss of muscular contraction in this region. Shedding of gravid
proglottids maybe facilitated by reduction in Ca con tent posteriorly, sinceits absence
or scarcity affects the integrity of intercellular cement substances (Heilbrunn 1952)
156 AM Nadakal and K Vijayakumaran Nair
Table 2. Percentages of Ca, P, Na, K, Cu and Zn in dry weights of whole
worms (Raillietina tetragona) and certain tissues of its host (Galltis gallus dottiest tons).
Tissues
Whole worms
Liver Intestine
Ca
mean ± SE
0-087 i -004
0-033 ± -004 O'Olli'
•002
P values
<0'001*
<0'001**
P
mean ± SE
0-139 ±'005
O'llOi-012 0'056i
•008
P values
<0-05*
<0'001**
Na
mean ± SE
0-112± -01
0-284 ±-012 0'098 ±
•Oil
P values
<0'001*
>0'1**
K
moin ± SE
Q-50l±-008
1-198 ±'192 0-261±
•029
P values
<Q-01*
<a-ooi**
Cu
mean ± SE
0-006 ±-0003
0-004 ±-0004 0-003 ±
•002
P values
<o-oi*
<0'001**
Zn
mean ± SE
0-031 i'002
0-018 ±-002 0-017 ±
•001
P values
<o-ooi*
<o-ooi**
Blood serum
Ca
mg/lOOml
11-588 ±-636
P
mg/lOOml
4- 866 ±-237
Na
mg/ml
3-210 ±-124
K
mg/ml
0-19Q±-012
Cu
/ig/ml
0-200 ±-014
Zn
MM
0-706 ±-028
Probability of significance of difference between : * worm and liver ; ** worm and intestine
The higher phosphorus content in the immature region may be attributed tc
higher metabolic activity in this region. Singh et al (1978) observed a significant])
higher level of phosphouis in the mature region of Thysaniezia giardi than in its
gravid region. The Ca : P ratios in the worms were higher than those in the
liver and intestine.
A gradual decrease in Ni content along the antero-posterior axis observed in
R. tetragona has also been noticed in H. diminuta (Goodchild et al 1962). The
reasons for this regional difference in distribution is not known. The immature
region contained the greatest amount of K. Potassium, being the major 'base'
of the body cells, may subserve the general functions relating to osmotic pressure
regulation and acid-base balance. The tissues of R. tetragona like liver and intes-
tinal tissues but unlike the serum, showed quantitative excess of K over Na and othei
cations. Goodchild et 07(1962) reported a similar situation in H. diminuta. The
K : Ca ratios in the worms were considerably lower than those in liver and intestinal
tissues, but higher than those in blood serum.
Copper and Zinc are co-factors associated with a number of enzymes including
oxidatiye ensymss, several dehydrogeiiases, phosphatases and cytochrome oxidases,
Mineral composition of the poultry cestode R. tetragona 157
Appreciable amounts of these enzymes in the cestode body have been demonstrated
(Smyth 1969 ; Enigfc et al 1976 ; Vasilev et al 1976). The higher concentration
of Cu and Zn in the immature region of R. tetragona may possibly be due to the
higher enzymatic activity in this region.
Eaigfc et al (1976) found considerably higher levels of electrolytes in the cyst
fluid than in the blood plasma of host and Greichus and Greichus (1980) observed
statistically different concentration of minerals in Ascaris lumbricoides and the
tissues of its host. The presence of higher amounts of cations in R. tetragona
than in the tissues of its host birds may be due to an efficient selective absorption
mechanism prevailing in this worm. Apparently an equilibrium between the
parasites and the host tissues with respect to the minerals was not discernible.
Acknowledgements
Thanks are due to the authorities of Mar Ivanios College, Trivandrum, for the
space and facilities provided and to Dr P K Joy, R & D Manager, Travancore
Titanium Products for permission to use the flame photometer and atomic
absorption spectrophotometer. The senior author is thankful to the University
Grants Commission (New Delhi) for financial assistance under USRT Scheme.
References
von Brand T 1966 Biochemistry of parasites (New York : Academic Press) 1-429
Chand K 1939 The effects of certain drugs and mineral deficiencies on helminths of ruminants;
Indian L Vet. Sci. 9 267-278
Chowdhury N and Singh A I 1978 Role of calcareous corpuscles in the organisation of egg
pouches in Raillietitta spp. ; Z. Parasitenkd. 56 309-312
Clark E P and Collip J B 1925 A study of the Tisdall method for the deter mination of blood
serum calcium with a suggested modification ; /. Biol. Chem. 63 461-464
Deo P G and Srivastava H D 1962 Studies on the effects of different deficient diets on the
natural resistance of chickens to Ascaridia galli (Schrank) Freeborn ; Indian J. Vet. Sci. 32
54-69
Entgk K, Feder H and Dey Hazra A 1976 Mineral content and enzyme activity of Cysticercus
tenuicollis in the sheep and pig ; ZbL Vet. Med. B23 255-264
Fiske C H and Subba Row Y 1925 The colorimetric determination of phosphorus ; J. Biol.
Chem. 66 375-400
Goodchild C G, Dennis E S and Moore J D 1962 Flame photometric studies of helminths :
Calcium, Magnesium and Sodium ia Hymenolepis diminuta ; Exp. Parasitol 12 107-113
Greichus A and Greichus Y A 1980 Identification and quantification of some elements in the
hog roundworm Ascaris lumbricoides suum and certain tissues of its host ; Int. J. Parasitol.
10 89-92
Heilbrunn L V 1952 An outline of general physiology (Philadelphia: W B Saunders).
Jakutowicz K and Korpaczewska W 1979 Determination of copper concentration in 7 parasite
species by atomic absorption spectropho tome try ; Bull. Acad. Pol. Sci. Ser. Sci. Biol. 21
67-70.
Mathur S C and Pande B P 1969 Raillietlna cesticillus and R. tetragona infections in chicks reared
on normal and deficient feeds— an experimental study ; Indian J. Anim. Sci. 39 115-134
158 A M Nadakal and K Vijayakumaraa Nair ..
Nadakal A M, Mohandas A, John K O and Muraleedharan K 1971 Resistance potential
of certain: breeds of domestic fowl exposed to Raillietina tetragona infection. 3. Species of
ants as intermediate hosts of certain fowl cestodes ; Poult. Sci. 50 115-118
Nadakal A M, Mohandas A, John K O and Simon M 1975 Resistance potential of certain
breeds of domestic fowl exposed to Raillietina tetragona infections. XII. Effects of calcium
deficient diet of the host on Raillietina tetragona infections ; Rev. Partsitol. 36 41-46
Salisbury L F and Anderson R J 1939 Concerning the chemical composition of Cysticercus
fasciolaris ; J. BioL Chem. 129 505-517
Scott D B, Nylen M V, von Brand T and Pugh M H 1962 Mineraiogical composition of the
cestode calcareous corpuscles of Taenia taeniaeformis ; Exp. Parasitol. 12 445--45#
Singh B B, Singh K S, Ghosal A K and Dwarakanath P K 1978 Inorganic calcium, magnesium
and phosphorus in Thysaniezia giardi ; Indian J. Parasitol 2 37-38
Smyth J D 1969 The physiology of cestodes (Edinburgh : Oliver and Boyd) 43-51
Vasilev I, Krusteva O and Gorchilova L 1976 Enzymo-histochemical studies of the tegument of
certain species of Raillietina genus ; Klieltnintohgiya 2 42-49
Wardle R A and Me Leod J A 1952 The zoology of tapeworms (Minneapolis : University
of Minnesota Press) 99-104
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91 , Number 2, March 1982, pp. 159-163.
© Printed in India.
A comparison of the electrophoretic haemoglobin pattern of the
commensal rodent species
M S PRADHAN
Zoological Survey of India, Western Regional Station, Pune 411 016, India
MS received 8 August 1981
Abstract. The preseot paper reports the haemoglobin pattern by paper electro-
phoresis of seven rodent and one insectivore commensal species collected from
Bombay-Pune region. Almost all the samples possess 1/1 type of haemoglobin
which is slower in mobility than that of the normal human type. While the genus
Bandicota possesses polymorphic haemoglobin types, it is quite surprising that
Surtcus murirtus has the haemoglobin of anodic mobility as against its Soricidae
counterpart's, Sorex's, haemoglobin showing cathodic mobility.
Keywords. Electroplioresis ; haemoglobin pattern ; commensal species.
1. Introduction
Use of haemoglobin due to its species specificity has been introduced in taxonomy
by modern workers. Of interest to the taxonomists, is the frequent occurrence
of genetically controlled multiple haemoglobins in wild species ; these may be
population, species or genus characteristics. While dealing with 324 mammalian
and 300 vertebrate species, Johnson (1974) and De Smet (1978) showed the simi-
larities and the differences in the mobility of the haemoglobin patterns of various
species. The polymorphism could be located even at the lowest level of the taxo-
nomic groups. In India haemoglobin studies have revealed many variants in man
and in domestic animals (Sukumaran 1975; Naik et al 1969 ; Naik 1975). Wild
rodent populations have yet not been touched so far by the Indian taxonomists
to study the comparative account of the haemoglobin patterns by the electropho-
retic techniques. The present article is an initial attempt to report the haemoglobin
types of some of the Indian commensal rodent species.
2. Materials and methods
Sixtytwo specimens belonging to seven rodent and one insectivore species were
collected from the various residential localities and godown areas of Bombay and
Pune cities. Live rats were caught by a number of methods, like trapping, cyno-
gassing, etc., with the help of workers of the Municipal Corporations. The animals
were sacrificed by cutting their heads on the spot of collection and the blood samples
159
160 MS Pradhan
were collected in the heparinised tubes. The identification of rats was done at
ZSI, WRS, Pune.
Haemoglobin solution was prepared and subjected to paper electrophoresis
following the method of Naik et al (1969) and Wright (1974) with some modifi-
cations. The buffer used for the studies was Barbitone (pH 8 • 6) supplied by M/s
Centron Research Laboratories, Bombay. The electrophoresis was run for four
hours and the strips were studied directly after drying. The anodic mobility of
the haemoglobins of different species was recorded and confirmed by repeated
runs.
Normal human blood samples (twentyfive in total) were provided, as and when
required, by ESIS Hospital Aundh Camp, Pune, for comparison.
3. Results and discussion
The diagrammatic representation shown in figures 1 and 2 of haemoglobins of com-
mensal rodent species clearly indicates the occurrence of Hb-1/1 type of haemo-
globin in these rats except in Bandicota bengalensis kok which shows subspecific
polymorphism. The nomenclature for haemoglobins is given according to John-
son (1974). No minor or trailing fractions could be located in these species.
Hb-1/1 type of haemoglobin has already been reported in the Euresian rodent
species except in those of the genera like Peromyscus and Apodemes (Johnson 1974).
It also appears from the present studies that the haemoglobins of most of the species
belonging to the genera, Rattus, Mus and Bandicota, show relatively slower mobi-
lity than that of the normal human haemoglobin (HbA). However, Johnson
(1974) has reported the equal mobility for normal human and European R. rattus
haemoglobins. R. r. wroughtoni possesses faster moving haemoglobin thatn that
of R. r. rufescens and has the same mobility as that of the normal human type.
That means there is a difference even at the protein level in these two sympatric
subspecies. Tiwari et al (1971) who awarded the specific status to rufescens get
the support from the different Hb patterns of these rats. However, the haemo-
globins in the species like R. norvegicus and Mus musculus show the s^rne mobility
as that of R. r. rufescens. Thus, further studies have become necessary for the
taxonomic confirmation of various species and subspecies of the genus Rattus.
Haemoglobin of the insectivore, Smcus murinus, quite surprisingly showed the
same mobility as that of the normal human type (figure 1). It is interesting to
note that while Johnson (1974) has reported cathodic migration of haemoglobins
for most of the insectivore species, including those of the genus Sorex, the present
observations show the anodic mobility for Swicas marinas. Confirmation and
further studies on the haemoglobins of the order Insectivora will also be interesting.
No common type of haemoglobin could be traced for any of the bandicoot species
under the present studies. All the species possess multiple haemoglobins.
Taking the present findings as sample drawn at random the probabilities of multi-
ple haemoglobins for the seven rodent and one insectivore species can be roughly
estimated to 50 %. De Smet (1978), while comparing the haemoglobins of approxi-
mately 300 vertebrate species, reported 40% occurrence of multiple haemoglobins
in the order Rodentia. He has also pointed out that the existence of intra-sub-
specific haemoglobin polymorphism is a common phenomenon. If the slow moving
Comparison of haemoglobin pattern of rodent species
161
Normal human control
J^ jr. r. Pune
R.JL-I- Bombay
•R.r. w.Bombay
R.n. Bombay
ML m. Pune
B. b. Pune
8. b. Bombay (Dadar)
B. b. Bombay (GhatUopar)
BJ. Pune + Bombay
S.m. Bombay
I
I
I
I
I
I
I
I
I
I
Figure 1. Diagrammatic representation of the haemoglobin pattern of, seven rodent
and one insectivore species collected from Bombay-Pane region.
haemoglobin band of B. b. kok is included, it will be seen that this type of haemo-
globin is a common type found in all the three genera under the present study. If
all the rodent species are studied, an evolutionary trend of rodent haemoglobin
could be unravelled
162 MS Pradhan
Normal human control Bombay
B.g. Bombay ('P ward )
B.jL Bombay CP'ward)
B.lxBombayfP'ward)
Figure 2. Diagrammatic representation of the genus Bandicota haemoglobins show-
ing the heterozygous form trapped from Malad, Bombay,
The B. b. kok populations caught from Bombay-Pune region possess the multi-
ple haemoglobins with a mixing of the two genotypes (figure 2). The populations
from Bombay city ward (Dadar) and Pune city possess slow moving haemoglobin,
while the other collected from a distant suburb (Ghatkopar) on NE side of the
Bombay city has the fastest moving haemoglobin. The animal with heterozygous
haemoglobin depicted in figure 2 was caught from another distant suburb (Malad)
on NW side of the Bombay city. Existence of the different homozygous a,Ueles
for the haemoglobins in the separate populations and also of the heterozygous
form in the subspecies indicates their genetic control over the two polymorphic
haemoglobins. So, if the allelic variation at the genetic loci controlling the struc-
ture of haemoglobin in the kok populations is studied further in detail, it might
be possible to estimate the degree of heterozygosity in the populations. This
evidence can be supported by estimating the degree of variations in the other pro-
teins also. Selander et al (1969) have reported a wide range of genetic variations
in the degree of differences in the wild populations of European house mouse. As
all the proteins are genetically controlled, the effect of these degree of differences
on the morphotaxonomy of the above-mentioned subspecies will be studied in
detail in future.
Acknowledgements
The author acknowledges his sincere gratitude to the Director, Zoological Survey
of India, Calgutta, for granting permission to publish this aritcle. He is also thank-
ful to Dr S N Naik, Cancer Research Institute, Tata Memorial Centre, Bombay,
for going through the manuscript critically and to Dr B K Tikader, Joint Director,
Zoological Survey of India, Poona, for providing the necessary facilities,
Comparison of haemoglobin pattern of rodent species 163
References
De Smet and Willem H C 1978 A comparison of the electrophoretic haemoglobin pattern of
the vertebrates ; Acta ZooL Pathol. Antverp. 70 119-131
Johnson M L 1974 Mammals. In : Biochemical and immunohgical taxonomy of animals (ed.) C A
Wright (London : Academic Press) pp. 1-87
Naik S N 1975 Haemoglobin polymorphism in Indian domesticated and wild ruminants ;
Indian J. Hered. 7 23-30
Naik S N, Sukumaran P K and Sanghvi L D 1969 Haemoglobin Polymorphism in Indian
Zebu cattle; Heredity 24 239-247
Selander R K, Grainger Hunt W and Suh Y Yang 1969 Protein polymorphism and genetic
heterozygosity in two European subspecies of the house mouse ; Evolution 23 379-390
Sukumaran P K 1975 Abnormal haemoglobins in India. In : Trends in haematology (eds.) N N
Sen and A K Basu, J B Chatterjee Memorial Committee, Calcutta, School of Tropical
Medicine, Calcutta, India
Tiwari K K, Ghosh R K and Chakraborty S 1971 Notes on a collection of small mammals
from Western Ghats, with remarks on the status of Rattus rufescens (Gray) and Bandicota
indica malabarica (Shaw) ; /. Bomb. Nat. Hist. Soc. 68 378-384
Wright C A 1974 (ed.) Biochemical and immunological taxonomy of animals^ 1st Edition
(London : Academic Press) pp. 490 -f xii.
Abbreviations
R. r.r. . . Rattus rattus rufescens
Rt r.w. . . Rattus rattus wroughtoni
R.n. . . Rattus norvegicus
M.m. . . Mus musculus
B.b. . . Bandicota bengalensis
B. i. , . Bandicota indica
B.g. .. Bandicota gigantea
S.m. . . Suncus murinus
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 2, March 1982, pp. 165-176.
© Printed in India.
Studies on egg and nymphal parasites of rice planthoppers,
Nilapavvata lugens (Stal) and Sogatella furcifera (Horvath)*
J S BENTUR, MANGAL SAIN and M B KALODE
All India Coordinated Rice Improvement Project, Rajendranagar , Hyderabad 500 030
India
MS received 28 April 1981 ; revised 16 January 1982
Abstract. Three species of egg parasites, viz. , Anagms Sp. , A. optabilis (Mymaridae)
and Oligosita sp. (Trichogrammatidae), and a nymphal/adult parasite Gonatopus sp.
of rice planthoppers were studied for their biology and control potential. Larger
number of adult mymarids emerged from host eggs between 8-30 a.m. and
12-30 p.m. of the day whereas trichogrammatid adults emerged between 12-30 p.m.
to 4-30 p.m. All the three species parasitised both brown planthopper (BPH) and
white backed planthopper (WBPH) but, in general, failed to parasitise rice leaf-
hoppers.
Developmental duration from oviposition to adult emergence noted for these
parasites indicated that males of mymarids, in general, developed faster (10-11 days)
than females (12-13 days) at 20-32° C prevailing during October, whereas Oligosita
females developed more slowly (14-15 days). However, both A, optabilis and Oligosita
developed three days faster at 30-38° C prevailing during April. Fecundity in
terms of number of eggs parasitised per female varied from 12-3 to 20-3. Under
greenhouse conditions, release of 1 and 5 pairs of mymarid parasites for 10 days
reduced the nymphal hatch of BPH by 60 and 85%, respectively. Nymphal/adult
parasite Gonatopus sp. completed its life cycle in 19-5 to 31 days OB both BPH and
WBPH. While the 4th and 5th instar nymphs of BPH were parasitised more fre-
quently, green leafhopper nymphs were not parasitised. Besides being endopara-
sitic, the adult females also predated on and killed as many as 5*2 nymphs a day.
Keywords. Anagrus spp. Gonatopus ; parasitoids ; rice planthoppers.
1. Introduction
Rice planthoppers have gained major pest status causing 'hopper burn' in several
rice growing Asian countries . Outbreaks of brown planthopper Nilaparvata lugen
(Stal) have been reported in different parts of India (Kalode 1974 ; Kulshrestha
et al 1974). Also, white backed planthopper, Sogatella furcifera Horvath, is
noted to cause dam age in northern India (Verma et al 1979), while smaller brown
planthopper, Laodelphax striatellus (Fallen) has been reported from the Punjab
(Shukla 1979).
* Al CRIP Publication No. : 231
165
166 / S Sentur, Mangat Sain and M B Kalode
Twentyfour species of egg parasites and 30 species of nymphal parasites have
been recorded mainly on brown planthopper (BPH), besides three species of nema-
tode parasites, 11 species of pathogenic fungi and 61 species of insect and spider
predators are also reported (Anonymous 1978 ; Manjunath 1978 ; Manjunath
et al 1978 ; Chiu 1979).
From India, except for a brief account of Anagrus sp. (Samal and Misra 1978),
no detailed study seems to have been made of egg or nymphal/adult parasites of
rice planthoppers. In the present investigation 3 species of egg parasites, viz.,
Anagrus sp., Anagrus ? optabilis (Perkins) (Mymaridae) and Oligosita sp. (Tricho-
grammatidae) and nymphal adult parasite, Gonatopus sp. (Dryinidae) were investi-
gated with respect to their biology, host range and biocontrol potential against
the planthoppers. A. optabilis has been reported here for the first time from India,
while genus Gonatopus on BPH is a new record.
2. Materials and methods
2-1. Rearing
2. la Egg parasites: Rearing was initiated with the parasitised eggs collected
from rice plants in the field and glasshouse. Leaf sheaths of plants were peeled out
and kept in glass jars containing a little water at the bottom and covered by an
inverted glass funneL A glass tube was kept inverted on the glass funnel. Adults
emerging from host eggs through leaf sheaths moved upwards and were collected
in the glass tubes. Populations of parasites were further built up by exposingnew
plants to parasites on which eggs had been freshly laid and later keeping such
plants in jars for adult collection (figure 1). All the three species of parasites were
reared together. Only for experimental purpose adult parasites were differentiated
under binocular microscope. Except when specified, rearing and biology studies
were carried out in the laboratory at room temperature ranging from 20-38° C
and, humidity varying from 30 to 80% RH.
2- Ib Nymphal parasite : Parasite pupae from affected brown planthopper
culture in glasshouse were collected in tubes to initiate rearing. Emerging Gona-
topus adults were fed with honey solution, and mated females were released on
T(N)1 rice plants along with the nymphs of BPH and WBPH, Adult parasites
were transferred every day to fresh plants with healthy nymphs kept in separate
cages to avoid nymphal mortality due to predation. All the studies were con->
ducted in the glasshouse with host insects reared on T(N)1 plants at 30 ± 5° C and
80 ± 10% RH.
2.2. Experiments
Studies on egg parasites were carried out using individual rice seedlings (15-20 day
old) kept in test tubes. Gravid females of BPH/WBPH were confined in such
tubes for 24 hr for oviposition prior to the release of parasites. After 6-7 days
plants were dissected to count number of eggs parasitised and such eggs were then
kept on moist filter paper in glass tubes with screw caps to observe adult emergence.
Nymphal parasites of rice planthoppers
167
Figure 1. Laboratory rearing method for the egg parasites of rice planthoppers
Nilaparvata lugens and Sogatella furcifera. Adult plantho-ppers are released on
T(N)1 plants for oviposition (pot 1), followed by parasite release for paras itisation
(pot 2). After healthy planthopper eggs hatch (pot 3), plants are cut to be kept
in adult emergence jars to collect the emerging adults.
Nymphal parasites of rice planthoppers
169
Life cycle of nymphal parasite, Gonatopus, was. studied by offering planthopper
nymphs to mated females confined on rice plants in mylar film cages. Similar
set-up was. used for experiments to determine suitable nymphal instar for parasiti-
zation, host range and to estimate predation by adults.
3. Results
3-1. Egg parasites
3. la Adults: Mymarid adults (Anagms ~spp.) are brown in colour with fringed
wings, body measuring 0-7 to 0-9 mm in length, while Ollgosita is greenish yellow
in colour with smaller body of about 0-5 mm with rounded wings. However,
Artagrus sp. has bulkier abdomen with short ovipositor whereas A. optabilis has a
slender abdomen with long and prominent ovipositor. In the laboratory popula-
tion of mymarids, females were more numerous than males (sex ratio of 5 females :
1 male). The virgin females parasitised host eggs normally thus indicating thely-
tokous development. Adults of myiuarids lived for 24-^36 hr without any food
and those of Ollgosita survived for 24-48 hr. Males of Ollgosita were not observed
and hence this species appears to be uniparental.
Adult emergence patterns were noted for the three species by recording the
number of adults emerging during different time intervals of the day. Results,
illustrated in figure 2, indicated more than 70% of mymarid adults emerged bet-
ween 8-30 a.m. and 12-30 p.m. whereas maximum percentage of trichogrammatid
adults emerged between 12-30 p.m. and 4-30 p.m.
emergirig
60
20
n
-
MYMAR
f Arvagrus
^""Jj
0
TRICHOC»RAMMA
1 8°
<2Ha2«±
. sp.)
1
£ ^o
a
20
0
_ ,
1
8.30am 1030am 12.30pm 4.30pm B.30am
Time o< the day
Figure 2. Adult emergence patterns for mymarid (Anagrus sp. and A. optabilis)
and trichogrammatid (Oligosita sp.) egg parasites of rice planthoppers Nilaparvata
lugens ard Sogatella furdfera. It may be noted that while mymarids emerge before
noon, trichogrammatids emerge during afternoon.
170 / S Bentur, Mongol Sain and M B Kalode
3. Ib Developmental duration and fecundity : The entire life cycle of egg parasites
was completed inside the host egg. All three species parasitised the eggs of both
brown planthopper and white backed planthopper. Eggs parasitised by mymarids
turned yellow by day 7, assuming orange to red-brown colour by day 8 and 9
when pupation occurred inside the eggs. Those parasitised by Oligosita turned
greenish yellow by 7-8th day without further change in colour.
The data on duration of development, presented in table 1, indicated that males
of mymarids, in general, developed faster (10-« 11 days) than females (12-43 days)
while the Oligostta females developed slowly (14-15 days) at 20-32° C prevailing
during October 1979. However, both A. optabilis and Oligosita developed 3 days
faster at 30-^38° C prevailing during April 1980. A female mymarid could para-
sitise 15-20 planthopper eggs whereas that of the Oligosita parasitised 12^18 eggs,
3. Ic Host range : As noted above the 3 species of parasites readily parasi-
tised both the species of rice planthoppers. However, they failed to parasitise
eggs of rice leafhoppers, viz., Nephotettix spp., Inazuma dorsalis and Tettigellc
spectra.
3. Id Biocontrol potential : An experiment was conducted under glasshouse
conditions to note the potential of mymarid parasites in checking egg hatching o1
brown planthopper. One pair of newly emerged brown planthopper was flrsi
caged on T(N)1 plants followed by daily release of one and five pairs of Anagrw
parasites into the cage from day 5 to day 15. As indicated in table 2, release
of 1 pair of mymarids reduced the egg hatch by about 60%, while release of 5 pain
reduced it by 85% indicating a high biocontrol potential of the egg parasites.
Table 1. Developmental duration and fecundity of egg parasites of planthoppci
Nilaparvata lugens (BPH) and Sogatella furcifera (WBPH).
Development duration* Fecundity
(days) (no. of eggs
Parasite Host parasitised/
Male Female female)
Anagrus sp.
BPH
13-5
12-6
20-3
WBPH
...
10-8
14-5
A. optabilis
BPH
11-0
12-1
17-5
9.2**
WBPH
10-4
12-1
19-0
Oligosita sp. BPH ... 13-8 18-0
10-6**
WBPH ... 15-3 12-3
* Development observed during October 1979 (temp, ranging 20-1 to 31-8° C) and
** April 1980 (29-1 to 38-0°C).
Nymphal parasites of rice ptanthoppers 171
Table 2. Influence of mymarid parasites (Anagrus spp.) on hatching of Nilapar-
vata lugens eggs.
Treatment No. of eggs hatched/ % reduction
female* in viability
MeaniSE
Control 439«3i50-3a
1 pair of parasites
released from day 5
to 15 199-3 ±25-7b 59-5
5 pairs of parasites
released from day
5 to 15 66-2 i 11-4° 84-9
* mean of 6 replications.
comparison of means: a-b ; a-c and b-c, p< 0-001 (Mest).
3.2. Nymphal parasite
3.2a Adults : Adult female Gonatopus resembles an ant in appearance but
can be distinguished from the latter by the presence of chela te fore tarsi adapted
for catching the prey. While the females are apterous (figure 3), males have mem-
branous wings and are more active than females. Body size is. smaller (2 to 3 mm
in length) in the case of males than in females which measure 4 to 5 mm in length,
and have a dark black body. Adult longevity ranged from 7 to 10 days for males
and 15 to 20 days for females when provided with honey solution.
Life cycle of Gonatopus was. studied by offering nymphs of both BPHand WBPH
to mated females for ovipositon and later observing these nymphs periodically.
The female parasite holds 4th or 5th instar planthopper nymphs with its forelegs,
bends its abdomen and thrusts eggs into the host body (figure 3) . The parasitised
nymphs are immobilised for 2-3 min before they move off. Since eggs are laid
internally, incubation period could not be exactly determined, but small black or
yellow sac-like structures (larval sacs) appear on the abdomen of nymphs 3-5 days
after oviposition (figure 3). The larval sacs, one or two per nymph, containing
larvae enlarge gradually. At the end of the larval period, ranging from 7 to 12
days, the larval sac bursts and a small 2-4 mm long yellowish white or sometimes
pinkish larva crawls, out killing the host. Prepupal stage lasts for 12 to 24 hr and
pupation takes place either on the rice plant or on the sides of cages. Prior to
pupation, the larva secretes an yellowish-white fluid to forma membranous, oval
shaped puparium (figure 3). Adults emerge after 9 to 12 days. The total life
cycle of Gonatopus takes 19-5 to 31 days on BPHand 24-5 to 31 days on WBPH
(table 3).
Since the dryinid normally selects only older nymphs or adult hosts for oviposi-
tion, suitability of different nymphal instars. for parasitisation was studied. As
presented in table 4, maximum parasitisation (larval sacs) per female was observed
172 J S Bentur, Mongol Sain and M B Kalode
Table 3. Life cycle of nymphal parasite, Gonatopus sp. on Nilaparvata lugens and
Sogatdhi fiifdfera.
Incubation
Larval
Prc-pupal
Pupal period
Total develop-
Host
period
period
period
(days)
mental duration
(days)
(days)
(hours)
(days)
N. lugens
3-5
7-12
12-24
9-13
19-5-31-0
S. furcifera
3-5
10-12
12-24
11-13
24-5-31-0
Table 4. Parasitisation of different instar nymphs and adults of Nilaparvata lugens
by Gojiatopus sp.
Host instar
No. of
insects
studied*
No. of
parasites
released
No. of
nymphs
parasitised
Av. No. of nymphs
parasitised/
jfemale/day
1st instar nymph
80
8
0
0
2nd instar nymph
80
8
0
0
3rd instar nymph
80
8
8
1
4th instar nymph
SO
8
40
5
5th instar nymph
80
8
48
. 6
Adults
SO ,
8
24
3
* based on four replications at 20 insects/replication.
in 5th instar nymphs (av. number 6) followed by 4th instar nymphs (5) and
adults (3).
3-2b Hast range : Host range and preference for parasitisation by Gonatopus
was noted by offering 4th and 5th instar nymphs of BPH, WBPH and green leaf-
hoppers (Nephotettix spp.). It is evident from table 5, that Gonatopus preferred
BPH to WBPH since 13 out of 30 BPH nymphs were parasitised as against 9 of
WBPH, while it did not parasitise green leafhopper nymphs.
3.2c Nymph predatioH : Besides parasitisation of older nymphs, Goftatopus
females were also observed to predate on younger nymphs. The observation on
the extent of nymphal predation showed that one female could kill on an average
5-2 nymphs per day. These findings revealed that dryinid, Gonatopus, had a good
biocontrol potential against planthoppers both as nymphal/adult parasite as well
as nymphal predator. :
Nymphal parasites of rice planthoppers
173
Figure 3. Nymphal/adult parasite (Gonatopus sp.) of rice planthoppers, Nilaparvata
lugens and Sogatella furdfera. a. Apterous adult female ; b. a prey being held
by a female for oviposition ; c. an adult planthopper with larval sac ; d. fully
grown larva (B) and pupa in puparium CA).
Nymphal parasites of rice planthoppers 175
Table 5. Host range and preference for parasitisation by Gonatopus sp.
Cumulative number of
insects parasitised after...
Host No. of No. of
host insects parasites 3 days 5 days
studied* released
Brown planthopper 30 15 10 13
White backed plant-
hopper
Green leaf hopper
30
30
15
15
5
0
9
0
* Total of 3 replications at 10 insects/replication.
4. Discussion
Adult emergence pattern has been noted for Paracentrobia andoi, a trjchogrammatid
egg parasite of the leafhopper, Nephotettix cincticeps (Vungsilabutr 1978). Most
of the adults emerged between 8 a.m. and 12 noon. In the present work, while
mymarids had a peak emergence before noon, trichogrammatid adults emerged
during afternoon. Samal and Misra (1978) noted the development period for
Anagrus sp. to be 12 to 14 days during April- May with temperatures ranging from
24-4 to 35° C. Our results for Anagrus sp. show that the developmental period
was. 11 to 14 days during October. The parasite took lesser time to develop
on WBPH than it did on BPH. It is not known, however, if these two species of
parasites are the same or different. Temperature effects on the rate of develop-
ment have been elaborately investigated for P. andoi (Vungsilabutr 1978). Though
in the present study constant temperatures were not maintained, differences in
developmental duration observed for A. optabilis and Oligosita sp. noted during
October (temp, ranged from 20*1 to 31-8°C) and April (29-1 to 38° C) essen-
tially reflect the effect of temperature.
Though all the three species readily parasitised both BPH and WBPH eggs,
mymarids failed to parasitise leafhoppers. The preference of mymarid parasites
among the planthoppers and host range of the trichogrammatid are yet to be
studied in detail. It is not uncommon, however, for an egg parasite to have both
leaf and planthopper hosts as noted for P. andoi (Vungsilabutr 1978) and many
other species (Anonymous 1978).
Studies on Gonatopus sp. substantiate the view that dryinids would make good
agents for biological control of injurious Cicadellidae and Fulgoridae (Olmi 1976).
Biological control of rice planthoppers through the use of natural enemies has
so far not been attempted in field scale. However, the only suggested candidate
for this purpose— the egg^ymphal predator Cyrtorhirtus Uvidipennis has a wider
range of predation (Bentur and Kalode 1980). In contrast, the egg parasites and
176 / S Bentur, Mangal Sain and M B Kalode
the nymphal parasite investigated in the present work have host range restricted
only to brown planthopper and white backed planthopper and also possess a good
degree of control potential. They can also be considered for use in field along
with C. lividipennis. Nevertheless, development of economical mass rearing
methods., information on the behaviour of released population under field condi-
tions and knowledge of mutual interaction of natural enemies are prerequisites for
any such attempts.
Acknowledgements
The authors are thankful to Dr R Seetharaman for providing necessary facilities
and encouragement. Thanks are also due to Dr (Mrs) Sudha Nagarakatti for
suggestions, and to Dr N C Pant, Director, Commonwealth Institute of Ento-
mology, British Museum, London, for identification of parasites.
References
Anonymous 1978 Prospects for biological control of rice hoppers— A status paper, Common-
wealth Institute of Biological Control, p. 12
Bentur J S and Kalode M B 1980 Biocontrol studies on leaf and planthoppers. Proc. 3rd
Workshop of AH India Coordinated Research Project on Biological Control of Crop Pests
and Weeds, Ludhiana, Indian Council of Agricultural Research, New Delhi, pp. 103-10$
Chtu S G 1979 Biological and cultural control of the brown planthopper pp. 335-355. In :
Brown planthopper : Threat to rice production in Asia, International Rice Research Institute,
Los Banos, Philippines
Kalode ML B 1974 Recent changes in relative pest status of rice insects as influenced by cul-
tural, ecological and genetic factors; Intern. Rice Res. Conference, April 1974, IRRI,
Los Banos
Kulshrestha J P, Anjaneyulu A and Padmanabhan SY 1974 The disastrous brown planthopper
attack in Kerala ; Indian Farm 24 5-7
Manjunath T M 197& Two nematode parasites of rice brown planthopper in India ; Int.
Rice Res. Newslett. 3 11-12
Manjunath T M, Rai P S and Gowda G 1978 Parasites and predators of Nilaparvata lugens in
India ; PANS 24 265-269
Olmi M 1976 Experience and prospects of biological control with Dryinidae (Hymenoptera :
Bethylaidea) In: Atti XI Congresso Nazlonale Italiano di Entomologia (English Abstract)
pp. 371-373
Samal P and Misra B C 197$ Notes on egg parasites of the brown planthopper Nilaparvata
lugens (Stal) in Orissa ; Oryza 15 96-98
Shukla K K 1979 Occurrence of a new insect, small brown planthopper, Loadelphax strlatellus
(Fallen) in India ; Curr. $ci. 48 548
Verma S K, Pathak P K, Singh B N and Lai M N 1979 Occurrence of brown and whitebacked
planthoppers in Uttar Pradesh, India ; Int. Rice Res. Ne\vslett. 4 20
abutr P 197$ Biological and morphological studies of Paracentrobia andoi (Ishii)
(Hymenoptera : Trichogrammatidae), a parasite of the green leafhopper, Nephotettix cincti-
ceps Uhler (Homoptera : Deltocephalidae) ; Esakia 11 29-51
Proc. Indian Acad. Sci. (Auim. Sci.), Vol. 91, Number 2, March 1982, pp. 177-187.
© Printed in India
New natural enemy complex of some fiilgoroids (Insecta: Homoptera)
with biological studies of three hymenopterous parasites
(Insecta : Hymenoptera)
S SWAMINATHAN* and T N ANANTHAKRISHNAN
Entomology Research Institute, Loyola College, Madras 600034, India
* Present address : Department of Zoology, Ramakrishna Mission, Vivekananda
College, Mylapore, Madras 600 004, India
MS received 4 January 19S2 ; revised 25 February 1982
Abstract. Natural enemy complex of the planthoppers, Dichoptera hyalinata F.,
Eurybrachys tomentosa F., and Ricania fenestraia F. includes two nymphal ecto-
parasites (Dryinus spp.), two egg parasites (Proleurocems fulgoridis F. and Tetra-
stichus sp.), an internal larval mermithid parasite, and a predator (Phidippus sp.).
Biological aspects of Dryinus spp. and P. fulgoridis are discussed.
Keywords. Fulgaroidea ; natural enemies ; parasite; predator.
1. Introduction
The planthoppers, an economically very important group as pests and vectors of
plant diseases, were investigated in relation to their association with crops as well
as their natural enemies. In India the bionomics and effectiveness of the natural
enemies of fulgoroids were studied extensively with reference to Pyrilla spp. (Rah-
man and Ramnath 1940 ; Rahman 1941 ; Sen 1948 ; Narayanan and Kundanlal
1953 ; Subba Rao 1957), Nilaparvata lugens Stal (Abraham et al 1973; Kalode
1976 ; Manjunath 1978 ;. Manjunath et al I978a,b ; Rai and Chandrasekhar
1979 ; Samaland Misra 1975, 1978a,b)and Sogatellafurciferallorvaih (Chaudhury
and Ramzan 1968 ; Israel and Prakasa Rao 1969). The present paper brings to
light the occurrence of new natural enemies of the fulgoroids, Eurybrachys tomen-
tosa F. (Eurybrachidae, Fulgoroidea), Ricania fenestrata F. (Ricaniidae, Fulgo-»
roidea) and Dichoptera hyalinata F. (Dictyopharidae, Fulgoroidea), the first two
being pests of important crops such as- Santalum album, Zizypus jujuba, Cajanus
indicus, Calotropis gigarttea, Camellia sinensis, Gossypium spp., Jasminum flexile,
etc. (Chatterjee 1933; Hutson 1919 ; Light 1929 ; Puttarudriah and Maheswariah
1958). Besides, the biology of two ectoparasites, Dryinus spp. (Dryinidae, Bethy-
loidea) and an egg parasite, Proleurocerus julgor idis F. (Encyrtidae, Chalcidoidea)
are discussed.
177
S Swaminathan and T N Ananthakrishnan
methods
tsitized eggs (in the case of egg parasites) and nymphs (in the case of ecto-
i) were brought from the field and reared in the laboratory for adult emer-
The emerged adult parasites were caged in small glass chimneys (110 ml) or
ials (10 ml, 15 ml) (figure 1A), and were fed with a dilute sucrose solution
\ cotton, the latter being fixed on a wire projecting inside the containers,
laid egg masses of E. tomentosa and fresh nymphs of E. tomentosa and
nata were provided in the cages for the egg parasites, and ectoparasites
rely to enable the parasites to lay their eggs. From the day of parasiti-
me egg from the same batch of parasitized eggs was dissected out daily to
e sequence of changes in the life cycle of the egg parasites. Parasitized
iphs. were caged separately and the larval development of the ectoparasites
erved. The larvae of the ectoparasites emerging from the hosts were
to pupate on a glass surface (figure 2C), which enabled observation of
ivelopmant. Laboratory conditions during the present study were 19° O
and 60%-90% (relative humidity).
cidertce of new parasites and predators
iphs of D. hyalinata were parasitized by an ectoparasite, Dryinus sp. (A)*
ae), while another dryinid, Dryimis sp. (R)*, was recorded on the nymphs
mentosa. The eggs of E. tomentosa were parasitized by an encyrtid,
cerus fulgoridis, and an eulophid, Tetrastiches sp., while the adults of this
ere parasitized by a larval mermithid. In the case of JR.. fenestrata a
predator, Phldippussp., was recorded as a natural enemy (figures IB to IF).
/ations on the seasonal cycles of the parasites (figure 3) revealed that
sp. (A) occurred in the field for 7 months, P. fulgoridis for 5 months,
sp. (R) for 3 months, and Tetrastichus sp. for a month. Dryinus sp. (A)
tie peak of its activity during November followed by the absence of the
unphal population during the four succeeding months, and their parasitic
was minimum during July. P. fulgoridis was very active during February
*ch, when all the egg masses of E. tomentosa collected from the field fvere
> be -parasitized by this parasite. Parasitization by Tetrastichus sp. was
, occurring only during April. Both P. fulgoridis and Tetrastichus sp.
;ed the same egg mass of E. tomentosa and in one instance the former parasi-
eggs of an egg mass, the latter 39 eggs. Occurring on its host only during
lon-ths, the ability of Dryinus sp. (B) to suppress the population of its host,
ifosa appeared to be less pronounced than that of Dryinus sp. (A) on its'
. hyalinata. Adults of E. tomentosa affected by a larval mermithid
isite were also identified and the parasitized adults appeared pale and
> Dryinus spp. have been designated as (A) and (B) as they have been identified to
*ew species (Dr Z Boucek, Commonwealth Institute of Entomology, Londpn—
communication). Being very host specific the identity of the species (A) and (B)
: be confusing.
D
I
Natural enemy complex of some fulgoroids
179
Figure 1. A. Rearing cages for the parasites, B. Proleurocems fulgoridis, C. Tetrastichus sp.,
D. Phidippus sp., E. Dryinus sp.(B). Male, F. Dryinus sp. (A)- Female (B, C, E- Scale = 1 mm ;
D, F— Scale = 5mm).
180
S Swammathan and T N Ananthakrishnan
Figure 2. A. Parasitized E. tomentosa nymph showing thalacium. B, C. Papa of
Dryinus $p. (B). D. Parasitized D. hyalinata nymph showing thalacium
E. Pupa of Dryinus sp.(A) (Scale = 5mm).
Natural enemy complex of some fulgoroids 181
Dry'mus sp.
(B)
Tetrastlchus sp.
Proleurocerus fulgoridis
Eurybrachys torr.entosct
tDryinus sp
(Al
Dichoptera hyalinata
Low Incidence
JFMAMJ J A S 0 H D
Figure 3. Seasonal cycle of hast and parasites.
seemed distinctly inactive. All the life stages of R. fenestrata except eggs were
actively predated upon by the spider, Phidippus-sp. (Salticidae, Arachnida) in the
field as well as under laboratory conditions. Besides feeding on R. fenestrata,
Phidippus sp. also fed on other insects found in the same habitat, but less
frequently.
3.2. Biology of parasites
3.2a Drymus.&p. (B) : — The adult parasites with a shining black body were
observed in the field actively moving around the plants in search of their host.
When the parasite actively chased the host for oviposition, a distinct parasitic
behaviour was noticed (Swaminathan and Ananthakrishnan 1981). Mostly second
and third instars of the host were preferred for parasitization by the females, while
in the laboratory they were able to parasitize the first, second, and third instars,
and sometimes the fourth instar nymphs as well. The fifth instar nymphs of the
hosts easily escaped from the attack of the parasite by kicking and jumping. In
all the first instar host nymphs examined, the egg-deposition and development of
the 'thalacium' were noticed only under the hind wing pad, while in the second
and third instar host nymphs, parasitization was under both the wing pads. In
the fourth instar nymphs, the parasite larva failed to develop after a certain stage ;
even in cases when it successfully completed its delayed larval life, and left the
host, it finally died before spinning the cocoon. The average time taken to complete
oviposition increased as the size of the host increased in the successive instars,
being 95 sec, 117-9 sec, 172-5 sec, and 235 sec in first, second, third, and fourth
instars respectively. The time interval between two successive acts of oviposition
was 5-40 min. Eggs were usually laid beneath the wing pads and in the laboratory
each host carried only one egg. Dryinus sp. (B) female laid more than one egg/host
under different wing pads if the same host was exposed for a long time and p.o
other hosts were available. A minimum of 1 egg/host and a maximum of 2 eggs/
host were recorded in the laboratory, but in the field the host nymphs generally
showed only one egg.
The eggs were elongate, cylindrical and translucent. In dryinids the first larval
instar is spent entirely within the host (Clausen 1940). Three to four days after
182 S Swaminathan and T N Ananthakrishnan
oviposition the parasite developed a bag-like structure (figure 2A) at the oviposited
site on the host's body. This bag-like structure the 'thalacium', was suggested
to be formed by a proliferation of the host integument (Subba Rao 1957). The
cyst membrane of the thalacium is found all over the parasite larva thus preventing
direct con tact of the larva with the body cavity of the host and the larva derives
all food material through this membrane (Clausen 1940). The whitish and trans-
lucent thalacium gradually turned brown after 3 days. The parasite larva deve-
loped inside the thalacium slowly sucking the haemolymph pf the host without
affecting the latter's life activities. With the establishment of the thalacium on
host, the planthopper nymphs lost their ability to moult further, particularly when
the nymphs were parasitized at the end of their stadial period. In the laboratory
only in three instances two larvae were found to develop on a single host. Such
parasites developed one on either side or on the same side under the wing pads.
If two thalacia developed from the same host, only one parasite larva fully completed
its. development and successfully pupated while the other showed partial develop-
ment ultimately perishing along with the host. Within the thalacium the larva under-
went three moults, and the mature larva sucked out most of the haemolymph of
the host before leaving it. The resultant enlargement caused a cleavage line antero-
posteriorly in the thalacium, through which the emerging parasite larva crawled out
and dropped down to the substratum. Owing to extensive feeding by the escaping
larva, the host suffered excessive shrinkage aoid died. The mature larva (2-5-4 mm
long and 0-75->l mm wide) appeared dull white in colour with a pointed anterior
end and bulging posterior end. It soon started building a white cocoon on the
leaves or stems (figure 2B). In ths laboratory, the cocoon spinning was also noticed
on the wall of the glass containers (figure 2C) and cloth, within 5 min of escape
from the host. While spinning the pupal case with white silken threads secreted
from the mouth, the larva entered the cocoon by peristaltic movements. The large
well-developed mandibles were efficiently used in cocoon building, particularly in
cutting the threads. The fully formed cocoon was generally oval, double-walled
— a tightly^spun inner and loosely-spun outer wall — measuring on an average 7 mm
long and 1\ mm wide.
Though the parasite larva entered the cocoon immediately after leaving the
host, the actual pupation took plaxre only after 4->5 days. During this period the
brownish larva showed peristaltic movements inside the cocoon, subsequently
turning reddish brown, and resulting in a complete pupa exhibiting swift and fre«
quent back and forth movements for 10 days. On the, 7th^8th day after spinning
cocoon, wings and limb buds developed and the demarcation of the head, thorax,
and abdoimn was evident. During the late pupal period there was a deposition
of black excretory material in the caudal end of the cocoon. On the 15th day after
spinning the cocoon, the pupa turned fully black and there was a cessation of
movements.
Theactive male and female adults emerged from the cocoon by making a hole at
the anterior end. Both parthenogenetic and sexual reproduction were observed, the
former always resulting in male offspring. Under lab oratory conditions the average
life span of adult female and male was* 25 days and 16 days respectively, the
average oviposition period of females being 20 days and the average total number
of eggs laid by a single female was 35. The average total duration of egg, larval
Natural enemy complex of some fulgoroids 183
Table 1. Duration of various stages in the life-cycle of parasites (in days).
Species Period between Duration of the
egg-laying atl(i larval stage spent
Period spent in
the cocoon
Total life-
cycle
thalacium formation
in thalacium
Dryinus sp.(A)
3
4
23
30
4
6
23
33
2
4
25
31
6
6
26
38
6
5
23
34
Mean
4-2± 1-6
5 ±0-89
24 ±1-27
33-2± 2-
79
Dryinus Sp. (B)
4
8
25
37
3
8
22
33
3
9
23
35
3
12
23
38
4
8
23
35
Mean
3-4±0-49
9 ±1-55
23-2iO-9*
35-6±l-
74
Egg
Larva
Pupa
Total
Proleurocerus
fulgoridis
2
3
4
9
2
4
5
11
2
4
4
10
2
4
9
15
2
4
10
16
Mean
2±0
3-8 :± 0-4
6-4±2-58
12-2±2
•7 9
and pupal periods was 35-6 ± 1-74 days and the parasite spent more time in the
cocoon (23-2 ±0-98 days) than in the thalacium (9 + 1-55 days) (table 1).
3-2b Dryinus sp. (A) : The life cycle appeared similar to that of Dryinus sp.
(B) with only some minor variations. The average total duration of egg, larva],
and pupal periods was 33-2 ± 2-79 days (table 1). Eggs were laid beneath the
wing pads, in the dorsal middle region of the thoracic segments, and on the
dorsal lateral region of the abdominal segments. Midthoracic region was highly
preferred for egg laying. Each host carried 1-2 parasite larvae both under
laboratory and field conditions.
The larvae developed inside the thalacium (figure 2D) which was gelatinous
white during the first day of its formation and turned brown after 2->3 days. As
in Dryinus sp. (B), when parasitized by two larvae, one developed faster than the
other and pupated, while the other died with the host. The larva underwent three
moults inside the thalacium. While leaving the host, the larva fed on most of the
184 S Swaminathan and T N Ananthakrishnan
host haemolymph and escaped from the thalacium by rupturing it while the host
was killed. Immediately after leaving the host, the larva formed an oval cocoon
with two walls of silken threads, on the bark of the host plant (figure 2E). The
colour of the cocoon varied with the surrounding and was brownish (on bark in the
field) or grey (on cloth in rearing cages in the laboratory). Both in the laboratory
and under field conditions the second, third, and fourth instar host nymphs were
susceptible to parasite attack. All the behavioural patterns and methods of
oviposition were similar to those of Dryinus sp. (B).
3-2c Proleurocems fulgoridis : The eggs of this encyrtid parasite were fusiform
and stalked. The average length and width of the eggs were 507-5 ^ and 160-2 //
respectively, while the stalk measured 267-0 //. The eggs were laid singly
within the host egg. The durations of egg and larval stages were 2 days and
3-8 ±0-4 days respectively (table 1). On the second and third day of larval
development the larva grows to a maximum size by consuming all the contents of
the host egg. The fully grown larva measured 1 • 78 mm long and 0 • 68 mm wide
with 12 segments (figure 4). During the development of the larva of the parasite
the host egg showed no colour change. Inside the host egg a constant peristaltic
movement of the parasite larva was noticed. Pupation resulted on the 5th or 7th
day of parasitization and the pupal period lasted 6-4 ±2-58 days (table 1). With
the pupation of the parasite the colour of the host egg changed to brown. The
host nymphs from unparasitized eggs in a partially parasitized egg mass always
hatched 1-2 days before the parasites emerged.
The adult parasites (figure IB) were shiny black and started ma ting as they emer-
ged from the host egg. The males chased the females and while moving, in front
of the females, vibrated their half-extended wings. The longevity of the adults
was 2-4 days when fed with 5% sucrose solution. The females started laying eggs
shortly after emergence. Young host egg masses (2-3 days old) were preferred
for oviposition. Usually 1-^2 females attacked a single egg mass under field condi-
tions. Each female took 5^6 hr to complete egg laying. As soon as a gravid
female located a fresh egg mass of E. tomentosa, it moved over it for sometime
and then started laying eggs. After making punctures on the waxy coat at many
places, the females inserted their ovipositors and laid eggs. Superparasitism was
not observed. In the egg masses of E. tomentosa 40-59%-100% of the eggs were
found parasitized under field condition (table 2).
0-5mm
Larva
Figure 4. Immature stages of Proleijrocerus fulgoridi§.
Natural enemy complex of some fulgoroids 185
Table 2. Percentage paras itization of individual egg masses of Eurybrachys
tomentosa by Proleurocerus fulgoridis under field condition.
Total number of
eggs in a mass
Number of para-
sitized eggs
Number of
unparasitized
eggs
Percentage of eggs
parasitized in a
mass
114
111
3
97/36
106
103
3
97-16
101
41
60
40-59
110
110
...
100-00
123
105
18
85-37
100
97
3
97-00
103
102
1
99-02
90
90
...
100-00
91
91
...
100-00
83
83
...
100-00
57
56
1
98-24
98il7-7 89-9±22-4 92-3 ±17-6
4. Discussion
A high, degree of host preference is exhibited by Dryinidae commonly found as
parasites of both adults and nymphs of Fulgoroidea and Cicadellidae and they
are known to be either solitary or gregarious (Clausen 1940). Observations of
Subba Rao on Lestodrylnus pyrillae Kieff (1957), Swaminathan and Anantha-^
krishnan (1981) on two Dryinus spp,, and the present study shows the following
characteristic features of these effective biological control agents: (i) The dryinids
are host specific, an important quality for effective biological control agents as
suggested by DeBach (1964) ; (ii) They show preference for nymphal stages ;
(iii) Opposition behaviour like chasing, pouncing, and paralysing is exhibited by
the females j (jv) The dryinids exhibit arrhenotoky ; and (v) The parasitized
nymphs are prevented from moulting to the next instar. Similarly other dryinids,
Pseudogonatopus hospes Perk. (Pagden 1934) and Dicondylus lindbergi Heikin-
heimo (Heikinheimo 1957) were found to prefer the last two instars of Delphacodes
furcifera Horvath and adults of Delphacodes pellucida F. respectively. The present
study was confined to the effects of parasitism by the dryinids. on nymphs of the
host. Hence the effects of parasitism on adult hosts such as deformities in repro-
ductive organs and external sex reversal in males (Clausen 1940) was not studied.
There are five larval instars in dryinids. (Clausen 1940), of which the first is seen
inside the host body, the following three are spent inside the thalacium, the fifth
one escaping and crawling away from the host for pupation. In the present
investigation also, similar larval instars were noticed including three moults
in the thalacium.
186 S Swaminathan and T N Ananthakrishnan
No natural enemies have so far been recorded from E. tomenttosa, R.fertestrata,
and D. hyalinata, the first two being pests of important crops (vide introduction).
In E. tomentosa all the life stages— eggs, nymphs, and adults are parasitized by the
natural enemies. In R. fenestrata the eggs are laid inside the plant tissue. Hence
the nymphs and adults are alone predated by the spider and in D. hyalinata only
the nymphal stages are attacked by the natural enemy. All the natural enemies
reported here appear to be new records. Under field condition the E. tomentosa
egg masses were parasitized to a maximum extent (100%) by P. fulgoridis which
shows the latter to be a promising biological control agent of the former.
Acknowledgement
Thanks are dhe to the Director, Commonwealth Institute of Entomology, London,
Di? Z Boucek, Commonwealth Institute of Entomology, London, Dr W R Nickle
Beltsville Agricultural Research Centre, Beltsville, USA, and Dr G J Phanuel
Department of Zoology, Madras Christian College, Madras, for identifying the
specimens of the natural enemies.
References
Abraham C C, Mathur K P and Das N M 1973 New record of Coccinella arcuata Fabricius
(Coleoptera : Coccinellidae) as a predator of Nilaparvata lugens Stal in Kerala ; Agric.
Res. J. Kerala 11 75
Chatterjee N C 1933 Entomological investigations on the spike disease of Sandal (12). The life
history and morphology of Eurybrachys tomentosa Fabr. Fulgoridae (Hompt.) ; Indian
Forest Rec. 18 1-26
Chaudhary J P and Ramzan M 1968 Pachygonatopus sp. (Dryinidae : Hymenoptera). A neW
parasite of Sogatella furcifera Horvath (Delphacidae : Homoptera) ; Indie n J. EntomoL
30317
Clausen C P 1940 Entomophagous insects (New York : McGraw-Hill) pp. 688
DeBach P 1964 Biological control of insect pests and weeds (London : Chapman and Hall)
pp. $44
Heikinheimo O 1957 Dicondylus lindbergi sp. n. (Hym. : Dryinidae) a natural enemy of
Delphacodes pellucida (F.) ; Ann. EntomoL Fennici 23 77-85
Hutsort J C 1919 Some minor insect pests in Ceylon in 1919; Trap. Agric. PeradeniyaL 3 No 2.
Israel P and Prakasa Rao P S 1969 Record of Anagms sp., a mymarid (Hymenoptera) parasite
ort eggs of Sogatella furcifera Horvath on rice in India : Curr. Sci. 38 320-321
Kalode M B 1976 Brown planthopper in rice and its control ; Indian Farming 27 3-5
Light S S 1929 Report of the Entomologist (for 192$) ; Bull, Tea Res. Inst. Ceylon No. 3
Manjunath T M 197$ Two nematode parasites of rice brown planthopper in India ; Int. Rice
Res. Newslett. 3 11-12
Manjunath T M, Rai P S and Gowda G 1978a Parasites and predators of Nilaparvata lugens
in India ; Pans 24 265-269, 3&7
Manjunath T M, Rai P S and Gowda G 197Sb Natural enemies of brown planthopper and
green leafhopper in India ; Int. Rice Res. Newslett. 311
Narayanan E S and Kundanlal 1953 Studies on the chalcid egg parasites of Pyrilla sp. occurring
in Delhi. Part I. Bionomics of Tetrastichus pyrillae Crawf., Ageniaspis pyrillae Mani, and
Chelloneurus pyrillae Mani ; Indian J. EntomoL 15 173-179
Natural enemy complex of some fulgoroids 187
Pagden H 1934 Notes on HymenopteroUs parasites of padi insects in Malaya ; Fed. Malay.
States. Dept. Agric. Bull. 15 pp. 13
Puttarudriah M and Maheswariah B M 1958 Ricania fencstrata Fabricius A potential pest of
Cotton ; Indian Cotton Growing Rev. 12 1
Rahman K A 1941 Parasites of the insect pests of sugarcane in Punjab ; Indian J. Agric. Set.
11 119-128
Rahman K A and Ramnath 1940 Bionomics and control of the Indian sugarcane leafhopper,
Pyrilla perpusilla Wlk. (Rhynchota, Fulg.) in the Punjab ; Bull. Entomol. Res. 31 179-190
Rai PS and Chandrasekhar H T 1979 The natural enemy complex of rice brown planthopper ;
Curr. Sci. 48 23-24
Samal P and Misra B C 1975 Spiders : The most effective natural enemies of the brown plant-
hopper in rice ; Rice Entomol. Newslett . 3 31
Samal P and Misra B C 197&a Notes on the egg parasites of the brown planthopper Nila-
parvata lugens (Stal) in Orissa ; Oryza 15 96-98
Samal P and Misra B C 1978b Casnoidea indica (Thunb) a carabid ground beetle predating
on brown planthopper, Nilaparvata lugens Stal of rice ; Curr. Sci. 47 688-689
Sen A C 1948 The Pyrilla pest of cane in Bihar and its control by utilization of its natural para-
sites : Indian J. Entomol. 10 45-50
Subba Rao B R 1957 The biology and bionomics of Lestodryinus pyrillae Kieff. (Dryinidae :
Hymenoptera) a nymphal parasite of Pyrilla perpusilla Walk, and a note on its role in the
control of Pyrilla ; J. Bombay Nat. Hist. Soc. 54 741-749
Swaminathan S and Ananthakrishnan T N 1981 Oviposition behaviour in two species of
dryinid parasites (Dryinidae, Hymenoptera) ; Proc. Indian Acad. Sci. (Anim. Sci.) 90
113-116
i>roc. Indian Acad. Sci. (Anirn. Sci.), Volume 91, Ho. 2, March 1982, pp. 189-191
(S) Printed in India. .
Transabdominal migration of ova in some freshwater turtles
P L DUDA and V K GUPTA*
Department of Biosciences, University of Jammu, Jammu 180001, India
* Present address : Department of Rural Technology, Regional Research
Laboratory (CSIR), Jammu 180001, India
MS received 9 July 1980 ; revised 18 July 1981
Abstract. The phenomenon of transabdominal migration of ova is fairly common
in all three fresh water turtles, Lissemys punctata punctata (70%), Kachuga tectum
tectum (4%) and K. smithi (73%), studied for the present work. Individuals 6f
Lissemys punztata punctata, Kachuga smithi, showed higher frequency of ovular
migration in smaller individuals. It is suggested that a better weight
balance is possibly achieved by ovular migration in these aquatic reptiles.
Keywords. Lissemys punctata punctata ; Kachuga tectum tectum ; Kachuga smithi ;
transabdominal ; ovaries ; corpus luteum ; ova ; oviduct ; ovulation.
1. Introduction ,
Upan rupture each ovarian follicle releases its contained egg into the body cavity
which is immediately engulfed by the infundibulum of the oviduct. Eggs ovulated
by left ovary normally pass into left oviduct and those of right ovary into right
oviduct. The collapsed follicular wall of the o.vulated follicle eventually gets
transformed into corpus luteum. The number of corpora lutea thus provides
a fairly accurate index to the number of eggs produced by ari ovaiy of a side
at a given time and also indicates the numb'er 01 eggs expected in the oviduct of
that side. Ordinarily, the total number of corpora lutea in the two. ovaries of
an animal corresponds to the total number of eggs in the two oviducts, except
in instances where either oviposition is extended ^beyond1 the resorption of corpus
luteum or clutching is multiple. Yet, it has often been observed that the counts
of corpora lutea in thek ovary of one side and the number of ovlducal eggs in
the ipsilateral oviduct differs, some times stiikingly. This difference is sougfct
.to be explained only by the phenomendn of transabdominal migration of eggs
during the short period that intervenes between the act of ovulation and encapsu-
lation by the oviduct.
Although reported in mammals too (Asdeil 19*46; Arey 1954), the phenomenon
of transabdominal* migration of eggs in reptiles was for the first time reported
by Weekes (1935). Ever since, the phenomenon has 'been reported for shakes
.(Tinkle 1957), lizards (Tinkle 1961 : Mayhewa96.3, 196.5, 1966, 1971 ;Telford 1969;
Cuellar. 1970; Goldberg 1972) -and turtles (Tinkle 1957; Legler 1958; Moll and
189
P,(B)-9
i96 P L Duda and V It Gupta
Legler 1971 ; White and Murphy 1973: Plummer 1977; Cox and Marion 1978).
To. obtain some more information on this very common phenomenon among
reptiles and therefore presumably of importance to them, three Indian
freshwater turtles, Lissemys punctata punctata, Kachuga tectum tectum, and
K. smithi were intensively studied from the standpoint of ovulation in them
from 1976 to 1978.
2. Materials and methods
Specimens were collected by hand, muddling, or by cast nets from two different
sources. Lissemys punctata punctata were collected from Lafce Mansar (about
65 fcm in the East of Jammu city, India) and Kachuga tectum tectum and K. smithi
were collected from a slow running stream, New Gho-Manasan Khul, situated
about 15fcm south-west of Jammu city. The taxonomy of the forms studied
was done after Smith (1931).
All linear measurements of the specimens were done in the laboratory with the
help of 1 meter flexible steel tape from live animals. Measurements were recorded
to the nearest millimeter. After preliminary weighing and measurements, the
turtles were dissected for examination of ovarian weight ; the number and size
of ovarian follicles and corpora lutea ; the number, size and weight of shelled
oviducal eggs, were noted separately for right and left side. The weights were
recorded to the nearest milligram.
3. Results and discussion
Daring their breeding season, which extends from August to October in Lissemys
punctata punctata, October to February in Kachuga tectum tectum and August
to November in K. smithi, 14 adults of Lissemys p. punctata (table 1, figure 1)
30 of Kachuga t. tectum (table 2, figure 2) and 17 individuals of K. smithi (table 3,
figure 3) were found to contain eggs in their oviducts. Of these 4 individuals of
Lissemys p. punctata, 25 of Kachuga t. tectum and 2 of K. smithi, showed more
corpora lutea than the number of eggs in their oviducts, representing more than
one series of ovulation and were thus of varying size and appearance. The
remaining individuals showed number of corpora lutea to be equal to the total
number of .eggs in two oviducts.
Of the remaining 10 turtles of Lissemys p. punctata, three showed the number
of eggs in one side oviduct to be equal to the number of corpora lutea in the ovary
of the same side. However, in the remaining 7, a striking disparity in their
number (table 1, figure 1) was observed. Four of these 7 turtles showed more
corpora lutea in the right ovary than in the left and three more corpora lutea in the
left ovary than in the right. The number of eggs in the oviducts of these seven
was equal on two sides in two individuals (5 and 3 in each oviduct), and unequal
in 5, being greater in the right oviduct in 2 and in the left oviduct in 3 individuals
In Kachuga t. tectum, only two individuals of the 5 animals (where number of
-corpora lutea was equal to egg number in the oviducts) showed the phenomenon
of ovular migration (table 2, figure 2). The remaining 3 individuals possessed
equal number of corpora lutea and oviducal eggs on each side. In these cases
it was impossible to determine whether ova had migrated from one side to the
Migration of ova in turtles
191
Table 1. Number of corpora lutea, eggs and ovarian weight (g) in Lissemys p.
punctata on the right and left sides of the body.
Total
Total
SI.
No.
Plastron
(mm)
Right side
Left side
number
of
corpus
luteum
number
of
shelled
eggs
Corpus
luteum
Shelled
eggs
Wt. of
ovary
Corpus
luteum
Shelled
eggs
Wt. of
ovary
1.
250
3
2
29-40O
4
5
48-900
7
7
2.
265
7
4
33-800
11
6
61-100
18
10
3.
225
4
3
10-600
6
2
24-250
10
5
4.
286
5
7
19-60O
8
6
30-300
13
13
5.
225
1
2
6-700
5
4
6-450
6
6
6.
245
6
4
56-400
5
7
42-900
11
11
7.
267
5
5
32-OOO
5
5
36-000
10
10
8.
235
3
3
43-100
5
5
21-050
8
8
9.
281
8
7
21-720
4
5
35-800
12
12
10.
244
6
5
42-000
4
5
31-100
10
10
11.
217
4
3
8-200
2
3
7-720
6
6
12.
282
15
8
18-20O
10
5
28-100
25
13
13.
258
9
5
27-200
9
4
20-900
18
9
14.
239
4
4
36-900
4
4
48-200
8
8
• OVIDUCAL EGGS
Q CORPORA LUTEA
Figure 1. Comparative counts of oviducal eggs and corpora lutea in Lissemys p.
punctata (represented by black and white bars, repectively).
192 PL Duda and V K Gupta
Table 2. Number of corpora lutea, eggs and ovarian weight (g) in Kachuga t.
tectum on the right and left side of the body.
si.
No.
Plastron
length
(mm)
Right side
Left side
Total
number
of
corpus
luteum
Total
number
of
shelled
eggs
Corpus
luteum
Shelled
eggs
Wt.of
ovary
Corpus
luteum
Shelled
eggs
Wt. of
ovary
1.
170
9
6
12-580
10
4
8-200
19
10
2.
173
3
4
6-600
4
3
19-950
7 ,
7 ,
3..
165
7
3
2-310
5 .
4
3-425
12
7
4.
166
3T
4
3-700
4
3
3-450
7
7
5.
161
5
4
2-900
8
3
2-750
13
7
6.
168
8
4
4-640
14
5
4-150
22
9
7.
160
7
3
2-995
3
3
2-725
10
6
8.
185
3
3
43-200
3
3
26-800
6
6
9..
167
7
2
3-670
4
3
3-810
11
5
10.
166
5
4
4-300
7
3
5-900
12
7
11.
161
7
2
3-995
5
3
6-800
12
5
12.
153
3
3
5-065
3
3
7-050
6
6
13.
173
9
2
5-900
13
2
3-910
22
4
14.
168
4
4
11-700
10
3
20-000
14
7
15.
155
5
4
7-600
8
3
2-820
13
7
16.
161
4
4
3-750
4
4
7-150
8
8
17.
143
2
2
2-190
6
2
2-220
8
4
18.
178
10
6
4-700
11
4
5-000
21
10
19.
152
9
2
3-480
3
3
5 • 850
12
5
20.
170
5
4
4-300
7
2
3-850
12
6
21.
154
6
3
6-610
5
4
4-115
11
7
22.
157
6
3
2-200
6
3
2-405
12
6
23.
165
9
3
4-425
6
4
5-110
15
7
24.
165
6
3
4-200
7
4
4-280
13
7
25.
183
13
4
7-300
8
4
3-200
21
8
26.
171
7
5
13-000
8
5
4-080
15
10
27.
155
4
3
3-245
5
2
3-225
9
5
28.
160
5
5
4-400
11
5
8-315
16
10
29.
171
7
6
4-425
13
4
9-000
20
10
30.
147
5
2
2-835
5
4
2-715
10
6
other. In the other two cases, more corpora lutea were seen on the left side when
oviducal eggs were more on the side opposite.
Eleven of 15 K, smthi showed an extrauterine migration of ova (table v 3
figure 3). Of the remaining four, three showed that the count of oviducal eggs
Migration of ova in turtles
193
I
•
!
1
I
o
o ?"»
II
194 P L Duda and V K Gupta
Table 3. Number of corpora lutea, eggs and ovarian weight (g) in Kachuga smith*
on trie right and left side of the body.
Total
Total
SI.
Plastroi?
Right
side
Left
side
number
number
f
VT./>
,
of
of
No.
length
(mm)
Corpus
luteum
Shelled
eggs
Wt. of
ovary
Corpus
luteum
Shelled
eggs
Wt. of
ovary
corpus
luteum
shelled
eggs
1.
173
1
26-750
2
3
46-900
3
3
2.
176
7
3
42-750
5
3
18-775
12
6
3.
191
6
3
18-500
...
3
12-950
6
6
4.
160
6
4
17-350
2
4
14-350
8
8
5.
194
4
2
74-350
1
3
38-000
5
5
6.
197
4
4
43-000
3
3
44-100
7
7
7.
190
2
3
23-100
5
4
39-500
7
7
8.
192
4
4
36-450
2
2
41-100
6
6
9.
195
5
3
19-500
1
3
26-100
6
6
10.
199
4
5
29-000
6
5
41-000
10
10
11.
195
4
2
48-210
2
4
30-380
6
6
12.
173
1
3
9-300
3
1
30-200
4
4
13.
180
3
4
43-700
3
2
41-800
6
6
14.
192
3
3
35-510
3
3
22-800
6
6
15.
187
4
5
13-000
7
6
4-600
11
11
16.
193
1
1
14-000
2
2
5-500
3
3
17.
177
6
3
6-580
4
2
4-750
10
5
• ovioucAt eoos
O CORPORA LUTEA
Figure 3. Comparative counts of oviducal eggs and corpora lutea in Kachuga
smithi (represented by black and white bars, respectively).
Migration of ova in turtles 1^5
differed from one another but corresponded well to. the total number of corpora
lutea as on their respective sides. One turtle had a balanced number of corpora
lutea and oviducal eggs on each side. In the remaining 11 individuals, 5 showed
more corpora lutea on the right side whereas the oviducal eggs were more on the
left in only two, 3 showing equal number of eggs on both the sides. Out of the
remaining six, 5 individuals showed more corpora lutea on left side but the
oviducal eggs were more on the left in three, on the right in one and the fifth
one had equal number of corpora lutea on the two sides, but the oviducal eggs
were more on the right side.
The present studies have thus revealed that in Lissemys p. punctata, Kachuga t.
tectwn and K. smithi, the phenomenon of transabdominal migration of ova is
of a relatively common occurrence. Seventy per cent of the Lissemys p. punctata
(N = 10), 4% of Kachuga t. tectum (N = 15) and 73% of K. smithi (N = 15)
studied for this phenomenon showed positive evidence of extra-uterine migration
of ova. There is no previous record of such a high percentage of transfer as has
been recorded presently in Lissemys p. punctata and Kachuga smithi. The
previous highest report of ovular migration (66-6%) has been recorded in Trionyx
muticus by Plummer (1977). Although reported in some other turtles as well
the magnitude of the phenomenon in all of them is rather low being 57% in
Terrapene ornata ; 13% in T. cerdina (Legler 1958) and 57% in Sternotherus
odoratus (Tinkle 1959).
The present observations reveal that in individuals of smaller size below 250 mm
(in plastron length) of Lissemys p. punctata (table 1), the transfer of ova is much
higher (87-5%) than in the larger individuals (of plastron length above 250mm)
of the species. In Kachuga smithi of a plastral length of 210mm or less, the
migration is again higher (83%, table 2) than in its bigger individuals, where the
transfer of ova was found to be only 49%. In Kachuga t. tectum, on the other _
hand, the sample size being very small did not provide sufficiently reliable data*
Thus our findings run counter to those of Tinkle (1959) who has reported for
Sternotherus odoratus, that the extent of transabdominal migration of ova is
higher in bigger individuals (62%) than in smaller ones (50%). Obviously the
phenomenon is unrelated to. size or age and could be a mere chance or an out-
come of an occasional positional shift of the oviduct or ovaries known in reptiles
(Cuellar 1970) during the act of encapsulation of the oocytes.
When viewed from the point of imbalance and differential weight of the ovary,
a definite relationship between the weight of the ovary and the oviducal eggs on
. the same side as that of the ovary is evident. In 10 turtles of Lissemys p. punctata
(table 1) with unequal number of eggs in the two oviducts, 7 showed lesser number
of oviducal eggs on the side of heavier ovaiy, the other three greater number of
oviducal eggs on the side of the heavier ovaiy. In Kachuga t. tectum (table 2)
only one of the two individuals, suspected of transabdominal migration, had
higher egg count on the side with heavier ovary. Of the 10 Kachuga smithi
(table 3) turtles with unbalanced number of oviducal eggs, 7 showed a higher egg
count in the oviduct on the side on which the ovary was lighter, the remaining 3
showing heavier ovary on the side with more eggs in the oviduct. After pooling
the data from the three turtles and subjecting these to X*9 it is found that the P
value stands between 0-05 to 0-20, which make deviation to be a matter of chance
196 P L Duda and V K Gupta
Table 4. Percental values of corpora lutea during the breeding season in the
two ovaries of Lissemys p. punctata, Kachuga t. tectum and Kachuga smttu.
Corpora lutea
Higher
number
Equal
Animal
number on
Total
Right
Left
both sides
number
(%)
(%)
(%)
Lissemys p. punctata
14
35-7
35-7
2S.-5
Kachuga t. tectum
30
26-6
56-6
16-6
Kachuga smithi
17
52*3
35-2
11-5
provided the assumption that the expected distribution of the eggs in the two
oviducts would be equal (I :A). Since it is not so^ the assumption stands unten-
able. It is therefore, suggested that the imbalance in number of eggs on the two
sides has some significance and may help in achieving a better -t weight balance by
having the greater number of ovulatory follicles on the side opposite the greater
number of oviducal eggs, particularly in aquatic vertebrates, as has also been
suggested earlier by Tinkle (1959).
A perusal of table 4 indicates that neither left nor right ovary in Lissemys p.
punctata is consistently more productive than the other, although in the emydid
turtles (Kachuga t. tectum and K. smithi) one of the two ovaries tends to be slightly
more productive than the other. But the data in tables I, 2 and, 3 indicate that
there is no positive relationship between greater productivity of any one ovary
and the migration of the eggs. Present findings however, do not support Legler's
(1958) view that in reptiles, the two ovaries show differential activity in. different
years, one being, more active during one breeding season than the other.
Nevertheless, a definite relationship appears to exist between the heavier ovary
and lesser number of eggs on a side, as shown above. Should the asymmetrical
position of the stomach in chelones have played any major role in the ovular
migration as maintained by Hoddenbach (1966),. then transabdominal would
have been of a. much wider and unfailing occurrence than reported or observed
in lizards also in nearly all of which asymmetrical disposition of this .stomach
has been amply documented (Duda 1965).
In conclusion, therefore, the imbalance in the number of ^ggs on the two sides
could be related in fair probably to the physiological necessity of achieving
balance at least in the aquatic forms. . » . ,
Acknowledgements
Authors are thankful to Dr Y R Malhotra, Professor and Head, -Department of
Biosciences, University of Jammu, for his constant encouragement and providing
Migration of ova in turtles 19 7
neeessafy facilities to conduct this work in the department. Financial assistance
from the CSIR, New Delhi, is also acknowledged.
References
Arey L B 1954 Developmental anatomy (Philadelphia: Saunders) 6th ed. V-XI -V- 680 pp.
Asdeil S A 1946 Patterns of mammalian reproduction (Ithaca : Comstock) XVI — 437 pp.
Cox W A and Marion K A 1978 Observations on the female reproductive cycle and asso -
elated phenomena in spiing-dwolling population of Sternothems minor in "North Florida
(Reptilia : Testudines) ; Herpetologica 34 20-33
Cuellar O 1970 Egg transport m lizards ; J. Morphol 130 129-135
Duda P L 1965 Functional morphology of Agama tuberculata Gray. Ph.D. thesis, Kashmir
University
Goldberg S R 1972 Reproduction in the southern alligator lizard Gerrhonotus multicarinatus ;
Herpetologica 28 267-273
Hoddenbach G 1966 Reproduction in western Texas, Cnemidophorus sexlineatus (Sauria; Teiidae):
Copeia 1966 pp. 110-113
Legler J M 1958 Extra-uterine migration of ova in turtles ; Herpetologica 14 49-59
Mayhew W W 1963 Reproduction in the granite spiny lizard, Sceloporus orcutti ; Copeia
1963 pp. 144-152
Mayhew W W 1965 Reproduction in the sand dwelling lizard, Vma inornata ; Herpetologica
21 39-55
Mayhew W W 1966 Reproduction in the arenicolous lizard, Vma notata ; Ecology 47 9-19
Mayhew W W 1971 Reproduction in the desert lizard, Dipsosaurus dorsalis ; Herpetologica
27 57-77
Moll E O and Legler J M 1971 The life history of neotropical slider turtle, Pseudemys scripta
(Schoepff) in Panama ; Bull Los. Ang. Country Mus. Nat. Hist. Sd. 11 1-102
Plummy M V 1977 Reproduction and growth in the turtle, Tnonyx muticus ; Copeia 1977
pp. 440-447
Smith M A 1931 The Fauna of British India, including Ceylon and Burma (London: Taylor
and Francis) pp. 188
Telford S R 1969 The ovarian cycle, reproductive potential and structure in a population of
tte Japanese lacertid Takydromous tachydromoides', Copeia 1969 pp. 548-561
Tinkle D W 1957 Ecology, maturation and reproduction of Thamnophis sauritus proximus ;
Ecology 38 69-77
Tinkle D W 1959 Additional remarks on extra-uterine migration of ova in turtles ; Herpeto-
logica 15 161-162
Tinkle D W 1961 Population structure and reproduction in the lizard Uta stansburiana steinogen ;
Am. Midi. Nat. 66 205-234
Weekes H C 1935 A review of plantation among reptiles with particular regard to the function
and evolution of the Placenta ; Proc. Zool. Soc. London pp. 625-645
White J B and Murphy G G 1973 The reproductive cycle and "^^gfjj °f thG «*"»"
snapping turtle, Chelydra serpentina serpentma ; Herpetologica 29 240-246
P.(B)-10
Proc. Indian Acad. Sci. (Anim. ScL), Vol. 91, Number 2, March 1982, pp. 199-206.
© Printed in India.
Sediment-polychaete relationship in the Vasishta Godavari estuary
D SRINIVASA RAO and D V RAMA SARMA
Zoology Department, Andhra University, • Waltair 530 003, India
MS received 3 April 1981 .
Abstract. A 16 km stretch of the lower Vasishta Godavari estuary (lat. 16° 18' N
long. 81° 42' E) was surveyed during October 1976-Januafy 1978 to -study the
polychaete-sediment relationship. Mean high mid- and low water, marks at 'six
permanent stations were sampled for studying polychaete distribution as well as
sediment characteristics. Sand fraction dominated stations I and II and the silt-
clay per cent increased higher up the estuary. Organic matter in the estuary
ranged from 0-1 to 4-2% and the amount is generally linked with the silt-clay
fraction of the sediment. Depending upon their tolerance to the sediment compo-
sition- polychaete species colonised different tidal levels. Carnivores were restricted
to sandy substrata. For the detritus feeders, the influencing factor appears to be
organic matter than the sediment composition.
Keywords. Sediment composition ; organic matter ; relationship ; carnivores ;
detritus feeders. . . .
1. Introduction
The importance of the substratum during settlement of polychaete larvae has been
documented by Wilson (1953). Sanders. (1958) successfully attempted to relate
the type of feeding of the organism and the sediment composition in Buzzards Bay,
Massachusetts . Thus he found detritus feeders restricted to the mud sediments and
filter feeders to the median grain size sediments. In contrast Muus (1967) stated
that in any estuary, with irregular or unfavourable fluctuating physical factors,
salinity and dissolved, oxygen are more important than the sediment composition
in influencing the species distribution. Moreover sediment particle size is- known
to be a function of the mixing and dilution of salt water by freshwater and
therefore particle size is dependent on salinity (Me Nulty etal 1962). Muus (1967)
therefore concluded that any attempt in that direction is fruitless. However;
later works in several other areas revealed the apparent relationship between the
substratum and the invertebrate fauna in general and polychaetes in particular.
In the present study an attempt has been made to establish the possible relation"
ship between the abundance of polychaete fauna and the intertidal sediments in
the Vasishta Godavari estuary;
199
200 D Srinivasa Rao and D V Rama Sarmd
2. Area of investigation
The area presently investigated is the intertidal habitat of the Vasishta Godavari
estuary, the southernmost branch of the river Godavari, opening into Bay of
Bengal at Antervedi (lat. 16° 18' N ; long. 81° 42' E). The geographical description
of the area and location of the stations have already been given by Srinivasa Rao
(1980).
3. Materials and methods
Collections were made from six stations at monthly intervals, from October 1976
to January 1978 exceptingin August 1977 due to fastcurrents associated with high
annual floods. At each station sampling was made from three tidal levels v/z.,
mean high water mark (MHWM), mean mid water mark (MMWM) and mean
low water mark (MLWM). Sediment was collected using a PVC corer while a
metal frame of 20 x 20 x 15cm dimensions was used/or polychaete collection.
Techniques employed for the collection and analysis of hydrographic parameters
were the same as described earlier (Srinivasa Rao 1980 ;. Srinivasa Rao and Rama
Sarma 1980). Sand, silt and clay fractions in the sediment were estimated by the
pipette method of Krumbein and Pettijohn (1938) whereas the organic matter in
the sediment was estimated, by the method of Gandette et al (1974).
4. Results
The nomenclature of Folk (1968) is adopted to classify the sediments of Vasishta
Godavari estuary and the sediment composition during different seasons is pre-
sented in figure 1. The sediments were generally sandy near the river mouth (stations
I and II) as the area is influenced by neretic waters and the silt-clay fraction
increased with the increasing distance from the river mouth (stations III to VI).
Along the transect, the sediment composition varied with increasing silt-clay
content down the transect. Generally the upper 3 cm layer of the substratum is an
unconsolida ted layer while below that is a closely packed silt-clay fraction. This
layering is the cumulative result of depositional and erosional factors operating
during the tidal cycles and the superimposed annual freshwater floods.
The maximum* minimum and average organic matter content for all the tidal
levels is presented in table 1. The organic matter content is significantly high in
the estuary as also observed by Dora and Borreswara Rao (1975). The low orga*
nic matter c ontent at the seaward stations (I and II) may be due to the sandy nature
of the substratum and sufficient aeration. The increased silt clay fraction, and
consequent compactness of the sediment and poor aeration resulted in the reten-
tion of a high amount of organic matter at stations III to VI. That the clay
minerals bind organic matter better than the loose sands is well-known (Sanders
1956). Similar relationship of the organic matter with fine sediments around the
Indian subcontinent was observed by Murthy et al (1969), Parulefcar et al (1976)
and Ansari et al (1977). The high organic matter content in the sediment during
summer is the result of high organic production characteristic of the estuaries.
Further the contribution from the adjoining mangroves and terrestrial sources is
Sediment polychaete relationship
201
SUT TOO*
CLAY 100* SILT i(?c*
CLAY 100* SILT 100*
CLAYttO*
CLAY 100*
Figure 1. Seasonal variations in the sediment composition during the period of
study.
Table 1. Organic matter content in the sediments daring the study period.
Station
Tidal level
Minimum
(%)
Maximum
(%)
Average
(%)
I
MHWM
0-11
1-20
0-61
I
MMWM
0-16
2-49
1-53
I
MLWM
1-04
2-64
1-71
II
MHWM
0-17
1-73
0-86
II
MMWM
0-66
3-20
1-85
II
MLWM
0-94
3-52 .
1-81
III
.MHWM
0-39
2-64
1-98
III
MMWM
0-62
3-66
2-25
III
MLWM
0.-72
3*20
2-11
IV
MHWM
0-16
3-20
1-99
IV
. MMWM
0-97
3-84
2-15
IV
MLWM
0-80
2-62
1-83
V
MHWM
0-27
2-14
1 -31
* V
MMWM
0-39
2-49
1-73
V
MLWM
0*15
2-57
1-77
VI
MHWM
0-41
4-20
2-34
VI
MMWM
0-38
3-32
2-43
VI
MLWM
0-17
3-74
2-39
202 D Srinivasa Rao and D V Rama Sarma
remarkably high. The high bacterial activity because of high temperature is yet
another factor by which the organic matter reduces in respect of particle size and
gets adsorbed onto the sediment.
5. Discussion
In estuaries, the sediment is of paramount importance in influencing the life in
general and the benthic fauna in particular. The importance of soil grade as a
factor in the distribution of polychaetes has long been recognised (Day and Wilson
1934 ; Southward 1957 J Bassindale and Clark I960 ; Clark and Haderlie I960,
1962 ; Bloom et al 1962 ; Williams 1962 ; Estcourt 1967 ; Nichols 1970 ;
Boyden and Little 1973 ; Wolff 1973 ; Gray 1974 ; Santos and Simon 1974 ;
Grassle and Grassle 1974 ; Buchanan 1963 ; Vietez 1976 ; Whitlatch 1977 and
Amaral 1979). Though the food and feeding habits of the polychaetes inhabiting
this estuary have not been worked out, the investigations of Sanders (1956) ;
McNulty et al (1962) and Brett (1963) show that a close relationship prevails
between the feeding habits of the infauna, gross organic matter content and the
texture of the sediment. Observations made on the morphological features of the
polychaetes of this estuary suggested that majority of them are detritus feeders.
This is due to the excessive silt-clay fractions in the sediments. The filter feeders
are absent up in the estuary as they need well aerated substrata. Such substratum
is available at MHWM at station I but prolonged period of exposure and high
temperature may be acting as deterrent factors preventing their settlement.
Depending upon their tolerance to the substratum composition, different
species occupied different positions along the transect, however in varying numbers.
The capitellid Heteromastus similis, nephtyd Nephtys oUgobranchia and nereid
Dendronereis arborifera appear to have great resistance for exposure, grain size
and salinity. They were represented equally at all the three tidal levels (figure 2)
and in almost all substrata except in places where the sand content was less than
10% (Rama Sarma and Srinivasa Rao 1980 ; Srinivasa Rao 1980 and Srinivasa
Rao and Rama Sarma 1980). Several other species which cannot tolerate hard
substrata at MHWM restricted themselves to the lower tidal levels (figure 2) where
the sediment comp oil tion was suitable (figure 3). But when forced by wave action
they survived there for certain periods.
It is interesting to note that though most species exhibited substratum preference,
individuals of each species appeared at times in substrata with different composi-
tion (figure 3). It may be because of the influence of the sediment in controlling
the abundance of the organisms but not their distribution (Holme 1949 ; Wilson
1953 ; George 1964 ; Sanders et al 1965). It also appears that mud dwelling
species were able to invade the substrata containing sand while the species inhabi-
ting sandy substrata failed to invade the muddy ones. This may be because of the
possible clogging of the feeding apparatus of the sandy inhabitants when they try
to invade the muddy sediments. ^ Similar observation was made by Johnson (1971).
Thus members of the genus Glycera, eunicids Diopatra neapolitana and Lumbri*
neris heteropoda which are known carnivores, restricted themselves to the sandy
substrata. They feed on the interstitial microfauna available in that habitat. For
several sedentarian species like Magelona cirtcta, Cossura coasta, Sternaspis
Sediment polychaete relationship
203
X
S
5
*
2
50'A.r
HETEROMASTUS SIMMS J
so%L
NEPHTYS OLIGOBRANCHIA
DENDRONEREfS ARBORfFERA
7INDONEREIS
NEREIS LAMELLOSA
NEREIS (N) CAPENS1S
PRIONOSPIO CIRRIFERA
NECTONEANTHES IJIMAI
ANCISTROSYU1S PARVA
50%r
°l
so%L
MAGELONA ClNCTA
COSSURA COASTA
STEftNASPIS SCUTATA
PRIONOSPIO PINNATA
PRIONOSPIO CIRROBPANCHLA
Figure 2. Transectwise distribution of polychaete species in the Vasishta Godavari
estuary.
scutata and members of the family Spionidae which are detritus feeders, there
appears to be no substratum preference, except the avoidance of sandy
substrata.
Nereis lamellosa occurred in good numbers at station I where grass was present
which not only provides stability to the substratum but also keeps the temperature
low, in addition to providing food material/in the form of detritus. The import-
ance of grass in the distribution and food patterns of intertidal organisms 'was
shown by MacGinitie as early as in 1939. ' '*
Organic matter in the sediment plays an important role in the abundance and1
distribution of benthic polychaetes especially in estuaries where the organic matter
content available in the shallow water sediments is usually very high. Buchanan
(1963) observed that the distribution of the organisms is generally related to the
temperature, salinity and grade of the soil and more closely with the organic-
matter. However the organic matter in the utilisable form in the sediment is*
reported to be important for the polychaete survival.
Organic matter in the Vasishta Godavari estuary is chiefly of plant origin
which is brought down by a multitude of small creeks, finally getting embedded
in intertidal sediments. . This material is played up and down the estuary and also
between MHWM and MLWM in the intertidal region. In addition the intertidal
organisms themselves contribute to the organic matter content apart from the
minute fraction swept in from the sea through tidal action.
204
D Srinivasa Rao and D V Rama Sarma
3000
2000
1500
1100
900
CD
500
300
100
• N-OLIGOBRANCHIA
o H-SIMILIS
X D.ARBORIFERA
• f.INOONEREIS
a M. C1NCTA
m A. PARVA
• P • CIRRIFERA
» G AONGIPINNIS
X *
a
o
o § o
00 20 60 60 80
SILT + CLAY */o — >•
100
Figure 3. Sediment composition and the density of polychaete species.
The c&piteti.id.Heteromastits similis was found to be cosmopolitan in distribution
(in respect of the nature of substratum) but the major factor which outweighed all
other factors is decidedly the organic matter content (Srinivasa Rao 1980). The
detritus feeders Dendronereis arborifera, Magefona cirtcta, Sternaspis scutata and
Cossura coasta were found in greater abundance in muddy areas where the organic
matter content was high.. On the other hand the carnivores of the family Glyce-
ridae and Enuicidae have shown no special relationship with the organic matter.
Similar is the case with Nephtys oligobranchia, a proven carnivore (Srinivasa Rao
and Rama Sarma 1978).
Acknowledgements
Thanks are due to the Head of the Department for providing necessary facilities
and to the Council of Scientific and Industrial Research, New Defti, for the
financial assistance given to DSR* ;
Sediment polychaeie relationship 205
References
Amaral A C Z 1979 The ecology and contribution of polychaetous annelids to the benthic bio-
mass of the tidal zones in the northern coast of Sao Paulo state ; Biol. Inst. Oceanogr. Sao
Paulo 28 1-52
Ansari Z A, Harkantra S N, Nair S A and Parulekar A H 1977 Benthos of the Bay of Bengal;
A preliminary account ; Mahasagar 10 55-60
Bassiandale R and Clark R B 1960 The Gann flat dale : Studies on ths ecology of a muddy
beach ; Field Studies I 1-22
Bloom S A , Simon J L and Hunter V D 1972 Animal sediment relationship and community
analysis of a Florida estuary ; Mar. Biol. 13 43-56
Boyden C R and Little C 1973 Faunal distribution in soft sediments of the Severn estuary ;
Esruar Coast Mar. Sci. I 203-223
*Brett E C 1963 Relationship between marine invertebrate infaunal distribution and sediment
type distribution in Bogue Sound, North Carolina. Ph. D. thesis, University of North
Carolina
Buchanan J B 1963 Bottom fauna communities and their sediment relationships off the coast of
Northumberland ; Oikos 14 154-175
Clark R B and Haderlie E C 1960 The distribution of N. cirrosa and N. hombergi on the
South Western coasts of England and Wales ; /. Anim. Ecol. 29 117-147
Clark R B and Haderlie E C 1962 The distribution of AT. californiensis and JV. caecoides on
the Californian coast ; /. Anton. Ecol. 31 339-357
Day J H and Wilson D P 1934 On the relation of the substratum to the metamorphosis of
Scolesolepis fulliginosa (Claparede) ; /. Mar. Biol. Assn. U.K. 19 655-662
Dora Y L and Borreswara Rao C 1975 Oragnic matter content of the Vasishta Godavari
river sediments ; Bull Dept. Mar. Sci-Univ. Cochin 7 945-952
Estcourt I N 1967 Ecology of benthic polychaetes in the Heathcote estuary, New Zealand ;
New Zealand J. Mar. Freshwater Res. 1 371-394
Folk R L 1968 Petrology of sedimentory rocks. (Texas : Hemphill's Austin) pp 170.
Gaudette H E, Wilson R F, Toner R and David W F 1974 An inexpensive titration method
for the determination of organic carbon in recent sediments; /. Sed. Petr. 44 249-253
George J D 1964 On some environmental factors effecting the distribution of Cirroformia tentacu-
lata (Polychaeta) at Hable ; /. Mar. Biol. Assn. U.K. 44 373-388
Grassle J F and Grassle J P 1974 Opportunistic life histories and genetic systems in marine
benthic polychaetes ; /. Mar. Res. 32 253-284
Gray J S 1974 Animal sediment relationships ; Oceanogr. Mar. Biol. Ann. Rev. 12 223-261
Holme N A 1949 The fauna of sand and mud banks near the mouth of the Exe estuary ; J. Mar.
Biol. Assn. U. K. 28 189-237
Johnson R G 1971 Animal sediment relations in shallow water benthic communities ; Mar.
Geol. 11 93-104
Krumbein W C and Pettijohn F J 1938 Manual of sedimentory petrology (New York : Appleton
Century Crofts) pp. 549
MacGinitie G E 1939 Ecological aspects of California marine estuary ; Am. Midi. Nat. 16
629-765
McNulty J K ,Work R C and Moore H B 1962 Some relationships between the infauna of
the level bottom and the sediments in South Florida ; Bull. Mar. Biol. Gulf Carib. 12
322-332
Murthy P S N, Reddy C V G and Varadachari V V R 1969 Distribution of organic matter
in the marine sediments off the west coast of India ; Proc. Nat. Inst. Sci. India 35 377-384
Muus B J 1967 The fauna of Danish estuaries and lagoons : distribution and ecology of domi-
nating species in the shallow water mesohaline zone; Medd. Danm. Fisk. ffavunders N S 5
147-174
* Not referred to in original.
206 D Srinivasa Rao and D V Rama Sarma
Nichols F H 1970 Benthic polychaete assemblages and their relationship to the sediment in
Port Madison, Washington ; Mar. BioL 6 48-57
Parulekar A H, Nair S A, Harkantra S N and Ansari Z A 1976 Some quantitative studies of
benthos off Bombay ; Mahasagar 9 51-56
Rama Sarma D V and Srinivasa Rao D 1980 Ecology of Dendroitereis arborifera (Polychaeta:
Nereidae) in the Vasishta Godavari estuary. Paper presented at V All India Congr. Zool.
Nov. 1980, Bhopal
Sanders H L 1956 Oceanography of Long Island Sound 1952-1954 Biology of marine bottom
communities ; Bull. Bingham. Oceanogr. Coll. 15 345-414
Sanders H L 1958 Benthic studies in Buzzards Bay. 1. Animal sediment relationships ; LimnoL
Oceanogr. 3 245-258 •
Sanders H L, Mangelsdorf Jr. and Hampson G R 1965 Salinity and faunal distribution in the
Pocasset river, Massachusetts ; Limnol Oceanogr. 10 216-228
Santos S L and Simon J L 1974 Distribution and abundance of the polychactous annelids in a
South Florida estuary ; Bull. Mar. Sci. 24 669-689
Southward E C 1957 The distribution of polychaeta in offshore deposits in the Irish Sea ; /.
Mar. BioL Assn. U.K. 36 49-75
Srinivasa Rao D 1980 Ecology of Heteromastus simllis Southern 1921 (Polychaeta : Capitellidae)
in the Vasishta Godavari estuary ; Proc. Indian Acad. Sci. (Anim. Sci.') 89 407-414
Srinivasa Rao D and Rama Sarma D V 1978 Food and feeding habits of Nephtys oligobranchia
Southern (Annelida : Polychaeta) ; Indian J. Mar. Sci. 7 193-195
Srinivasa Rao D and Rama Sarma D V 1980 Ecology of Nephtys oligobronchia Southern from
the Vasishta Godavari estuary ; Indian J. Mar. Sci. 9 218-221
Vieitez J M 1976 Ecologia de Poliquetos y Moluscos de la playa de Meira (Ria de Vigo) 1-
Estudio de las communidades ; Inv. Pesq. 40 223-248
Whitlatch R B 1977 Seasonal changes in the community structure of the macrobenthos inhabit-
ing the intertidal sand and rmid flats of Barnstable Harbor, Massachusetts ; BioL Bull. Mar.
BioL Lab. Woodshole 152 275-294
Williams G B 1962 The effects of extracts of Fiicus serratus in promoting the settlement of larvae
of Spirorbis borealis (Polychaeta) ; /. Mar. BioL Assn. U.K. 44 397-414
Wilson D P 1953 The settlement of Ophelia bicornis Savigny larvae. The 1951 experiments;
, J. Mar. BioL Assn. U.K. 31413-438
Wolff W J 1973 The estuary as a habitat, an analysis of the data on the soft bottom macro-
fauna of the estuarine areas of the rivers Rhine, Meuse and Scheldt ; Zool Verhandelingen
1261-242
Proc. Indian Acad. Sci. (Aitim. Sci.), Vol. 91, Number 3, May 1982, pp. 207-215.
© Printed in India.
The form-function relationship of vertebrates : A selected review
HIRAN M DUTTA
Associate Professor of Biological Sciences, Kent State University, Kent, Ohio 44242,
USA
MS received 30 September 1981
Abstract. A selected literature dealing with, the relationship between vertebrate
structures and functions has been reviewed. Published literature in this field gene-
rally relates to three approaches: evolutionary, ontogenetic and holistic. This paper
explains the salient features of these approaches and how their findings can be,
verified experimentally. Evolutionary approach can only make use of theoretical
explanation, whereas, in both ontogenetic and holistic approaches experimentation
is possible.
Keywords. Vertebrates; form-function relationship; evolutionary approach; ontoge'aetic
approach ; holistic approach.
1. Introduction
The study of the relationship between vertebrate structures and functions, also'
referred to as "form-function relationship analysis", is becoming more a part of
morphological research than gross anatomy. Published literature in this field
generally adheres to three approaches: evolutionary, ontogenetic and holistic.
The purpose of this paper is to review selected literature, that explains the iihportaiit
aspects of those approaches and how their findings can be verified experimen tally .
^ 1.1. Evolutionary approach
Evolutionary approach to the form-function relationship is not a new one. - Boker
(1935, 1936) establishes that form is derived from function; thus accordingly,
function always precedes form. Consequently, he defines that the aim of research
should be to describe the functional series, and that along with the phylog^netic
development of function concurrent development of form occurs. But, Boker's
views are not comprehensive because he considers only the functional aspects i of
form while neglecting the influence of genetics and convergence. Most of the
functional anatomists started as evolutionists (Eaton 1935; Hofer 1948; Gans
;f 1952, 1960; Bock 1959, 1964; Davis 1949, 1958, 1964; Gutmann . 1966V 1967,
1968). Their research is mainly based on the shape or structure of living orga-
nisms. They consider the function or change .ia tjie function sas parameter *of .the
207
208 Hiran M Dutta
structure, therefore, any change in the structure causes parallel development in the
function or evolution.
Bock and DsWitt (1959), in a study on the position of the toes in birds in rela-
tion to thiir locomotion, distinguished six types of toes which perform two func-
tions, climbing and perching. Those six types of toes are all irregularly distributed
over the taxonomic groups. Bock and DeWitt are of the opinion that the various
typss of toes have developed under the influence of selective forces of function
(functional requirement). Bock (1960) considers the supplementary joint between
the lower jaw and the cranium to be a pre-adapted structure which is a bracing
mechanism that withstands the strong force exerted on the lower ja\v during prey-
catching. Gans (1952, I960, 1966) has given similar evolutionary approach to
functional anatomy. According to him a general body plan is formed genetically,
upon which the functional influences are logically superimposed to develop the
modified structures. Another evolutionary approach considered by Liem (1967a, b,
1970) combines comparative and deductive methods. Greenwood (1965) has
correlated the environmental effects on the pharyngeal gills of cichlid fish. His
work researches the adaptive strategies in the pharyngeal jaws based on the effects
of the natural environment. Additionally, mosaic evolutionary approach has
been postulated by DiBeer (1954) in which he indicates that the transitional changes
do not involve a single organ-system but are rather functionally integrated struc-
tural complexes. For example, modifications in the feeding mechanism generally
include changes in the skull, jaw musculature, and circulation. In describing the
evolution of bony fish* several authors have emphasized the specializations and
adaptations of the skull (jaws) and the muscles. The bony fish tend to optimize
these structures of the head for food intake (Schaeffer and Rosen 1961).
1.2. Ontogenetic approach
Several functional morphologists have interrelated the developing elements at
spssi&c ontogenetical stages. Even in their developing stages, the elements are
integrated in a pattern by their properties. These.properties have been subdivided
into coherence, presence, position, size and shape (Dullemeijer 1974).
It has been suggested that the individual parts of living organisms must develop
in coherence with each other. To illustrate this, Milaire (1963) and Landsmeer
(1968) have established a coherent system of phalangi with their surrounding
tendons and muscles in a developing hand. Accordingly, the parts are arranged
in specific positions in a limited space. The specific position and spatial coherence
are needed for the parts to realize their function.
The simultaneous occurrence of several elements or organs seems to fulfil the
functional demand of a growing organism. The functional interdependence of
two specific elements like muscles and jaws in the amphibian larvae has been
confirmed by Gaupp (1905), Sedra (1950) and Dejfongh (1968).
Elements develop as a result of the differentiation of homogeneous materials
into heterogeneous structures. Heterogeneity of structures evolves at different
stages of development. Wolpert (1968) has suggested the process by which a
heterogeneous area evolves from a homogeneous one.
Elements may be divided into dominant and subordinate sub-groups. The
dominant elements have a strong influence in early development. In the head the
The form-function relationship of vertebrates 209
central nervous system seems to be dominant in all stages and is followed by the
sense organs and the pharyngeal cavity (Duilemeijer 1971). Based on the domi-
nance of surrounding mesenchymal tissue in the formation of mouth and the
middle ear cavities, Goedbloed (1964) suggested that the formation of those cavities
is controlled by the shifting of the epithelial border in relation to the mesenchyme.
Thus, mesenchyme seems to be the dominant structure in the development of
mouth and middle ear cavities. However, an opposite viewpoint has been put
forth by Moss (1971), who suggests that oral and middle ear cavities influence the
formation of mesenchyme. Moreover, the importance of the presence of the
surrounding elements has been observed by Blechschmidt (1955) in the descent. of
a male gonad. Tnus, a certain morphological arrangement is essential for a male
gonad to descend.
The position of the elements in the process of ontogeny is also significant to
their functions. Werner (1958", 1959) states that the position of the elements
shifts greatly during ontogeny in order to carry out their activities. The specific
position for the specific element is essential to carry out certain functions in a parti-
cular spatial arrangement (Landsmeer 1968). Therefore, position of the growing
elements is related to their functions. As some elements are dominant they
influence the position, form and structure of other elements. Such influences have
been indicated by DeJongh (1968) and Moss and Salentijn (1969a,b). Moss
(1968c) suggests that the positions of many growing skeletal elements are passively
displaced because of the growth of other elements. Positional changes of the
elements influence the determination of the general body plan (Moss and Young
I960;. Moss and Salentijn 1969b).
Size also influences the process of development. Balinsky (1965) establishes a
correlation between the amount of yolk and the process of gastrulation. The size
or the amount of yolk determines the process of cleavage and gastrulation. The
change in the size of an element such as a muscle will have a functional effect
(exertion) on the other element (e.g., on a bone). Moreover, the change in the
size will have impact on the shape of the local elements (Duilemeijer 1974).
' The shape of the skull changes under the influence of muscle attachments- and
the weight it carries. The skull of pigs and elephants may be cited as examples
(Duilemeijer 1974). The shape of a developing jaw is influenced by the size of
the muscle attached to it. Moss and Salentijn (1969b) indicate that the general
shape and position of an element depends on the position and size of Other elements
(e.g., the position ctf the jaws depends on the oral cavity and the position of the
calvaria bones depends on the size and shape of the growing brain).
A switchover in the properties of an element has been observed by Claes (1964,
1965). He suggests that the chorda carries out the inductive function at an early
stage but the same structure transforms into a supporting bar at a later stage. Such
structural and functional switchovers in the elements are not infrequent. For
example, during the endochondral ossification the cartilaginous structure is
transformed into a bone.
Thus, during the process of ontogeny one can observe a spatially coherent system
and interdependence of the developing elements with respect to their position,
size and shape. The most important requirement of the elements is to carry out
their functional demands.
210 Hiran M Dutta
1.3. Holistic approach
Recently, the fractional anatomists have applied a holistic principle to the func-
tional analysis of form. The holistic principle in its most modern form has been
initiated by VaJi der Klaauw (1945, 19485 1951, 1952). He was the first to intro-
duce the concept of holism m modern morphology and was followed by Moss
(1958, 1959, 1960, 1961b, 1968a,b), Dullemeijer (1956, 1958, 1959, 1974), Goss
(1964), Dutta (1968, 1975, 1979a,b, 1980), and Osse (1969). There is, however, a
difference of opinion amongst the functional morphologists regarding holism in
relation to form and function. For example, Russell (1936), Smit (1961) and
Goss (1964) believe that the specific structures develop after the influence of the
function, while Bock (1959) postulates that function is caused by structures. On
the other hand, Rensch (1948, 1958, 1960, 1972) formulates that there may have
been structures without a function and, in turn, the non-functional structures may
ac.quire new functions. He believes that a causal relationship exists between
function and form though most other modern functional anatomists reject suclj
a relationship (Barge 1919, 1936).
In order to correlate form and function, the head has been considered to be com-
posed of several functional components which form a totality (Dutta 1975). The
-'functional component" h?s been described by Van der Klaauw (1948, 1951,
1952) and Dullem.ijer (1956, 1958, 19^9, 1&71) as a morphological structure of
an element which performs a certain function. According to Klaauw (1945),
Dullemeijer (1956) and Dutta (1968, 1975) the components have a well-defined
individuality which is determined by the components themselves and by the pat-
tem of the skull. The components of the head are in turn composed of closely
related elements such as bony elements, ligaments, muscles and other tissues which
perform one or more functions together as mentioned by Bock (1964), Liem
(1967a, b) and Dutta (196.8, 1975, 1979, 1980). These functional units (elements)
are connected and form couplings (Liem 1967a, b; Dutta 1975) which conduct
the function of the animals.
, Dutta (1975) has illustrated two such couplings in Anabas testudineus and Ciena-
poma acutirostre. They are: (a) the levator pperculi-opercular apparatus-
mandibular coupling (regarded to cause depression of the lower jaw during ftsh
respiration) and (b) the sternohyoideus-hyoid apparatus-interopercular-mandibular
coupling (wjbich collaborates with the former coupling during feeding).
A functional component cannot maintain its separate entity because in order to
carry out its functions, it becomes involved with the elements of its neighbouring
component(s) (Dutta ,1975). This was further illustrated by Moss and Young
(1960) who have conceived that the maxilla, which forms the orbit, is somehow
related to vision while it also relates to the function of biting along the pajatine.
The interdependence of functional units is also emphasized by Gans (1969) when
he states that, " Tne structures tend to be affected by the influence of multiple
functions and any function will almost certainly affect multiple characteristics of
an animal," Tais overlap between two or more functional components is not
only limited to function, but also to structure as well as space.
The form-function relationship of vertebrates 211
2. Experimental
Based on the philosophy of form-function relationship, several scholars have
studied anatomy since the turn of the century. As early as 1903, Allis worked
on the functional aspects of the skull, cranial, first spinal muscles, and nerves in
Scomber scomber. Takahasi (1925), Tchernavin (1953), Holmquist (1910) and
Edgeworth (1935) have also investigated functional aspects of structures. Their
interpretations and conclusions were based on anatomy and visual observations.
A new approach to the form-function relationship was established by Klaauw
(1945, 1963) and followed by Dullemeijer (1974), Gans (1969), Liem (1967a, b),
Barely/ (1976), Young (1969), Osse (1969), Sarkar (I960), Dutta (1968, 1973,
1974, 1975, 1977, 1978, 1979a, b, 1980). EishoudmOldenhave and Osse (1976),
Lauder (1979, 1980a,b), and Lauder and Liem (1980). This philosophy in turn has.
become more apt to empirical experimentation with the introduction of electro"
myographic techniques and high-speed cinematography.
Within the last one and a half decades functional anatomists have begun to ana-
lyse experimentally the feeding and respiratory mechanisms of vertebrates. These
experimental studies involve high-speed cinematography (Dutta 1968, 1975, 1979,
1980; Liem 1967b, 1970; Lauder 1979;. Nyberg 1971).
It is well known that in many families of the bony fish, food is obtained by
sucking (Alexander 1970). These movements are very fast (20-^50 m second) and
negative pressures from 100 to 400 cm of water have been registered (Hughes
1970). Therefore, a high speed, movie camera of 500-1000 frames/second is
essential to make a precise recording of movements of the bony elements as well
as the entire mechanism of prey intake offish and other vertebrates. Food intake
is the dominating function in fish populations and depends on the rapidness of
movements of box y elements as well as the activity of their related muscles.
Synchronized electromyo graphic (EMG) and cinematographic techniques have
been adopted by Ballintijn et al (1972), ElshcudOldenhave and Osse (1976),
Lauder and Liem (1980a,b), Liem (1973, 1978), Liem and Osse (1975) and Osse
(1969). Some authors have also used the cinematographic-electromyographic
technique in higher forms of vertebrates (Kallen and Gans 1972; Weijs and
Dantuma 1975). The usual cinematographic technique has been improved through
the use of x-ray movies (Anker et al 1967). The x-ray cinematography is being
extensively used by the researchers in Anglo-America as well as in Europe for the
analysis of bone movements.
3, Conclusions
To establish form-function relationship all three approaches (evolutionary, onto-
genetic and holistic) may be considered scientific, but there are relative advantages
and disadvantages in their experimentation. Since verification of evolutionary
findings is experimentally impossible, this approach deals with theoretical explana-
tion. As different stages of development of an organism (including very minute,
embryonic structures) are involved in on to genetic studies, microscopic analysis,
in addition to the application of electromyographic and cinematographic tech-
niques, is necessary wherever required. Nonetheless, electromyographic technique
is almost impossile to apply in small sizje specimens at the early stages of verte-
212 Hiran M Dutta
brate development. However, since the holistic approach normally involves
mature organisms it is generally possible to apply all three techniques (microscopic,
electromyographic and cinematographic) without much difficulty.
References
Alexander R Men 1970 Mechanics of the feeding action of various teleost fishes ; /. Zool. 162
145-156
Allis E P 1903 The skull and cranial and first spinal muscles and nerves in Scomber scomber ;
/. Morphol 18 45-328
Anker G C, Simons J and Dullemeijer P 1967 An apparatus for direct x-ray cinematography
exemplified by analysis of some respiratory movements in Gasterosteus ctculeatus ; Experi-
entia 23 74-77
Balinsky B I 1965 An introduction to embryology (Philadelphia : Sanders) p. 725
Ballintijn C M, Burg A Van Der and EG-Erink B P 1972 An electromyographic study of the
adductor mandibulae complex of a free swimming carp (Cyprinus carpiol) during feeding ;
J. Exp. Biol 57 261-283
Barel C D N, Witte F and Vanoijen M J P 1976 The shape of the skeletal elements in the head
of a generalized Haplochromis species : H. elegans trewavas. 1933 (Pisces, Cichlidae) ;
Neth. J. Zool 26 163-265
Barge J A J 1919 Vorm en functie (Leiden : Rede Publishers) p. 29
Barge J A J 1936 Forme et fonction. La nature du probleme ; Folia Biotheoretica I 12-26
Blechschmidt E 1955 Embryologische untersuchungen unter funktionellen gesichtspunkten ;
Acta Anat. 24 339-392
Bock W J 1959 Preadaptation and multiple evolutionary pathways; Evolution 13 194-211,
Bock W J 1960 Secondary articulation of the avian mandible ; Auk. 77 19-55
Bock W J 1964 Kinetics of the Avian Skull ; /. Morphol. 114 1-42
Bock W J and DeWitt M 1959 The scansorial foot of the Woodpeckers with comments on the
evolution of perching and climbing feet in birds ; Am. Mus. Novit. 1931 45
Boker H 1935 Vergleichende biologische Anatomie der Wirbeltiere ; /(Jena: Gustav Fisher)
p. 228
Boker H 1936 Form und Funktion im Lichte der vergteichenden biologischen Anatomie ; Folia
Biotheoretica 1 27-41
Claes H 1964 Das volumenwachstum des korpers, seiner blasteme and organe wahrend den
embryonalund fruhen Larvalentwicklung von Rana temporaria ; Acta Anat. 59 229-281
Claes H 1965 Histogische dirTerenzienung Anderung des volumenverteilungsmusters und morpho-
genetische bedeutung der chorda dorsalis Wahrend den Embryonal und Larvalentwicklung
von Rana temporaria L. ; Acta Anat. 62 104-156
Davis D D 1949 Comparative anatomy and the evolution of vertebrates ; Genetics Paleontol
Evolution pp. 64-89 . . • -
Davis D D 1958 Tarsal ligaments of the spectacled bear, Tremarctos ornatus ; Fieldiana, Zool
Mem. 39 19-105
Davis D D 1964 The Giant Panda ; Fieldiana Zool. Mem. 3 339
DeBeer G R 1954 Archaeopteryx and evolution ; Adv. Sci. 42 1-11
DeJongh H J 1968 Functional morphology of the jaw apparatus of larval and metamorphosing
Rana temporaria. L. (Leiden : E J Brill) p. 103
Dullemeijer P 1956 The functional morphology of the head of the common vipera Viper a berus
(L.) ; Arch. Neerl Zool 11 386-497
Dullemeijer P 1958 The mutual structural influence of the elements in a pattern ; Arch, Neerl,
Zool. 13 Suppl. pp. 174-188
The form-function relationship of vertebrates 213
Dullemeijer P 1959 A comparative functional-anatomical study of the heads of some Viperidae ;
Morphol. Jaheb. 99 881-985
Dullemeijer P 1971 Comparative ontogeny and cranio-facial growth. In : Cranio-facial growth
in man (eds.) R E Moyers and W Krogman (Oxford ; Pergamon Press) pp. 45-75
Dullemeijer P 1974 Concepts and approaches in animal morphology \ 1-120. vanGorcum, Assen.
The Netherlands, p. 264
Dutta H M 196& Functional morphology of the head of Anabas testudineus (Block) (Leiden,
Netherlands : Drips Repro. N. V. Meppel), p. 146
Dutta H M 1973 Functional correlation between the structure of the head and feeding mechanisms
in Ctenopoma acutirostre ; Anat. Rec. 175 310
Dutta H M 1974 Structural analysis of the hyomandibula in Macropodus opercularis, Ctenopoma
acutirostre, and Anabas testudineus ; Anat. Rec. 178 348-349
Dutta H M 1975 The suspensorium of Ctenopoma acutirostre : A comparative functional
analysis with Anabas testudineus (Bloch) ; /. Morphol 146 457-478
Dutta H M 1977 Functional basis of Micropterus salmoides : An electromyographic and
cinematographic analysis ; Anat. Rec. 187 570-571
Dutta H M 197S Feeding mechanism of largemouth bass; Am. ZooL 18 622
Dutta H M 1979a An Electromyographic and cinematographic analysis of the feeding
mechanism in Ictarulus melas (Catfish) ; Anat. Rec. 190 387
Dutta H M 1979b Form and function of jaws during feeding. Ctenopoma acutirostre, Anabas
testudineus and Macropodus opercularis ; Acta Morphol. Neerl-Scand. 17 119-132
Dutta H M 1980 Comparative analysis of the hyomandibula during respiration in Anabantoid
teleost fishes : Macropodus opercularis in relation to Ctenopoma acutirostre and Anabas
testudineus ; Zoomorphol 94 185-202
Eaton T H 1935 Evolution of the upper jaw mechanism in teleost fish ; /. Morphol. 58 157-172
Edgeworth F H 1935 The cranial muscles of vertebrates (Cambridge Univ. Press)
Elshoud-Oldenhave M and Osse W M 1976 Functional morphology of the feeding system in
the 'Ruff—Gymnocephalus cerna (L. 1758) (Teleostei, Percidae) ; J. Morphol 150 399-422
Gans C 1952 The functional morphology of the egg-eating adaptations in the snake genus
Dasypeltis ; Zoologica 37 209-245
Gans C 1960 A taxonomic revision of the Trogopophinae and a functional interpretation of
the Amphisbaenid adaptive pattern ; Bull Am. Mus. Natl Hist. 119 129-204
- Gans C 1966 Some limitations and approaches to problems in functional anatomy ; Folia
Biotheoretica 6 41-50
Gans C 1969 Functional components versus mechanical units in descriptive morphology ; /.
Morphol 128 365-368
Gaupp E 1905 Die entwicklung des kopfskelettes. In : Handbuch der vergleiehenden und
experimentellenentwicklungslehrederwirbeltiere. (ed.) OttertwigII(Jena: G Fisher) 573-890
Goedbloed J F 1964 The early development of the middle ear and the mouth cavity. A study
of the interaction of process in the epithelium and mesenchyme; Arch. Biol 75 207-729
Goss A N 1964 Adaptive growth (London, New York : Academic Press) p. 350
Greenwood P H 1965 Environmental effects on the pharyngeal gill of a cichlid fish, Astatoreo-
chromis alluaudi, and their taxonomic implications; Proc. Linn. Soc. London 176 1-10
Gutmann W F 1966 Funktionsmorphologische Beitrage zur * Castraea-Coelomtheorie ' ; Senck.
Biol 47 225-250
Gutmann W F 1967 Die Entstehung des Coeloms und seine phytogenetische Abwandlung in
Deuterostomier-Stamm ; Zool Anz. 179 109-131
Gutmann W F 1968 Die funtionelle Beziehung von Achsenskelett und Stamm-Muskel-Apparat
der fischartigen Vertebraten ; Senck Biol. 49 265-272
Hofer H 1948 Untersuchungen tiber den Bau des Vogelschadels, besonders uber den der
Spechte und Steiszhuhner ; Zool Jahrb. Abt. Anat. 69 1-158
, Holmquist O 1910 Der Musculus Protractor Hyoidei (Geniohyoideus Auctt.) und der
Senkungmechanismus des Unterdiefers bei den Knochenfischen. Lunds Univ. Ar&., N. F.
Afd. 2, Bd. 6, Nr. 6 1-24
Hughes G M 1970 Respiration in an air-breathing fish, the climbing perch, Anabas testudineus ;
/. Exp. Biol 53 281-29$
214 Hiran M Dutta
Kallen F C and Cans C 1972 Mastication in the little brown bat, Myotis lucifugus ; J. MorphoL
136 385-420
Klaauw C J Van Der 1945 Cerebral skull and facial skull. A contribution to the knowlegde of
skull structure ; Arch. Neerl. Zool. 7 16-37
Klaauw C J Van Der 1948, 1951, 1952 Size and position of the functional components of the
skull. A contribution to the knowledge of the architecture of the skull base on data in
the literature ; Arch. Neerl Zool. 9 1-176 ; 177-368 ; 369-559
Klaauw C J Van Der 1963 Projections, deepenings and undulations of the surface of the skull in
relation to the attachment cf muscles (Amsterdam : N. V. Noord Hollandsche Uitgebers
Maatschappii) p. 247
Landsmeer J M P 196& Les coherences spatiales et Tequilibre spatial uans la region carpienne ;
Acta Anat. 70 Suppl 54 1-84
Lander G V 1979 Feeding mechanism in primitive teleosts and in the halecomorph fish, Amia
calva ; J. Zool. London 187 543-578
Lauder G V 1980 The role of the hyoid apparatus in the feeding mechanism of the coelacanth
Latimeria chalumnae ; Copela 11-9
Lauder G V and Liem K F 1980a The feeding mechanism and cephalic myology of Salyelinus
• fontinalis : form, function and evolutionary significance on Charm (Salvelinus). (ed.) E K
Balon (The Netherlands : Junk Publishers) pp. 365-390
Lauder G V and Liem K F I980b Evolution of the feeding mechanism in primitive Actinoptery-
gian fishes : A functional anatomical analysis of Polypterus lepisosteus and Amia ; J.
MorphoL 163 283-317
Liem K F I967a A morphological study of Luciocephalus pulcher, with notes on gular elements
in other recent teleosts ; /. MorphoL 121 103-133
Liem K F 1967b Functional morphology of the head of the anabantoid teleost fish, Helostoma
temmincki ; /. MorphoL 121 135-158
;Liem K F 1970 Comparative functional anatomy of the Nandidae (Pisces: Teleostei) ;
Fieldiana, Zool. 56 166
Liem K F 1973 Evolutionary strategies and morphological innovations : Cichlid pharyngeal
jaws ; Syst. ZooL 22 425-441
Liem K F 1978 Modulatory multiplicity in the functional repertoire of the feeding mechanism
in Cichlid fishes ; J. MorphoL 158 323-360
Liem K F and Osse J W M 1975 Biological versatility evolution, and food resource exploitation
in African Cichlid fishes ; Am. Zool. 15 427-454
Milaire J 1963 Etude morphologique et cytochimique du developement des membres chez la
sowris et chez la taupe ; Arch. Biol. 74 131-317
Moss M L- 1958 Rotations of the cranial components in the growing rat skull and their experi-
mental, alteration ; Acta Anat. 32 65-86
.Moss M L 1959 Disorders of the temporomandibular joint (Philadelphia : Saunders)
Moss M L 1960 Functional analysis of human mandibular growth ; /. Prosthet. Dent. 10 1149-
1159
Moss M L 1961a Rotation of the otic capsule in bipedal rats ; Am. J. Phys. AnthropoL 19
301-307
Moss M L I968a A theoretical analysis of the functional matrix ; Acta Biotheoretica 18 195-202
Moss M L 1968b Functional cranial analysis of mammalian mandibular ramal morphology ;
.Acta Anat. 71 423-447
Moss M L I968c The primacy of functional matrices in orofacial growth ; Trans. Br. Soc. Study
Orthod. and Dental Pract. 19 65-73
Moss M L 1971 Ontogenetic aspects of cranio-facial growth. In Cranio-fadal growth in man
(eds.) R E, Movers and W Krogman (Oxford and New York: Pergamon Press)
pp. 109-124
Moss M L and Salentijn L 1969a The primary role of functional matrices in facial growth ;
Am. J. Orthod. 55 6 556-577
Mass M L and Salentijn L 1969b The capsular matrix; Am. J. Orthod. 56 474-49Q
Moss M L and Young R. 1960 A functional approach, to craniology ; Am. J. Phys. AnthropoL
18 281-292
Nyberg D 1971 Prey capture in the largemouth bass ; Am. Midi. Nat. 86 128-144
The form-function relationship of vertebrates 215
Osse J W M 1969 Functional Morphology of the head of the perch (Perca fluviatilus L.) ; An
electromyographic study (Leiden : E J Brill) pp. 289-392
Osse J W M, Elshoud-Oldenhave M and Vanshie B 1972 A new method for insertion for wire
electrodes in electromyography ; Electromyography 12 59-62
Rensch B 1943 Histological changes correlated with evolutionary changes of body size ; Evolution
2 218-230
Rensch B 1958 Die Abhangigkeit der Struktur und der Leistungen tierischer Gehirne von ihrer
Grosse ; Die Naturms sens chaf ten 45 145-154 ; 175-180
Rensch B 1960 Trends toward progress of brains and sense organs ; Cold Spring Harbor Symp.
Quant. Biol. 24 291-303
Rensch B 1972 Neuere Probleme der Abstammungslehre. Die transspezifische Evolution. 3e,
durch einen Anhang erweiterte Auflage, XI + 468 p. Stuttgart, Ferdinand Enke Verlag
Russell E S 1936 Form and Function. A historical note ; Folia Biotheoretica 1 1-12
Sarkar S 1960 A study of the functional morphology of the head of an Indian puffer fish,
Sphaeroides oblongus (Bloch). Thesis, Leiden, p. 119
Schaeffer B and Rosen D E 1961 Major adaptive levels in the evolution of the actinopterygian
feeding mechanism ; Am. Zool. 2 187-204
Sedra S N ±950 The metamorphosis of the jaws and their muscles in the Toad, Bufo regularis
Reuss, correlated with the changes in the animals' feeding habits ; Proc. ZooL Soc. London
120 405-449
Smit P 1961 Ontogenesis and phylogenesis-— their interrelation and their interpretation ; Acta
Biotheoretica 15 1-103
Takahasi N 1925 On the myology of the cranial muscles of the cypriniform fishes ; /. Morphol.
40 1-103
Tchernavin V V 1953 On the mechanical working of the head in bony fishes ; Proc. ZooL
Soc. London 118 129-143
Weijs W A and Dantuma R 1975 Electromyography and mechanics of mastication in the
albino rat ; J. Morphol. 146 1-34
Werner C L F 1958, 1959 Relative grosze und lage der organe als faktoren der ontogentischen
und phylogenetischen formbildung ; Wiss. Z. Karl Mann Univ. J. 8 7-16
Wolpert L 1968 The French flag problem. In Towards a theoretical biology 1, Prolegomena,
(ed.) C H Waddington Edinburgh University Press, pp. 125-133
adian Acad. Sci. (Anim. Sci.), Vol. 91, Number 3, May 1982, pp. 217-223.
in India.
ibolic rates and quotients in the cichlid fish,
« mossambka (Peters) in relation to random activity
M PEER MOHAMED
Central Inland Fisheries Research Substation, 24, Pannalal Road,
Allahabad 211002, India
MS received 21 February 1981 ; revised 31 October 1981
Abstract. Oxygen consumption, carbon dioxide production and NH3-N excretion
increased with increase in random (spontaneous) activity in Tilapia mossambica in
air-saturated water in tests at 30 and 35 °C. The random activity change did not
affect the RQ which remained near unity at adequate ambient oxygen. But, the
AQ decreased with increase in activity at both 30 and 35° C, suggesting that
increased protein utilization in quieter fish when adequate ambient oxygen is
available. The routine and standard metabolic rates at 35 °C are slightly higher
than at 30° C, but, in general, the overall metabolic rates and quotients are signi-
ficantly in close proximity, suggesting that the temperature range (30-35° C) does
not seem to cause a marked metabolic difference in Tilapia mossambica.
Keywords. Standard metabolic rate ; routine metabolic rate ; respiratory quotient ;
ammonia quotient ; random activity ; Tilapia mossambica.
introduction
gy utilization for the various biological activities of the whole animal can be
J only if the values of metabolism truly reflect the standard (basal), activity
;her activity-spelt-out (e.g., swimming speed in fish) state of the animal. The
snce of random activity on metabolic rates and quotients (Respiratory
tient, RQ = volume of COa produced/volume of O2 consumed; Ammonia
tient, AQ = the volume or mole : mole relation of NH3-^N excreted to Oa
umed) has been extensively studied only in a few fish (Kutty 1968; Peer
tamed 1974;; Kutty and Peer Mohamed 1975). Under aerobic conditions
iom activity did not appear to have any effect on the RQ of goldfish and
y ow trout (Kutty 1968), but the AQ might change with random activity (Peer
lamed 1974; Kutty and Peer Mohamed 1975). Since Tilapia mossambica
l>een subjected to metabolism studies in relation to several factors (Rutty et al
jaj Kutty 1972; Karuppannan 1972; Peer Mohamed and Kutty 1980; Peer
lamed 1981), there is lack of information on its standard (basal) metabolic
s and quotients. Present observations deal with metabolic rates — Oa con-
ption, CO2 production and NH3-N excretion— and quotients (RQ and AQ)
"*, mossambica during random activity at high ambient oxygen.
217
218 M Peer Mohamed
2. Material and methods
Tilapia mossambica (Paters) were collected from freshwater tanks in and around
Madurai and acclimated to freshwater at 30 ± 0-5° C and 35 ± 0-5°C for
at least 15 days before the experiments. The fish were fed ad lib with a formu-
lated food : fish muscle, goat liver and wheat hearts pro rata 2:1:1 (Karuppannan
1972). Experimental fish were starved for 24 hr (Peer Mohamed and Kutty
1980) and subsequently left in the respirometer overnight with a continuous
flow of water. Tests were performed at the temperature of acclimation.
The apparatus used was a modification of Fry's respirometer (Kutty et al 1971b)
in which simultaneous measurements of metabolic rate and random activity can
be made. Decarbonated tap water, adjusted to a pH of 8-2, was used as
explained by Kutty et al (1971a).
2 . L Experimental procedure
Each experiment consisted of 7-# rims of 1 hr in duration, when the respiro-
meter remained closed. Water samples for analyses of dissolved oxygen, total
carbon dioxide and ammonia were collected just before and after the closure
of the respirometer for individual runs. The respirometer was flushed for 30 min
with air-saturated decarbonated water between runs so as to bring the ambient
oxygsn content near air saturation at the beginning of each run. The random
activity of the fish was counted by the difference between the initial and final
figure of the activity counter, which was noted immediately after each sampling.
2.2. Methods of water analyses
Dissolved oxygen was measured by using the unmodified Winkler technique
(American Public Health Association 1955). The sample used for titration was
25ml.
Total carbon dioxide was estimated by Maros-^Schvlefc technique (Maros et al
1961) modified for fish metabolism studies by Kutty et al (197la) was followed.
Fifty ml of water sample was used for each determination.
Ammonia was measured by the method of Stroganov (1962) as described by
Kutty (1,972). Fifty ml of water sample was distilled, the distillate nesslerised and
the optical density read in Bausch and Lomb spectrophotometer (Spectronic-20)
at a wavelength of 420 ju. Ammonia-free water (American Public Health Associa-
tion 1955) was used for blank and to prepare the reagents.
3. Results
Plots of routine oxygen consumption, carbon dioxide production,, KH3^N excre-*
tion, RQ and AQ against random activity of T. mossambica (63*4 g; 16-8 cm)
acclimated to and tested at 30° C at ambient oxygen concentration near air satu^
ration are shown in figure 1. Similar plots for T. mossambica (64- Ig, 17-0 cm)
acclimated to and tested at 35° C are . given in figure 2. The present plots for
Tilapia are simple plots through which a single regression line could easily be
fitted (table 1). Mean values of routine metabolism and randoin activity of
T. mossambica at 30 and 35° C are given in table 2. Tlie standard metabolic
Metabolic rates and quotients in T. mossambica
219
o
cr
I
ro
X
CM
O
O
0. I 5
0. I 0
0.07
I. 2
KO
ON
:R
x
UJ
2
2
I-
O
ID
O
O
<r
a.
T
a:
x
0
0.8
0.6
I 0
5
I 00
50
40
30
100
50
40
1.
J_
0 10 20 30 40
RANDOM ACTIVITY (COUNTS/HOUR)
Figure 1. Oxygen consumption;, carbon dioxide production, NHa-N excretion,
RQ and AQ in relation to random activity in Tiktpia mossambica acclimated to and
tested in air-saturated water at 30° C. The lines fitted through the plots are
according to the regression equations given in table 1.
rates and quotients (extrapolated values at 'zero ' activity) are also included in
table 2.
The routine metabolic rates (O2 consimption, COa production and NH3-*N
excretion) at 35° C are slightly higher but the metabolic quotients (RQ and AQ)
are remarkably close to each other at 30 and 35° C.
.* Regression coefficients of metabolic rates and quotients at 30 and 35° C were
statistically tested by applying /-test and it was observed that the regression
coefficients are not significantly different (P > 0-05), suggesting that the
temperature range (30^35° C) does not cause a marked metabolic difference in
220
M Peer Mohamed
0. 15
0 I0 20 30 40
RANDOM ACTIVITY (COUNTS/HOUR)
Figure 2. Oxygen consumption, carbon dioxide oroduction MH M
4. Discussion
nullet,
(Kutty and Peer
The
Metabolic rates and quotients in T. mossambica
221
Table 1. Regression equations (log 7= a + bX) of oa consumption, CO2 produc-
tion, NH8-N excretion (ml/kg/hr in each case), RQ and AQ (Y) on random activity
(counts/tor) (A") in Tilapia mossambica at ambient oxygen concentration near air
Saturation.
Acclimation and test at 30° C
Acclimation and test at 35° C
O3 log Y = 1, • 69888 -4- 0 • 00677 X
COa log F== 1/60628 -f 0/00852 X
NH3-N log Y = 0-75565 +• 0-00342 X
RQ log F = -0-08831 -f-0'00128 JT
AQ log r = -0- 92576 - 0-00451 X
log Y = 1 '79633 -f 0-00644 X
log Y = 1 • 68797 + 0 * 00877 X
log Y = 0- 87070 -f- 0- 00308 X
log F = -0-09903+0-00203 X
log y = -0-92552 - 0*00335 X
Table 2. Routine and standard O2 consumption, CO2 production, NH3-N excretion
(ml/kg/hr in each case), RQ, AQ and random activity (count$/hr) in Tilapia
mossambica at ambient oxygen concentration near air saturation. Results of
experiments at 30 and 35° C are Shown separately. In the case of routine values
mean arid one Standard error (N = 15 and 1 6 for 30 and 35° C respectively) is
indicated in each casjo. The Standard values are estimates obtained by extrapolation
of the regression lines to zero activity through the plots in figures 1 and 2.
30° C
35° C
JLYJLC KB LfUUV^ JL « «7/ C£ «U HCJJ,t
Routine
(Mean ± SE)
Standard
Routine
(MeaniSE)
Standard
Oa consumption
64-4 ±2-1
50-0
81-8 ±3-7
62-6
COa production
55-6 ±2-4
40-4
70-6 ±4-3
48-7
NH3-N excretion
6-5 ±0-06
5-7
8-4 ±0*27
7-4
RQ
0-86 ±0-02
0-82
0-87 ±0-02
0*80
AQ
0-101 ±0-003
0-119
0-105 ±0-003
0*119
Random activity
1.5-7 ±2-0
••
17-0 ±2-8
"*
yield regressions which have negative slopes (figures. 1 and 2, table 1) thereby
suggesting that lower random activity was. associated with higher AQ, i.e., the less
active the fish, the proportionally higher its protein use and/or a greater involve-
ment of protein degradation. This observation is in agreement with earlier reports
on R. corsula (Rutty and Peer Mohamed 1975) and also on smolting Atlantic
salmon (Saunders and Kutty 1973). But there is also a variance in the results
of study on the influence of forced activity on AQ which increased with increase
in activity (Kutty 1972; Karuppannan 1972; Sukumaran and Kutty 1977). In
!T. mossambica, it was observed that the initial AQ (first hour of 5-hour exercise)
at lower swimming speed was. even less than the routine AQ (Kutty 1972 j
Karuppannan 1972) and it was suggested that in this case there might be a
222 M Peer Mohamed
protective action of carbohydrates on protein. This might be the reason why the
steady AQ (after 2-3 hours swimming) is correlated well with the initial RQ of
fish under long-term exercise. It is possible that random activity (spontaneous
random movement) and forced activity (intense exercise) have different relations
to protein degradation and utilization, as indicated by NH3-^N excretion. A
quieter flsh would utilize more proteins but with increase in random activity rela-
tively more energy is required by the feh for several breaks and starts in swimming
overcoming inertia each time (Smit 1965; Kutty 1969). In this case utilization of
carbohydrates could be higher as long as adequate oxygen is. available, thereby
indicating lower AQ at higher random activity, more or less similar to the low
initial AQ during the beginning of intense activity (continued exercise). In both
these cases, fish under high random activity and initial phase of intense swimming
(forced activity), the protective action of carbohydrates on proteins may be
operative (Phillips 1969).
The validity of the present observations lies in the fact that the estimations of
metabolic rates and quotients are made in single fish as was usually done in earlier
studies (Kutty et al 1971b; Peer Mohamed 1974). It is clear from the statistical
tests of the regressions that the estimates at 30 and 35° C are remarkably signi-
ficant, suggesting that the temperature effect is minor. This, is possible, probably
because these temperatures are close to each other and are within the upper range
to which T. mossambica is normally exposed during a good portion of the year.
The present method described here, which enables separation of the interfering
influence, of activity on metabolism is of much importance in organismal
physiology.
Acknowledgements
The author is grateful to Dr M N Kutty for his. supervision and guidance, to
Professor S Krishnaswamy for providing facilities and to Mr R K Tyagi for
statistical analyses.
References
American Public Health. Association 1955 Standard methods for the examination of water, sewage
and industrial wastes 10th edn. (New York ; Am. Publ. Health Org.) pp. 522
Karuppannan N V 1972 Studies on the locomotory metabolism of Tilapia mossambica (Peters) ;
Ph.D. Thesis, Madurai Kamaraj University, Madurai, India
Kutty M N 1968 Respiratory quotient in goldfish and rainbow trout ; /. Fish. Res. Bd. Can. 25
16S9-172S
Kutty M N 1969 Oxygen con-sumption in the mullet Liza macrolepis with special reference to
swimming velocity ; Mar. Biol. (Berlin : Springer- Verlag) 4 239-242
Kutty M N 1972 Respiratory quotient and ammonia excretion in Tilapia mossambica ; Mar.
Biol (Berlin : Springer-Verlag) 16 126-133
Kutty M N, Karuppannan N V, Narayanan M and Peer Mohamed M 197la Maros-Schulek
technique for measurement of carbon dioxide production in fish and respiratory quotient in
Tilapia mossambica', J. Fish. Res. Bd. Can. 28 1342-1344
Kutty M N and Peer Mohamed M 1975 Metabolic adaptations of mullet Khinomugil corsula
(Hamilton) with special reference to energy utilization ; Aquaculture 5 253-270
Kutty M N, Peer Mohamed M, Thiagarajan K and Leonard A N 1971b A modification of Fry's
fish activity counter and respirometer ; Indian J. Exp. BioL 9 218-222
Metabolic rates and quotients in T. mossambica 223
Maros L, Schulek E, Molnar-Perl I and Pinter-Szakacs M 1961 Einfaches Destinations ver-
fahren zur titrimetrischen Bestimmung von Kohlendioxyd . (A simple distillation method
for the titrimetric determination of carbon dioxide). Anal. Chim. Acta 25 390-399
(Trans, from German, Fish. Res. Bd. Can. Trans. Ser. No. 596, 1965)
Peer Mohamed M 1974 Influence of hypoxia on fish metabolism and activity ; Ph.D. Thesis,
Madurai Kamaraj University, Madurai, India
Peer Mohamed M 1981 Metabolism of Tilapia mossambica (Peters) with emphasis on hypoxia ;
Indian J. Exp. Biol. 19 1098-1100
Peer Mohamed M and Kutty M N 1980 Respiratory quotient and! ammonia quotient in Tilapia
mossambica (Peters) with special reference to hypoxia and recovery ; Hydrobiologia (In
press)
Peer Mohamed M, Nath D, Srivastava G N and Gupta R A 1978 Influence of sublethal DDT
on standard (basal) metabolism of the freshwater fishes Cinhina mrigala (Hamilton), Labeo
rohita (Hamilton) 'and Colisa fasciata (Bloch and Schneider) ; Indian J. Exp. Biol. 16
385-386
Phillips A M 1969 Nutrition, digestion and energy utilization; Fish Physiology (eds.) W S Hoar
—- and D J Randall (New York and London : Academic Press) 1 391-432
Saunders R L and Kutty M N 1973 Oxygen consumption and ammonia excretion m Atlantic
salmon smolts (Ms)
Smit H 1965 Some experiments on the oxygen consumption of goldfish (Carassius aurotus L.)
in relation to swimming spc ed ; Can. J. Zool. 43 623-633
Siroganov N S 1962 Methods of study of respiration of fish and methods for ammonia determi-
nation, used in studies on fish metabolism ; Techniques for the investigation offish physio-
logy pp. 106-111, (ed.) E N Pavlovskii Izd. Akad. Nauk SSSR (Transl. Natn. Sci. Fdn.,
Washington, D.C., PST Cat. No. 1130 by the Israel Programme for Scientific Translation,
Jerusalem, 1964)
Sukumaran N and Kutty M N 1977 Oxygen consumption and ammonia excretion in the- cat-
fish Mystus armatus, with special reference to swimming speed and ambient oxygen ; Proc.
Indian Acad. Sci. B3 195-206
P.(B)~2
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 3, May 1982, pp. 225-234.
© Printed in India.
Microanatomy of the 7th abdominal ganglion and its peripheral
nerves in the scorpion Heterometrus fulvipes
K YELLAMMA, K SUBHASHINI, P MURALI MOHAN and
K SASIRA BABU
Department of Zoology, S.V. University, Tirupati 517 502, India
MS received 17 November 1981 ; revised 20 April 1982
Abstract. Microanatomical studies on the 7th abdominal ganglion of the scorpion
were carried out by htstological methods. The ganglion revealed a total of approxi-
mately 2000 nuclei, most of them belonging to medium-sized cells. The connec-
tive and peripheral nerves revealed fibres of varying number in each, with fibres of
3 tim contributing largely to the total content. The fibres included the axons of
motor, sensory and interneurons.
Keywords. Heterometrus fulvipes ; histological techniques ; microanatomy ; cells ;
abdominal ganglion ; peripheral roots.
1. Introduction
Microanatomical knowledge of arthropod abdominal ganglia has been based on
highly selective methylene blue stains (Retzius 1890 ; Bethe 1897) and silver-
stained serial sections (Kendig 1967). These procedures provided information
concerning the course of fibre groups or the location of cell bodies. The avail-
able literature on the anatomy of the arachnid central nervous system comes
mainly from the studies of Babu (1965). The present investigation attempts to
resolve the microanatomy of the 7th abdominal ganglion and its peripheral roots
in the scorpion H. fulvipes at the light microscopic level.
2. Materials and methods
Freshly collected adult scorpions were anaesthetized with chloroform and care-
fully dissected under binocular microscope to expose the 7th abdominal ganglion
with all its branches intact. The connective tissue around the ganglion and its
branches was cleared without any damage to the nervous tissue and the entire
preparation was submerged in Bouin's fluid in a Petridish (Babu 1965) for 24 hr.
After 24 hr the connective, 4th and 5th segmental and telsonic nerves were cut
to the ganglion and used for sectioning.
225
226 K Yellamma et al
The method employed for sectioning was Peterfi's celloidin double embedding
method, through which satisfactory results were obtained. After dehydration in
methanol series, the material to be sectioned was led through two or three changes
of paraffin maintained at 60-62 °C. Use of mixtures of paraffin with different
melting points and addition of fresh wax each time before making the block
minimized the impediments during sectioning. Cross-sections (10 /mi) were
taken from the ganglion, connective, 4th and 5th segmental a«nd telsonic nerves.
Serial sections of the ganglion were stained by making use of Palmgren's (1948)
silver-staining method. The silver method of Holmes (1952) was used for staining
the nerve trunks.
After clearing and mounting the sections, the counting of cell number and the
measurement of the cell diameter were done as suggested by Abercrombie (1946).
The diameter of the nerve fibres was measured under high magnification. The
cells and nerve fibres were categorized into different groups based on their diameter,
and tabulated according to their size and number.
Preliminary studies to trace the anatomical organization of the 7th ganglion
.and its nerves were also made using the cobalt chloride back-filling technique
(Babu and Subhashini 1981).
3. Results
3-1. Microanatomy of the 1th abdominal ganglion
The 7th abdominal ganglion, being double in its nature, consisted of relatively
larger number of cells compared to other abdominal ganglia of the cord. In
its cross-section (figure 1A) the 7th abdominal ganglion showed the cellular peri-
pheral rind region where the somas of the neurons were located, and a central
fibrous neuropile where synapses would occur. The size of the ganglion in its
cross-section measured approximately 980-1000 #m wide and 650 /on thick. The
cellular rind constituted 1/3 of the total area of the ganglion and the remaining
2/3 was occupied by the neuropile. The ganglion was enclosed in a thick enveloping
sheath of 12 /on which was found to be heterogeneous, containing 4-6 layers of
tissue which were closely packed. There was no cellular perineurium beneath
this neural lamella. Gross-section of the 7th abdominal ganglion revealed the
presence of numerous- cells, with their nuclei staining dark in colour. They
(the nuclei) ranged from 3-20 jam in diameter and they were found to be distri -
buted ventro-laterally and no cells were found on the dorsal side of the ganglion.
The nuclear counts were made by applying Abercrombie's formula. The nuclei
were distinguished as belonging to three sizes, viz., large, medium and small. The
7th abdominal ganglion comprised of a total of approximately 2033 nuclei on
average: The nuclei measuring between 3-9 /nn were around 609 in number
and "they constituted nearly 30% of the total cells. The medium-sized (10-15 /an
nuclei ranged between 1176-1205 and constituted about 58-8% of the total
nuclear content. The rest of the nuclei (large 20-22 /^m) on average were found
to be only about 229 in number and contributed relatively less (11 -2%), to the
total nuclear content of the ganglion. The large nuclei were confined to the peri-
phery of the ganglion, while the small ^nd medium-sized nuclei were distribute4
Microanatomy of scorpion nervous system
227
Figure 1. A. Transverse section of the seventh abdominal ganglion showing the
peripheral rind (R) with different categories of cells, and the central fibrous core
of neuropile (NP) x 40. B. Transverse section of the connective between 6th and
7th abdominal ganglia, showing fibres of different diameters. Note the presence
of large fibres (LA) along with medium (MA) and small (SA) fibres x 160.
K Yellamma et al
Figure 1. C. Transverse section of the 4th segmental nerve after its bifurcation, sho.w-
ing the two roots and their fibre content. Nate the presence of large fibres (LA)
along with medium (MA) and small (SA) fibres x 160. D. A combined transverse
section of 5th segmental (5N) and telsonic(TN) nerves, exhibiting fibres of different
diameters. Note the presence of giant fibres (LA) along with medium (MA) and
small (SA) fibres x 400.
Microanatomy of scorpion nervous system 229
at random. The arrangement of the cells was so compact that the boundary of
each cell could be resolved only under high magnification. By subtracting the
number of motor and interneurons from the total cell count, the number of intra-
ganglionic interneurons was obtained. Their number ranged from 1800-1830.
3 • 2. Microanatomy of the connective and peripheral nerves
Fibre counts and analyses were made for the connective and peripheral nerves
such as the 4th and 5th segmental nerves and the telsonic nerve originating from
the 7th abdominal ganglion. There was no significant bilateral difference between
counterparts of either side. This similarity in composition was reinforced by the
finding of consistent spatial positioning within the root of identifiable axons and
certain medium and large fibres.
3-2a. Connective : The connective in its cross -section (figure IB) exhibited a
thick enveloping sheath (neural lamella) of 8 /on and its diameter ranged between
280-300 /mi. Table 1 shows the number of fibres that occurred in each of the
four different diameter groups, and the fibre diameters used in counting were
arranged in four descriptive groups, viz., fine fibres (less than 5 /on), small fibres
(6-10 /mi), medium fibres (11-1 5 /mi) and large fibres (greater than 16/rai).
Seven axons were above 16 /mi, with the largest axon having a diameter of 18 jum
(table 1). These axons were not arranged into separate distinguishable bundles,
but tended to occur dorsally, centrally, and a few distributed through the entire
area of the connective. In contrast to this, the fine fibres along with the small
fibres were found to be distributed uniformly in the connective whereas the medium
sized fibres were sometimes arranged in pairs and in triplets and occupied various
regions in the connective. The connective comprised of approximately 1460
fibres, falling into different sizes. The fine fibres were relatively larger in number
(about 1259) and constituted 86% of the total fibre population. About 154
fibres were found to be in the diameter range of 6-10 /on and they contributed to
Table 1. Numerical distribution of fibres of different diameter ranges in the con-
nective between the 6th and 7th. abdominr.l ganglia of H. fulvipes.
Fibre diameter
range in nm
Fibre
population
, Per cent in
total fibre
population
0-5
1259±ll-3
86
6-10
154±1M
10
11-15
41 ± 2-6
2-8
16-20
7± 1-5
0-5
Each, value i$ an average of 4 counts of the Same Section, ± standard deviation ,
230 jE Yettamma et al
10% of the total population. The medium sized fibres were about 38 in number
and they contributed to about 2-8%. The large fibres, about 7 in number, fall
into the 4th category of 16-20 /«n range, by far the lowest contribution to the
total axon number.
3-2b. 4th segmented nerve: Cross-section of the 4th segmental nerve (figure 1C)
showed two separate and distinct roots, viz., the dorsal and ventral roots. The
dorsal root measured 120-130 /an in diameter and was enclosed in a 6 /mi thick
neural lamella and the diameter of the ventral root ranged between 90-100 jum
with a 5 /on thick covering sheath. The differences between the two roots were
with regard to the size, the distribution and number of fibres in the nerve. The
dorsal root comprised of about 10 large fibres and the ventral root consisted of
about 7 large fibres. The two roots totally consisted of 290 fibres of which
about 15 were large fibres and represented 5%. The rest of the fibre content
consisted of about 20 medium-sized fibres constituting 7 %, about 30 small fibres
occupying 10% and the remainder (79%) represented by fine fibres (table 2).
3 • 2c. 5th segmental nerve : The total diameter of the 5th segmental nerve close
to its root measured 150 /mi and was enclosed within a neural lamella of 5 jam
diameter. This nerve in its cross-section (figure ID) exhibited a total of approxi-
mately 620 fibres of different sizes. About 550 of these fibres were less than 5 jum
and formed relatively a major contribution (87-4%) to the total axon number.
Fibres of 6-40 /mi contributed about 8%. Medium fibres ranging from 11-15 jum
in diameter were about 10-12 in number and they represented 1 • 9 %. The
large fibres were about 8 in number and measured 16 jum and above in diameter
(table 3). A majority of the large fibres were seen confined to the dorsal region
and at the ventro-lateral region facing towards the telsonic nerve. The fine and
small fibres were distributed uniformly throughout the nerve and the medium-
sized fibres were observed to be located at various regions of the nerve and did not
show specific pattern in their distribution.
Table 2. Numerical distribution cf fibres of different diameter ranges in the 4th
Segmental nerve arising from the 7th abdominal ganglion of H. fulvipes.
Fibre diameter
range
in Aim
Fibre Percent in
population total fibre
population
0-5
231 ±12- 5 79
6-10
30± 8-3 10
11-15
18±l-2 6
16-20
10±1'2 3-5
Each value is an average of 4 counts of the Same section, ± standard deviation,
Micro anatomy of scorpion nervous system 23 1
Table 3. Numerical distribution of fibres; of different diameter ranges in the 5th
Segmental nerve, arising from the 7th abdominal ganglion of JET. fulvipes.
Fibre diameter
range in j^m
Fibre
population
Per cent in
total fibre
population
0-5
540 ±6-1
87-4
6-10
50 ±4- 8
8-0
11-15
12±1'5
1-9
16-20
8±0-1
1-3
Each value is an average of 4 counts of the Same section, ± Standard deviation.
Table 4. Numerical distribution of fibres of different diameter ranges in the telso-
nic nerve, arising from the 7th abdominal ganglion, of H. fulvipes.
Fibre diameter Fibre Percent in
range in ^m population total fibre
population
0-5 757±10-7 87
6-10 61±ll-4 7
11-15 48db 3-1 5-5
16-20 20± 3-0 2-3
Each value is an average of 4 counts of the same section, ± standard deviation.
3-2d. Telsonic nerve: The telsonic nerve (220 jam diameter) (figure ID) like
other peripheral nerves possessed an 8 /an thick enveloping sheath. It revealed
a total fibre number of about 890, the majority of which belonged to the first
category, the size ranging between 2-3 /on. These fine fibres represented 81 %
of the total fibre content. Fibres of 6-10 /on were about 50 in number and
formed 7% of the total fibres. The fibres measuring 10^1 5 jam formed 5-5%
of the total population of fibres. In contrast to other peripheral nerves the
telsonic nerve comprised of more number of large (giant) fibrest, approximately
20, and they measured more than 16 /mi, and constituted about 2-3% (table 4).
These large fibres occupied dorsal, mid-central and ventral regions of the nerve.
The rest of the fibres were distributed at random.
K Yellamma et al
4. Discussion
The general organization of the 7th abdominal ganglion as elucidated by the
present study is in conformity with the pattern of organization of other inverte-
brate ganglia (Bullock and Horridge 1965), with a peripheral cellular rind and a
central fibrous neuropile. The neuropiie, regarded as the terra incognita of neuro-
anatomy (Bullock and Horridge 1965), is one of the most important regions of
neural processing, integrating information from a variety of sources and effecting
patterned outputs.
The sheathing around the ganglion and its nerves resembles that of other arthro-
pods like cockroach (Pipa et al 1959 ; Wigglesworth 1959), locust (Cook 1951),
etc., in being visible only under high magnification and also in the absence of a
cellular perineurium beneath the neural lamella.
The occurrence of a relatively low number of large-sized cells and axons compared
to the number of medium and small-sized neurons again goes well with the
general plan of invertebrate neural organization. These relatively small number
of * giant ' neurons are known to subserve the function of faster conduction of
impulses (Bullock and Horridge 1965), and so very useful in quicker reflexes of
the stinger in the present context. A role for the 7th ganglion in stinger reflexes
in this scorpion was suggested earlier (Yellamma et al 1980). Further, the overall
count of cells in the 7th ganglion in the present study has been found to be fairly
larger than, that reported from several other ** arthropods (Zawarzin 1924 ;
Wiersma 1957 ; Backer 1962 ; Kendig 1967). The occurrence of larger number
of neurons in any system naturally facilitates greater number of synaptic contacts
and thus documents a high degree of integration (Bullock and Horridge 1965).
The fibre count of the connective between the 6th and 7th ganglia in the present
study has been found to be fairly less compared to that in the cockroach (Nunne-
macher et al 1974) and locust (Rowell and Dorey 1967). In keeping with the
organization of the ganglion, the connective also showed only a few fibres larger
than 15 /an and the majority of the fibres were less than 5/jm. This observation,
however, coincides with that in the cockroach (Nunnemacher et al 1974) where
majority of the fibres were less than 3 /on, and large fibres were relatively sparse.
Fibres of less than 2 /*m and 1 /m were reported in wax-moth pupa (Pipa and Wool-
ever 1965) and in locust (Rowell and Dorey 1967) respectively. However, in the
present study attempts to resolve fibres less th$n 1 /mi by cobalt chloride back-filling
were unsuccessful, since cobalt chloride could not diffuse into these axons so easily
as to be feasible for compound microscopic studies. Basing on the work on cray-
fish connectives (Wiersma and Hughes 1961 ; Kennedy and Mellon 1964), it may
be presumed here that many of these small fibres could be sensory, running through
the connective.
Histological observations on peripheral nerves such as the 4th and 5th segmental
and telsonic nerves also corroborated with those on the connective, in that majority
of the fibres were less than 3 /on, and large fibres were relatively less.
These observations were further strengthened by cobalt chloride back-fillings
showing only few fibres larger than 12 /«n, with the remaining ranging between
3-10 /an in diameter. The total fibre count of these peripheral nerves, however, was
far less compared to that of the 1st, 2nd and 3rd roots in the crayfish abdominal
cord (Michael 1970).
Microanatomy of scorpion ntrwiis system
Thus the present investigation on the 7th abdominal ganglion and its nerves
in the scorpion H. fulvipes demonstrates that despite variations in the number
of their different components and possibly accompanying subtle modifications in
physiological functions, they conform in general to the organization met within
the nervous systems of other invertebrates, especially arthropods.
Acknowledgements
The authors thank Prof. K S Swami for providing necessary facilities for this
Work. The financial help rendered by the CSIR through research fellowships to
KY and KS is gratefully acknowledged*
References
Abercrombie M 1946 As cited by Mirrable A W 1062 the counting of cells arid niiciei itf
microtome section ; Q. J. Microsc. Sci. 103 331-347
Babu K S 1965 Anatomy of the central nervous system of arachnids ; ZooL Jb. Ariat. 82 1-154
Babu K S and Subhashini K 1981 Morphology of soma and dendrites of the giant fibre system
in the sixth abdominal ganglion of the cockroach ; /. MorphoL 169 351-355
Backer H W 1962 The number of neurons in an insect ganglion ; Experientia 21 719
Bethe A 1897 Vergleichende Untersuchungen iiber die Funktionen des Zentralnervensystems der
Arthropoden ; Pflugers Arch. ges. PhysioL 68 449-545
Bullock T H and Horridge G A 1965 Structure and function in the nervous system of inverte*
brates (San Francisco : W H Freeman)
Cook P M 1951 Observations on giant fibres of the nervous system of Locusta tnigrantia ;
Q. J. Microsc. Sci. 92 297-305
Holmes W 1952 A silver method for staining nerve axons, its application to sections and whole*
mount preparations; Dept. of Zoology, University Museum, Oxford Neurophysiological Club
Kendig J J 1967 Structure and function in the 3rd abdominal ganglion of the crayfish* P
clarkii; J. Exp. ZooL 164 1-20
Kennedy D and Mellon D Jr 1964 Synaptic activation and receptive fields in crayfish inter-
neurons ; Comp. Biochem. PhysioL 13 275-300
Michael C R 1970 Brain behaviour ; Brain Behaviour Evol. 3 205-209
Nunnemacher R F, Fiske W J and Sherman R O 1974 Size and number of nerve fibres in
the central connectives of the cockroach B. craniife ; /. Insect PhysioL 20 2123-^2134
Palntgren A 1948 A rapid method for selective silver staining of nerve fibres and nerve endings
in mounted paraffin sections; Ada ZooL 29 377-392
Hpa R L and Woalever P S 1965 Insect neurometamorphosis ; Z. Zellftrsch. Mick. Anat 68
80-101
Hpa R L, Cook L F and Richards A G 1959 Studies on the hexapod nervous system. II. The
histology of the thoracic ganglia of the adult cockroach, Periplaneia americana (L):
/. Comp. NeuroL 113 401-433
Retzius G 1890 Zur Kenntniss des Nervensystems der Crustacean, in Biologische Untersuchungen
von Prof. Gustaf Retzius ; NF. I 1-50 Central Druck, Stockholm
Rowell CH F and Dorey A E 1967 The number and size of axons in the thoracic connectives
of the desert locust, S. gregaria ; Z. Zellforsch. Mick. Anat. 83 288-294
Wicrsma C A G 1957 Or* the number of nerve cells in a crustacean central nervous system •
Acta PhysioL Pharm. NeerL 6 135 *
P.(B)~-3
234 & Yellamma et at
Wiersma C A G and Hughes G M 1961 On the functional anatootiy o.f neural units in the
abdominal cord of the crayfish, P, clarkii (Girard) ; /. Comp. NeuroL 116 209-228
Wiggleswoi'th V B 1959 The histology of the nervous system of an insect, Rhodnius prolixus
(Hemiptera). II. The central ganglia ; Q. /. Microsc. Sci. 100 100-299 ,
Yellamma K, Murali Mohan P and Babu K S 1980 Morphology and physiology of giant
fibres in the seventh abdominal ganglion of the scorpion Heterometrus fulvipes ; Proc.
Indian Acad. Sci. (Anim. Sci.) 89 29-38
Zawarzin A 1924 Zur Morphologic der Nervenzentren des Bauchmarks der Insecten ; ein
Beitrag zur vergleichenden Histologie (Histologische Studien iiber Insecten VI) ; Z. Wiss*
Zool 122 323-424
Proc. Indian Acad. Sci. (Anim. ScL), Vol. 91, Number 3, May 1982, pp. 235-241.
© Printed in India.
Branchial protein metabolism of freshwater fish Tilapia mossambica
(Peters) during acute exposure and acclimation to sublethal
alkaline water
M BHASKAR, G VEMANANDA REDDY, V KRISHNA MURTHY,
P REDDANNA and S GOVINDAPPA
Department of Zoology, Sri Venkateswara University, Tirupati 517 502, India
MS received 29 December 1980 ; revised 11 March 19S2
Abstract. Freshwater fish Tilapia ntossambica (Peters) were exposed to sublethal
alkaline water (pH 9 • 0) and the branchial protein metabolism was studied on acute
exposure and acclimation. Branchial tissue had elevated structural and soluble
protein fractions on acclimation which was correlated towards the gill hypertrophy.
Proteolytic activity of the tissue was elevated on both acute exposure and accli-
mation. A/O ratio which forms a measure of ammonia formed per unit Oa
consumption was lesser on acclimation and hence mobilization of tissue free
ammonia towards glutamine formation was suggested. The metabolic modu-
lations have been correlated towards the positive survival value of the fish in
alkaline waters.
Keywords. pH acclimation ; branchial metabolism ; structural proteins ; soluble
proteins ; glutamine ; A/O ratio.
1. Introduction
Fish encounters abnormal pH levels of freshwater due to several factors like
environmental pollution, addition of industrial effluents, hot springs, volcanic
lakes, mine drainage and geological pattern of natural changes (McKee and
Wolf 1963 ; Beamish 1972 ; Cairns etal 1972 ; Dovland etal 1976 ; Oden 1976 ;
Dillon etal 1978). These pH changes affect Wild fish populations in many fresh-
water lakes, streams and rivers (Anderson etal 1971 ; Beamish and Harvey
1972 ; Jensen and Snekvik 1972 ; Aimer etal 1974 ; Schofield 1975 ; Karuppa-
samy 1979).
Studies on fish in altered pH media have been undertaken regarding tolerance
levels (Bandt 1936 ; Trama 1954 ; Carter 1964 ; Jordan and Lloyd 1964 ;
Beamish 1972 ; Daye and Garside 1975), O2 consumption (Packer and Dunson
1972; Krishna Murthy etal 1980) survival and development of embryos and
histopathological changes of tissues (Daye and Garside 1976, 1980a, b) and physio-
logical aspects (Packer and Dunson 1970, 1972 ; Lievestad and Muniz 1976).
235
236 M BJtaskar et al
However there has been little information on tissue metabolism of fish exposed
and acclimated to alkaline media. Our previous studies revealed that the pHs
10*5 and 3-5 were lethal limits to the fish Tilapia mossambica (Krishna Murthy
etal 1980) and tissue metabolism was drastically altered (Bhaskara Haranath
et al 1978).
The animals develop compensatory changes in tissue metabolism under stress
conditions (Precht 1958 ; Kanungo and Prosser 1959 ; Das and Prosser 1967 ;
Govindappa and Rajabai 1976). Hence it was felt desirable to understand the
possible tissue metabolic modulations during acute exposure and acclimation to
sublethal alkaline waters. Since the branchial tissue participates in immediate
ionic regulations, it will be worthwhile to study the metabolic changes of this
tissue under induced alkaline stress.
2. Materials and methods
Freshwater fish, T. mossambica (Peters) of 10 ± 1 g weight, were acclimatized
in glass aquaria with flowing dechlorinated water to the laboratory conditions
(25° C, pH 7- 0 ± 0- 2 ; and light period of 12 hr). The fish were fed with formu-
lated diet of commercial fish pellets. The test fish were exposed to extreme sub-
lethal alkaline pH medium (pH 9) which was prepared by adding 10"1 N NaOH
as suggested by Krishna Murthy etal (1980) and the pH was checked with pH
meter (Elico model LI-10 Hyd.).
The fish were divided into three groups, viz., controls, acute exposed and accli-
mated. The control fish was maintained in normal tap water at pH 7 ± 0-2
and the second and third groups of fish were exposed to pH 9 ± 0- 1 for one day
(acute exposed) and for 15 days (acclimated) (Krishna Murthy etal 1980). The
fish were sacrificed separately, the gill was isolated, rapidly chilled and employed
for biochemical analysis.
Total, soluble and structural proteins were estimated by the method of Lowry
et al (1951) and protease activity (neutral) and free amino acid levels by the
method of Moore and Stein (1954). The rate of tissue respiration was measured
using the conventional Warburg constant volume respirometric apparatus (Umbreit
etal 1959). Glutamate dehydrogenase (E.G. 1.4-1-3) activity was estimated by
the method of Lee and Lardy (1965) as modified by Reddanna and Govindappa
(1979). Free ammonia, urea and glutamine levels were estimated by the methods
described by Bergmeyer (1965), Natelson (1971) and Colowick and Kaplon
(1957) respectively. A/O ratios were calculated by dividing free ammonia
contents with tissue O2 consumption.
3. Results
Data are presented in tables 1 and 2. On acute exposure to sublethal alkaline
medium the branchial tissue had depleted total protein (TP) content (table 1).
The soluble protein (SP) fraction was considerably elevated while structural
protein (StP) was depleted. Protease activity was highly elevated with significant
increase in free amino acid content. The ratios of soluble proteins to structural
Protein metabolism of freshwater fish
237
. Table 1. Levels of total, Soluble and structural proteins, free amino acid content,
protease activity and ratios of SP/TP, StP/TP, SP/StP in tl%e branchial tissue of
control, acute exposed and acclimated fish. -
Parameter (mg/g/ wetwt) Control
Acute exposed
Acclimated
Total proteins (TP)
Soluble proteins (SP)
Structural proteins (StP)
Protease
tyrosine/mg
protein /hr
Free ammo acids
SP/TP
StP/TP
SP/StP
94-60
±8-21
32-15
±2-85
62-45
±4-86
0-0064
±0-00071
17-91
dbl'84
0-340
0-66
0-515
-11-24
P< 0-001
+34-06
P<0-001
-31-56
P<0-001
+509-4
P<0-001
+40-93
P< 0-001
+50-20
-25-86
+105-28
83-97
±6-4
43-10
±3-84
40-87
±4-21
0-039
±0-0028
25-2
±2-32
0-51
0-49
1-055
^17-92
+31-01
P<0-001
+11-18
+275
p<o-ooi
+92-91
P<0-001
+18*81
— 6-09
+17-74
111-55
±8-95
42-12
±4-18
69-43
±5'62
0-024
±0-0016
34-65
±3-85
0'38
0<62
0-606
The values are mean of 6 observations ; Mean ± S.D. ; + and — indicate per cent increase
and decrease respectively on the control values. P denotes statistical significance.
proteins (SP/StP) and soluble proteins to total proteins (SP/TP) were higher and
StP/TP was lesser than controls. The tissue oxygen consumption was considerably
high (table 2). A/O ratio was slightly lesser and GDH activity was highly inhi-
bited. Free ammonia and urea contents had non-significant changes while
glutamine content was depleted. However, on acclimation, the levels of total,
soluble and structural proteins were significantly elevated. Tissue protease was
activated ^nd free amino acid content w^s elevated. The SP/TP and SP/StP
238
M Bkaskar et al
Table 2. Levels of O3 consumption, ammonia/Os ratio, glutamate de^ydrogenase,
free ammonia, urea and glutamine and ratios of urea /ammonia, glutamine/
ammonia in the branchial tissue of control, acute exposed and acclimated fish.
Parameter
Control
Acute exposed
Acclimated
Tissue oxygen
309-15
397-96
324-05
consumption
±25*12
±29-85
±26-52
ni of Oa/g.wt/hr
4-28-73
4- 4-82
P<Q-001
NS
A/O ratio
0-0082
0-0077
OT055
- 6-1
-32-95
GOH
0-029
0-012
0-033
jM mole of formazan/
±0-001
±o-roi
±0-002
mg protein/hr
-58-62
4-13-79
P<0-001
P<0-001
Free ammonia
2-55
2-65
3-11
fj, tnoles/g. wt.
±0-35
±0-28
±0-34
4-3-94
4-21 -96
KS
P<0-01
Urea
4-15
4-11
2*81
/* moles/g. wt.
±0-18
±0-25
±0*14
- 1-44
-25-86
NS
P< 0*001
Glutamine
113-83
96-57
134-24
ft rnolss/g. wt.
±10-25
±2-19
±13-12
- 8-83
±26-74
p<o-ooi
P<0-01
Urea/Ammonia
1-486
Qlutaminc/Ammonia 44- 64
4- 8-51
-15-23
1-612
37-87
-39-33
- 3-33
0-904
43-16
Each value is mean of 6 observations; Moan±S.D.; 4- and — indicate percent increase
and decrease respectively from controls. P denotes statistical significance and ' NS ' is non-
significant.
ratios were considerably higher while that of StP/TP were lower than controls.
Tissue oxygen consumption had non-significant change with low A/O ratio as
compared to control. GDH activity level was elevated. While the free ammonia
and glutamine levels were significantly increased, urea content was depleted.
The urea/ammoni^ ratio was considerably lesser than the control value^
Protein metabolism of freshwater fish 239
4* Discussion
The branchial protein metabolism showed differential pattern during acute
exposure and acclimation to sublethal alkaline waters.
Acute exposure of fish to sublethal alkaline waters depleted total protein content
of the gill. In view of highly elevated protease activity, depleted protein content
in the tissue can be envisaged. However, soluble protein fraction was elevated
while the structural protein fraction was depleted, suggesting possible alterations
in the solubility properties of the proteins in the tissue. Higher SP/TP and SP/
StP ratios indicate that the soluble protein fraction was elevated, probably a
prerequisite for proteolysis in the tissue. Consequently, the structural proteins
of the tissue depleted suggesting proteolysis at structural level of organization of
gill. These observations agree with earlier reports where high tissue proteolysis
was recorded in liver and muscles of fish exposed to alkaline waters (Bhaskara,
Haranath et al 1978) and histopathological changes in tissues of fish exposed to
extreme pH of the medium (Daye and Garside 1976). Since protease activity was
high, the tissue free amino acid content increased considerably. The oxygen
consumption was elevated with lesser A/O ratio suggesting possible suppression
of oxidations of protein components in the tissue. In view of the highly inhi-
bited GDH activity, which forms an index of amino acid oxidations, the decreased
mobilization of amino acids into oxidations can be envisaged. Consequently,
free ammonia and urea levels had non-significant change from the controls.
However, on acclimation the branchial total protein content was significantly
elevated suggesting the onset of either enhanced protein biosynthetic mechanisms,
or decreased proteolysis in the tissue. Since protease activity was also elevated,
increased protein content might be due to stepped-up protein biosynthesis, with
active turnover of tissue proteins. In the light of widely reported mucification
and hypertrophy of the branchial tissue in altered pH media (Daye and Garside
l980b) active protein synthesis can be visualised. Since both soluble and structural
protein fractions were elevated, accumulation of proteins at structural and dynamic
levels of organization of gill can be expected. Free amino acid content was
elevated, which may be due to increased proteolysis.
Tissue oxygen consumption had non-singificant change, while A/O ratio was
far lower than the control, suggesting lesser mobilization of protein components
into oxidations or mobilization of ammonia into other components. However,
GDH activity was considerably elevated indicating the involvement of amino
acids in oxidative reactions. Free ammonia content was increased due to high
oxidative deamination reactions in the tissue. Glutamine content was considerably
high with decrease in urea, suggesting the mobilization of tissue ammonia towards
the formation of glutatnine, which may be responsible for the lower A/O ratiot
In general it can be concluded that the branchial tissue, on acclimation in sub-
lethal alkaline waters, accumulates proteins leading to hypertrophy of the tissue
which might provide positive survival value for the fish in imposed alkaline stress.
Acknowledgements
The authors express their gratitude to the University Grants Commission for
financial assistance,
240 M Bhaskar et al
References
Aimer B, Dickson W, Ekstrom C, Hoinstrom E and Miller U 1974 Effects of acidification of
Swedish lakes ; Ambio 3 30-36
Anderson G, Gustafsoa K J and Lindstrom T 1971 Rodingen I Rosjoarna Pa Fulufjall.
Drottningholm : Freshwater Research Laboratory Sweden Information 8 9
Bandt H J 1936 Der Fur Fische " todliche pH Wert " in alkalischem Bereich ; Z. Fisch. Deren
Hilfwiss 34 359-361
Beamish R J 1972 Lethal pH for the white sucker Catastomus commersani (Lacepede) ; Trans.
Am. Fish Soc. 101 355-358
Beamish R J and Harvey H H 1972 Acidification of the Lacloche mountain lakes, Ontario and
resulting fish mortalities ; J. Fish. Res. Board Can. 29 1131-1143
Bergmeyer H U 1965 Methods of biochemical analysis (New York and London : Academic
Press)
Bhaskara Haranath V, Reddanna P and Govindappa S 1978 Effects of exposure to altered
pH media on tissue proteolysis and nitrogenous and products in a freshwater fish Tilapia
mossambica (Peters) ; Indian J. Exp. Biol. 16 1088-1090
Cairns J, Dickson K L and Grossman J S 1972 The response of aquatic communities to
spills of hazardous materials ; Proc. 1972 Natl. Conf. Hazardous materials spills pp. 179-197
Carter L 1964 Effects of acidic and alkaline effluents on fish in sea water ; Effluent water Treat. J.
4 484-486
Colowick S P and Kaplan N O 1957 Methods in enzymology (New York : Academic Press)
3 501
Das A B and Prosser C L 1967 Biochemical changes in tissues of gold fish acclimated to high
and low temperatures. I. Protein synthesis ; Comp. Biochem. Physiol. 21 449-467
Daye P G and Garside E T 1975 Lethal levels of pH for brook trout, Salvelinus fontinalis
(Mitchill) ; Can. J. Zool. 53 639-641
Daye P G and Garside E T 1976 Histopathological changes inWfacial tissues of brook trout,
Salvelinus fontinalis (Mitchill) exposed to acute and chronic levels of pH ; Can. J. Zool
54 2140-2155
Daye P G and Garside E T 1980a Structural alterations in embryos and alevins of the Atlantic
salmon, Salmo salar L. induced by continuous or short-term exposure to acidic levels of
pH ; Can. J. Zool. 58 27-43
Daye P G and Garside E T 1980b Development, survival and structural alterations of embryos
and alevins of Atlantic salmon, Salma salar L. continuously exposed to alkaline levels of
pH from fertilization ; Can. J. Zool. 58 369-377
Dillon P J, Jeffries D S, Snyder W, Reid R, Yan N D, Evans D, Moss J and Scheider W A
1978 Acidic precipitation in South-Central Ontario : recent observations ; /. Fish. Res.
Board Can. 35 809-815
Dovland H, Joranger E and Semb A 1976 Deposition of air pollutants in Norway ; in Impact
of acidic precipitation on forest and freshwater ecosystems in Norway ; (ed) F H Breekke
SNSF Project FR 6/76 14-35
Govindappa S and Rajabai B S 1976 Some aspects of protein metabolism in crab Paratelphusa
hydrodromus (Kterbt) during cold acclimation ; /. Aitim. Morphol. Physiol. 23 76-84
Jensen K W and Snekvik E 1972 Low pH levels wipe out salmon and trout population in
southernmost Norway ; Ambio 1 223-225
Jordan D H M and Lloyd R 1964 The resistance of rainbow trout [Salmo gairdneri (Richardson)]
and roach (Rutilis rutilus L.) to alkaline solutions ; Int. J. Air Water Pollut. 8 405-409
Kanungo M S and Prosser C L 1959 Physiological and biochemical adaptation of gold fish to
cold and warm temperatures. I. Standard and active oxygen consumptions of cold and
warm acclimated gold fish at various temperatures ; /. Cell Comp. Physiol 54 259-263
Karuppasamy P 1979 Pollution and fish mortality in Chaliyar river, Mavoor (near Calicut)
from 7-3-1979 to 16-3-1979 ; Marine Fisheries Information Service 7 11-13
Protein metabolism of freshwater fish 9 241
Krishna Murthy V, Reddanna P and Govindappa S 1980 Hepatic carbohydrate metabolism in
Tilapia mossambica (Peters) acclimated to low environmental pH; Can. J. Zoo!. 59 400-404
Lee Y L and Lardy H A 1965 Influence of thyroid hormones on L-glycerophosphate dehydro-
genases and other dehydrogenases in various organs of the rat ; /. BioL Chem. 240 1427-
1432
Lievestad H and Muniz I P 1976 Fish Kill at low pH in a Norwegian river ; Nature (London)
259 391-392
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with
Folin phenol reagent ; /. BioL Chem. 193 265-275
McKee J E and Wolf H W 1963 Water quality criteria (Sacramento, Calif. : Water Quality
Control Board Publ. 3-A)
Moore S and Stein W H 1954 A modified ninhydrin reagent for the photometric determination
of aminoacids and related compounds ; J. BioL Chem. 211 907-913
Natelson S 1971 Techniques in clinical chemistry (Illinois : Charles C. Thomas, Publishers)
pp. 146
Oden S 1976 The acidity problem— an outline of concepts : Proc. 1st Int. Symp. on acidPrecip.
Forest Ecosystems ; in special issue ; /. Water Air Soil Pollut. pp. 1-36
Packer R K and Dunson W A 1970 Effects of low environmental pH on blood pH and Na+
balance of brook trout ; /. Exp. Zool. 174 65-72
Packer R K and Dunson W A 1972 Anoxia and sodium loss associated with death of brook
trout ; Comp. Biochem. Physiol. A41 17-26
Precht H J 1958 Concept of temperature adaptation of unchanging reaction systems of cold
blooded animals ; In Physiological adaptation (ed.) CL Prosser (Washington D.C. : American
Physiological Society)
Reddanna P and Govindappa S 1979 Effects of in vivo muscular stimulations. II. Influence on
hepatic carbohydrate metabolism ; /. Anim. Morphol. PhysioL 26 156-161
Schofield C L 1975 Lake acidification in the Adirondack Mountains of New York : Causes and
consequences ; Proc. 1st Int. Symp. Acid Precip. Forest Ecosystems ; Ohio State University
Trama F B 1954 The pH tolerance of the common bluegill (Lepomis machrochirus Rafinesque) ;
Not. Nat. Acad. Nat. Set. Philadelphia 256 1-13
Umbreit W W, Burris R H and Stauffer J F 1959 Manometric techniques (Minneapolis :
Burgess Publishing)
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 3, May 1952, pp. 243-247.
© Printed in India.
Temperature-related chromosome polymorphism in Drosophila
ananassae
D P DASMOHAPATRA, N K TRIPATHY and C C DAS
Department of Zoology, Berhampur University, Berhampur 760007, India
MS received 26 March 1981
Abstract. Correlated studies on the influence of temperature in the frequency
of inversions in the D. ananassae population of Golabandha shows that tempe-
rature fluctuation has a positive bearing on 2LA inversion while negatively so
with respect to SLA and 3RA inversions.
Keywords, Drosophila ananassae ; inversion ; 2LA ; 3LA ; 3RA.
1. Introduction
Clear evidences exist to sustain the evolution of differential gene arrangements in
species of Drosophila to meet the adaptive needs in a dynamic environment. In
as much as the adaptive values of different genomes differ considerably, the fit-
ness of certain kinds of gene arrangements may, therefore, decrease or increase
with fluctuation in environmental mileu. D. ananassae, a cosmopolitan domestic
species, is known to be invested with a large number of inversions in its natural
population (Kaufmann 1936 ; Kikkawa 1938 ; Dobzhansky and Dreyfus 1943 ;
Shirai and Moriwaki 1952 ; Seecof 1957 ; Freire-Maia 1961 ; Ray-Chaudhuri
and Jha 1966 ; Singh and Ray-Chaudhuri 1969 ; Sreeram Reddy and Krishna-
murthy 1969, 1970 ; Sajjan and Krishnamurthy 1970 ; Singh 1970 ; Siddaveere
Gowda and Krishnamurthy 1971). Again, of the several paracentric inversions,
2LA, 3LA and 3RA (Rajeswari and Krishnamurthy 1969), or their equivalent
alpha, delta and eta (Ray-Chaudhuri and Jha 1966) are common to all popu-
lations of this species (Singh 1970), while all other inversions are selectively
restricted to these populations. Certain populations of Drosophila undergo
seasonal changes with respect to their chromosomal composition which, however,
varies in intensity (Carson and Stalker 1949 ; Spiess 1950). For instance, Levitan
(1951, 1957) reported marked seasonal fluctuation in the frequency of inversions
in D. robusta population of Virginia while Carson's (1958) data on the same
species endemic to Missouri are quite contradictory in being insignificant. Epling
et al (1953) have argued that seasonal changes of gene arrangement in the chromo-
somes promote the adaptive values of the inversions which in turn influence the
243
244 D P Dasmohapatra, N E Tripathy and C C Das
nature and frequency of the polymorphism itself. In an attempt to assess the
correlation if any, between the frequency of different inversions and the environ-
mental temperature, the present study has been undertaken.
2. Materials and methods
The flies were collected from the natural population of D. ananassae of Gola-
bandha situated at an altitude of 17- 5m and about 6km to the south of the
University campus. Collections were made in the first week of every month
on fermented banana bait in glass bottles. Fertilised females collected from
nature were transferred to independent vials with wheat cream agar media.
Chromosomal polymorphism was studied from the salivary glands of a hundred
larvae from lacto-aceto-orcein squash preparations.
3, Results
Table 1 represents the percentage of homo- and hetero-karyotypes of D. ananassae
in different months, i.e., January to December. The percentage of homokaryo-
types were more than 50 every month while that of the heterokaryotypes ranged
between 31 and 48. The frequency of the three commonly occurring inversions,
as found in this population, are repre ented in figure 1. It is observed that the
frequencies of 2LA inversion vary between 4% (in January) and 13% (in June
and July) and that of 3RA between 2% (in February, March, April and September)
and 5 % (in May) while the percentage of 3LA inversion varies between 23 % (in
April) and 44% (in December) in a year. Moreover, it has been marked that
3RA inversion is completely absent in the population in the month of July.
4. Discussion
Extensive qualitative chromosomal variability has no doubt been reported in
D. ananassae but unfortunately the information on the frequencies of these
qualitative chromosomal variabilities and their correlation, if any, with the
fluctuation of environmental factors is extremely meagre (Dobzhansky 1947 ;
Stalker and Carson 1948 ; Carson and Stalker 1949 ; Spiess 1950 ; Battaglia and
Birch 1956 ; Carson 1967). Curiously however the data of Dobzhansky
(1956) on D. pseudoobscura while indicating seasonal fluctuations in the
frequency of chromosomal composition, those of Battaglia and Birch (1956) on
D. wittistoni deny such correlation. In our studies, what is still more intriguing,
the annual temperature fluctuation has positive bearing on 2LA inversion but
negatively so with respect to SLA and 3RA inversions (figures 2, 3 and 4). Indeed
if this is proved to be a widely occurring phenomenon, then we must conclude
that the inversions in their very nature confer such * position effects ' as seemingly
contribute to the homeostatic mechanism of the species,
Polymorphism in Dros&phila anwassae
245
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NQV DEC
40
30-
20
Q.
J 10
r = * o«64
P <0-01
OD O
10 20 30
2, Frequency of 2LA inversion
r= -Q-322
r = + 0-193
40
40
P,NS
°oo °
oo6
o 30
o o o « 30
L^o°
I2'
V
•- 10
0
0
*- 20
Q.
£
u 10
o
— 1 »
10 20
3 frequency of 3 UA inversion T* Frequency of 3RA inversion
Figures 1-4. 1. Histogram showing the frequency of different types of inversions
in D. ananassae. 2-4. Correlation between inversion and environmental tempe-
rature. 2. 2LA inversion. 3. SLA inversion. 4. 3RA inversion.
246 D P DasMohapatfa, tf K Tripathy and C C Das
Table 1. Number of heterokaryotypcs of Drosophila ananassae in different months.
Months Average temp. % Heterokaryo-
in°C types*
January
24
42
February
28
31
March
28
36
April
32
34
May
36
43
June
34
35
July
32
48
August
34
37
September
34
38
October
31
42
November
31
45
December
28
47
* 100 larva© were examined every month.
Acknowledgement
The award of a fellowship to one of the authors (DPD) by the Council of Scientific
and Industrial Research, New Delhi, is thankfully acknowledged.
References
Battaglia B and Birch L C 1956 Nature (London) 178 1005 (from Science 1966) 1J2 (3723) 111
Carson H L 1958 The population genetics of Drosophila robustai Adv. Genet. 9 1-40
Carsoa H L 1967 Chromosomal polymorphism in altitudinal races of Drosophila ; Proc. Jpn.
Soc. Syst. Zool. 3 10-16
Carson H L and Stalker H D 1949 Seasonal variation in gene arrangement frequencies over a
three year period in Drosophila robusta Sturtevant ; Evolution 3 322-329
Dobzhansky Th 1947 Genetic structure of natural populations ; Yearb. Carnegie Inst. 46
155-165
Dobzhansky Th 1956 Genetics of natural populations. XXV. Genetic changes in populations
of Drosophila pseudoobscura and Drosophila persimilis in some localities in California ;
Evolution 10 82-92
Dobzhansky Th and Dreyfus A 1943 Chromosomal aberrations in Brazilian Drosophila ananassae ;
Proc. Natl. Acad. Sci. U.S.A. 29 301-305
Epling C, Mitchell D F and Martoni R H T 1953 On the role of inversions in wild popu-
lations of Drosophila pseudoobscura ; Evolution 7 342-365
Freire-Maia N 1961 Peculiar gene arrangements in Brazilian natural populations of Drosophila
ananassae i Evolution 15 486-495
Kaufmann B P 1936 A terminal inversion in Drosophila ananassae ; Proc. Natl. Acad. Sci. USA
22 591-594
Kikkawa H 1938 Studies on the genetics and cytology of Drosophila ananassae ; Genetica 20
458-516
Polymorphism in Drosophila ananassae 247
L vitan M 1951 Selective differences between males and females IB Drosophila robusta ; Am.
Nat. 85 335-388
Levitart M 1957 Natural selection for linked gene arrangements ; Anat. Rec. 127 430
Rajeswari P and Krishnamurthy N B 1969 Inversion polymorphism in different populations
of Drosophila awnassae from Mysore State ; Indian J. Heredity I 143-147
Ray-Chaudhuri S P and Jha A P 1966 Studies on the salivary gland chromosomes of Indian
Drosophila ananassae ; Proc. Int. Cell BioL Meet. Bombay pp. 352-383
Sajjan S N and Krishnamurthy N B 1970 Report on two new translocations in a natural popu-
lation of Drosophila ananassae from Heriyar (Mysore State, India) ; DIS 45 166
Seecof R L 1957 Cytological analysis, section III from XXII genetic studies in irradiated
natural populations of Drosophila ; Univ. Texas Publications No. 5721 pp. 261-281
Shirai M and Moriwaki D 1952 Variations of gene sequence in various strains of Drosophila
ananassae ; DIS 26 120-121
Siddaveere Gowda L and Krishnamurthy N B 1971 Inverted gene arrangements in Drosophila
ananassae population of Western Ghats ; Science J. Mysore Univ. 24 115-119
Singh B N 1970 Distribution of most common inversions of Drosophila ananassae in- different
parts of India including Andaman and Nicobar Islands ; Indian BioL 2 78-81
Singh B N and Ray-Chaudhuri S P 1969 Geographic differentiation: in natural populations of
Drosophila ananassae ; Abstr. HI Cell BioL Conf. Delhi pp. 32
Spiess E 1950 Experimental populations of Drosophila persimilis from an altitudinal transect
of the Sierra Nevada ; Evolution 4 14-33
Sreeram Reddy G and Krishnamurthy N B 1969 Inversion heterozygosity in two populations
of Drosophila ananassae ; Proc. 56th Indian Sci. Cong. Assn. pp. 451
Sreeram Reddy G and Krishnamurthy N B 1970 Studies with variants of the gene arrange-
ments in Goan and Karwar populations of Drosophila ananassae ; Proc. 51th Indian Sci.
Cong. Assn. pp. 362
Stalker H D and Carson H L 1948 An altitudinal transect of Drosophila robusta Sturtevant ;
Evolution 2 295-305
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 3, May 19S2, pp. 249-257.
© Printed in India.
Life and fecundity tables for the longicorn beetle borer,
Olenecamptus bilobus (Fabricius) (Coleoptera : Cerambycidae)
T N KHAN and P K MAITI
Zoological Survey of India, 34, Chittaranjan Avenue, Calcutta 700 012, India
MS received 18 July 1981 ; revised 30 December 1981
Abstract. The present paper deals with the life and fecundity tables for the
cerambycid borer, Olenecamptus bilobus (Fabricius). Under a given set of condi-
tions and food supply, the population of this species increased with an infinitesimal
and a finite rate. The population increased by 20-41 times between two successive
generations and 86*42 days were taken to complete one generation. The adults
constituted only 1-29% to the population of the stable age, while eggs, larvae,
pupae and dormant adults contributed to the extent of 24-95, 68-22, 4-38 and
1-16% respectively.
Keywords. Life and fecundity tables; Olenecamptus bilobus (Fabricius); Artocarpus
chaplasha Roxb. ; stable age-distribution.
1. Introduction
The longicorn beetle borer, Olenecamptus bilobus (Fabricius), is found predomi-
nantly in the Oriental region with an extended distribution up to the Papuan and
Malagasy subregions. The species is one of the most common sap-wood borers
of a number of dead or dying timber yielding plants. The bionomics and life-
history of this species have been dealt with by Khan and Maiti (1980). The
observations, reported here, are concerned with the life and fecundity tables,
including the stable age-distribution of the species. The infinitesimal (rm) and
finite (X) rates of increase, net reproductive rate (RQ) and the mean' generation time
were the basic parameters used in the present communication to assess the popu-
lation growth in the laboratory.
2. Material and methods
The present study was based on the material collected during the period of 1978-80
under a research project on the " Ecological interaction and economic status of the
xylophagous insects of the Islands of Andaman and Nicobar ", under the guidance
of one of us (P K Maiti). During the course of the study, a number of logs
of Artocarpus chaplasha Rpxb., infested with the immature stages of O. bilobus,
249.
256 f N Kftan and P K Haiti
was collected from several field sites of South Andaman and held in galvanized
iron cages (70cm x 37cm x 37cm) for the emergence of the adult beetles in the
laboratory. The newly emerged beetles were sexed and 50 pairs of males and
females were each separately kept in glass breeding-cages (36 cm x 22 cm x 22 cm),
containing a layer of moist sandy soil at the bottom. Moist sandy soil was
however, kept to minimise the loss of moisture from the breeding-cages. The
beetles were provided with fresh green leaves and twigs of Ficus religiosa L. for
food and freshly cut billets, measuring 25-30 cm in length and 8-42 cm in dia-
meter, of Artocarpus chaplasha Roxb. for opposition, both of which were renewed
everyday between 900 and 1000 hr 1ST. The number of oviposition slits on the
billets was counted and the total number of eggs laid thereon was recorded.
Each day, the infested billets from the breeding-cages were assigned a batch-
number and placed in a galvanized iron cage (similar to those used for rearing the
immature stages collected from the field sites), containing a layer of moist sandy
soil at the bottom. In this case, however, the moist sandy soil was used to pre-
vent over desiccation from the infested billets, so that they could retain the mois-
ture content for a longer period for proper development of the progeny. Beginning
from the third day following oviposition up to the completion of development of
the progeny, three sample billets were taken out and dissected every alternate day
between 1200 and 1400 hr 1ST, to study the development of egg, larva and pupa
and other relevant phenomena. The adults, which emerged on a particular day,
were transferred to separate cages for oviposition to determine the age-specific
fecundity. The average fecundity of the females on subsequent days was recorded
uatil all the females died. Since, the sex-ratio was 2? : 1(? (based on 1600
adults), the number of eggs laid per female was multiplied by 2/3 to get the number
of female births (mf). Life and fecundity tables were constructed according to
Birch (1948), elaborated by Howe (1953), Laughlin (1965) and Atwal and Bains
(1974). The innate capacity of increase (rm) and finite rate GO were calculated.
The values of % (pivotal age in days), lx (survival of females at different age inter-
vals), and m, (number of female progeny per female) were worked out. Observa-
tions were also made on the stable age-distribution (per cent distribution) of
various age groups by calculating the birth-rate and death-rate when reared in, a
limited space.
During the course of these studies in the laboratory, the maximum and
minimum temperatures recorded were 29- 7° C and 26-5° C respectively, while
the relative humidity prevailed between 67-5% and 93-0%.
3* Results and discussion
The maximum duration of incubation, development of larva, pupa and dormant
adult has been observed to be 5, 50, 15 and 7 days respectively. Present observa-
tions, on the duration of different developmental stages correspond strikingly with
those .observed by Khan and Haiti (1980). The number of individuals survived
between different developmental stages is presented in table 1. From the data
presented in the table the developmental survival tote has been estimated at 0 • 44 L
The figures of the mature females emerged from the immature stages have been
pooled and a grouping of a day interval of age is enjoyed. The results ar*e
Life and fecundity tables for Olenecamptus bilobus (Fabr) 251
Table 1. Survival of different developmental stages of Olenecamptus bilobus (Fabr.)
on Artocarpus chaplasha Roxb.
Number survived
TtAteh NTn "Mn nf
JJvt lWi|. AXU • - J-^tU « wl
eggS
Egg period
Larval period
Pupal period
Dormant
(0-5 days)
(6-55 days)
(56-70 days)
adult period
(71-77 days)
1 379
334
235
197
173
2 456
390
274
230
205
3 503
434
303
263
233
4 412
348
244
201
175
5 517
429
29J)
24?
204
6 450
396
280
233
206
7 391
341
241
20Q
178
8 342
286
203
169
149
9 401
341
238
199
H6
10 279
237
171
143
J31
Total 4148
3536
2479
2077
1830
Table 2. Observed, as well as, smoothed distribution of mortality amo&g the
mature females of Olenecamptus bilobus (Fabr.).
Age of the
mature females
in days
X
Observed
Smoothed
Observed
Smoothed
1
854
854-00
24
45-68
7
830
808-32
60
70-63
3
770
737-69
100
88-70
4
670
648*99
95
98*55
5
575
550-44
94
100-44
6
481
450-00
101
95-21
7
380
- 354-79
S9
85-17
a
291
269-62
78
72*05
9
213
197-57
60
58-25
10
153
139-32
50
44-32
11
103
95-00
36
32-63
12
67
62-37
2O
22-90
13
47
39-47
17
15-39
14
30
24-C8
16
9-92
15
14
14-46
14
14-46 *
854
854-00
: 869-77e-0*0188ool*2T
252 T N Khan and P K Main •
presented in table 2, where the customary symbols have been used, i.e., I, being
the number of individuals which survive to the age x and dx the number dying
between the ages x and x + 1, so that in the present table,
The raw data have then been smoothed. For smoothening of the raw data,
there are many a satisfactory method of which the following one seems
to be most convenient :
Defining /** as the force of mortality at the age x ;
_ ~
/ dX ~ ^'
Now, if the force of mortality is assumed to be directly proportional to the age
and iJL9 = 2ft2 3, where 2ft2" is a constant, then by integration we get
/, = /o<r*v- (1)
Patting IJk = ?•> and fi, = 1 - P«
and, by differentiation we have,
dQx = 2/z* Xe~~K*~ dX ;
the probability of dying between the ages SS and 21 + dX being given by the
right-hand member of the last equation. Then if M be the mean age at death,
M = 2ft2 f" &er**dX9
0
from which, by integration we get,
.
2h
The observed and the smoothed values of 19 have been presented in table 2, and
in testing the agreement between the smoothed and observed /„ Chi-Square test
is employed. It has been observed that J*?2 = 15-86 and for n = 12, P = (0-20),
0- 10, which shows that the St is satisfactory. As a matter of interest, it might
be worth mentioning that the same type of equation has been found to give an
excellent fit in the case of 524 female vestigial Drosaphila{A^i^, from Pearl and
Parker 1924) and in case of 119 voles (Microtus agrestis) (Leslie and Ranson 1940).
The life and fecundity tables for 0. bilobus on Artocarpus chaplasha Roxb.
based on the smoothed /„ values, is given in table 3, which has been calculated
from tl e following equation ;
lm = i-ooe-0'0183001*, (2)
considering the 83rd, 84th, 85th, . . ., 98th day of the pivotal age as the Oth, 1st,
2nd, . . ., 15th day of the age of mature females (vide table 2). In the present
table, the /, column presents the adult survival rate only, while m, gives the number
Life and fecundity tables far Olenecamptus bilobus (Fabr.)
253
Table 3. Life table and fecundity schedule for Olenecamptus bilobus (Fabr.) on
Artocarpus chaplasha Roxb.
Pivotal age in
days
Survival of
females at
different age
intervals
Age specific
fecundity
($ births)
Actual <j>
births Per
time unit
(*•**)
(*)
0,)
0-77
**
Immature
Stages
78
1-000
*
0*441
0-441
34-398
79
1-000
*
0-441
0-441
34-839
80
1-000
*
0-441
0-441
35*280
SI
1-000
*
0-441
0-441
35-721
82
1-000
*
0-441
0, -441
36-162
83
1-000
2-52
1-111
1-111
92-213 '
84
0-982
4-31
1-901
1-867
156-828
85
0-929
5-98
2-637
2-450
208-250
86
0-848
7-05
3-109
2-636
226-696
87
0-746
7-67
3-382
2-523
219-501
88
0-633
8-73
3-691
2-336
205-568
89
0-517
7-49
3-303
1-7C8
152-012
90
0-408
7-06
3-113
1-270
114-300
91
0-310
6-19
2-730
0*846
76-986
92
0-227
5-68
2-505
0-569
52-348
93
0-160
5-68
2-505
0-401
37-293
94
0-109
4-59
2-024
0-221
20-774
95
0-072
4-34
1-914
0-138
13-110
96
0-045
4-07
1-795
0-081
7-776
97
0-028
3-34
1-473
0-041
3-977
98
0-016
1-08
0-476
0-008
0-784
99
o-ooo
o-oo
0-000
o-oao
o-aoo
Net reproductive rate = R0 = Z k» = 20- 411 ; £ x • kx = 1746- 81 6.
* Pre-oviposition period. ** Developmental survival rate =0-441.
of female eggs laid by the average female per day. For presenting the results
in a more convenient way, an additional column (Dltt • /w^has been proposed,
which gives the product of the number of female eggs laid by the average female
per day and the developmental survival rate. Another column gives the product
of Dh • mg and the adult survival rate (4 in the present table), whose product is
designated by k, in the present communication.
The total number of female eggs laid per female of tho original cohort (R0) has
been estimated at 20-411, which indicates that 20-411 females are produced per
female per generation. The maximum pre-oviposition period has been observed
to be 5 days, i.e., from the 78th to 82nd day of the pivotal age, and oviposition
c ontinues almost throughout the life span of the females . Maximum c ontribution
(Wjp = 8-37) in the life-cycle is observed to be made by the females of tlje 88th
254
T N Khan and P K Haiti
day of the pivotal age (vide table 3). The first female mortality within the cohort
occurs on the 7th day (/, = 0-982) after the emergence of the adult female and
mortality increases gradually thereafter, as shown in figure 1.
97
PIVOTAL Afif IN DAYS
Figare 1. Age-specific survival and fecundity of Olenecamptus bilobus (Fabr.) on
Artocarpus ckaplasha Roxb.
Table 4. Mean iergth of generation, innate capacity for increase in numbers and
finite rate of increase in numbers in Olenecamptus bilobus (Fabr.) on Artocarpus
chaplasha Roxb.
Particulars
1 . Cohort generation time (T^
Zx'k« 1764-816
20-411
2. Innate capacity for increase in numbers (rm)
__ In R9 __ log eRo __ 3-0161
r<w = "rT "" ~ 86-464
3. Fir.ite rate of increase in numbers
(A) = Natural antilog of rw
= Natural antilog of 0-0349
4. Corrected generation time (T)
^ In It* log e^o 3-0161
Tsss , = s= .
rm rm 0-0349
5. Weekly multiplication of popvlation
86*464 days
0-0349
1-03552
86-42 days
1-2767
Life and fecundity tables for Olenecamptus bilohustfabr.')
TableS. Stable age-distribution of Olemcamptus bilobus (Fabr.) on Artocarpus
chaplasha Roxb.
(rw.= 0-0349)
Pivotal age
in days,
1
L.
2
3
4
Percentage age-d&tributioo
100 0L, • <rrm<«+D
5
0
1-000
0-9657
0-9657
4-6874
1
1-000
0-9326
0-9326
4-5268
2
1-000
0-9006
0-9006
4-3714
Total eggs
3
1-000
0« 8697
0-8697
4-2214
24-95%
4
0-930
0- 8399
0-7811
3-7914
5
0-850
0-8111
0-6894
3-3463
6
0-850
0-7833
0-6658
3-2317
7
0-840
0-7564
0-6354
3-0842
8
0-830
0-7304
0-6062
2-9424
9
0-825
0-7054
0-5820
2-8250
10
0-820
0-6812
0-5586
2-7114
11
0*815
0-6578
0-5361
2-6022
12
0-810
0-6352
0-5145
2-4973
13
0-805
0*6135
0-4939
2-3974
14
0-800
0-5924
0-4739
2-3003
15
0-800
0-5721
0-4577
2-2216
16
o-goo
0-5525
0-4420
2-1454
17
0-795
0-5336
0-4242
2-0590
18
0-790
0-5153
0-4071
1-9760
19
0-785
0-4976
0-3906
1-8959
20
0-780
0-4805
0-3748
1-8192
21
0-765
0-4640
0-3550
1-7231
22
0-750
0-4481
0-3361
1-6314
23
0-745
0 -4327
0-3224
1-5659
24
0-740 J
0-4179
0-3092
1-5008
25
0-735
0-4036
0-2966
1-4397
26
0-730
0-3897
0-2845
1-3809
27
0-725
0-3764
0-2729
1-3246
28
0-720
0-3635
0-2617
1-2703
29
0-710
0-3510
0-2492
1-2096
30
0-700
0-3390
0-2373
1-1518
31
0*695
0-3273
0-2275
1-1043
Total larvae
32
0-690
0-3161
0-2181
1-0586
68-22%
33
0-690
0-3053
0-2107
1-0227
34
0-690
0-2948
0-2034
0-9873
35
0-680
0-2847
0-1936
0-9397
36
0-670
0-2749
0-1842
0-8941
37
0-665
0-2655
0-1766
0-8572
38
0-660
0-2564
0-1692
0-8213
39
0-650
0-2476
0-1609
0-7810
40
0-640
0-2391
0-1530
0*7426
41
0-630
0-2309
0*1455
0-7062
42
0-620
0*2230
0-1383
0-6713
43
0-615
0-2153
0-1324
0*6427
44
0-610
0*2079
0-1268
0-6155
45
0-610
0-2008
0-1225
0-5946
46
0-610
0-1939
0-1183
0-5742
T N titan and P K Matti
1
2
3
4
5
47
0-610
0-1873
0-1143
0-5548
48
0-610
0-1808
0-1103
0-5354
49
0-605
0-1746
0-1056
0-5126
50
0-600
0-1687
0-1012
0-4912
. 51
0-600
0-1629
0-0977
0-4742
52
0-600
0-1573
0-0944
0-4582
53
0-600
0-1519
0-0911
0-4422
54
0-595
0-1469
0-0874
0-4242
55
0-590
0-1416
0-0835
0-4053
56
0-585
0-1368
0-0800
0-3883
57
0-580
0-1321
0-0766
0-3718
58
0-580
. 0-1276
0-0740
0-3592
59
0-580
0-1232
0/0715
0-3471
60
0-580
0-1190
0-0690
0-3349
61
0-575
0-1349
0-0661
0-3208
62
0-570
0*1109
0-0632
0-3068
Total pupae
63
0-565
0-1071
0-0605
0-2937
4-38%
64
0-560
0-1035
0-0580
0-2815
65
0-545
0*0999
0-0544
0-2641
66
0-530
0-0965
0-0511
C-2480
67
0-515
0-0932
0-04O)
0-2330
68
0-500
0-0900
0-0450
0-2184
69
C-495
0-0869
0-0430
0-2087
70
0-.490
0-0839
0-0411
0-1995
71
0-480
0-0810
0/0389
0-1*88
72
0-480
C-0781
0-0375
0-1820
73
0-475
0-0756
0-0359
0-1743
Total
74
0-470
0- 0730
0-0343
0-1665
dormant
75
0-455
0-0705
0-0321
0-1558
adults
76
0-441
r-068l
0-0300
0-1456
1'16%
77
0-441
0-0657
0-0290
0-1408
78
0-441
0-0635
0-0280
0-1359
79
0-441
0-0613
0-0270
0*1311
80
0-441
0-0592
0-0261
0-1267
81
0-441
0-0.572
0-0252
0-1223
82
0-441
0-0552
0-0243
0-1180
83
0-437
0/0533
0-0233
0-1131
84
0-422
0-0515
0-0217
0-1053
85
0-392
0-0497
0-0195
0-0947
86
0-352
0-0480
0*0169
0-0820
87
0-304
0-0464
0-0141
0-0684
88
0-254
0-0448
0-0114
0-0553
89
90
91
0-204
0-156
0-119
0-0432
0-0418
0-0403
0-0088
0-0065
0-0048
0-0427
0-0316
0-0233
Total adults
1-29%
92
0-086
0-0389
0-0033
0-0160
93
0-060
0-0376
0-0023
0-0112
94
0-040
0-0363
0-0015
0-0073
95
0-026
0-0351
0-0009
0*0044
96
0-016
0-0339
0-0005
0-0024
97
o-oio
0-0327
0-0003
0-0015
98
0-004
0-0316
0-0001
0-0005
1/J? = 20-6019
Life and fecundity tables for Olenecamptus bilobus (Fabr.) 257
The present investigation suggests that the innate capacity of increase (rm) is
0-0349 per female per day, while the daily finite rate of increase (X) is 1-03552
(table 4). The mean time for completing a generation (T) has been calculated at
86-42 days. It appears, therefore, that under a given set of conditions in the
laboratory, the daily finite rate of increase (A = 1-03552) enables the borer insect
to multiply by 1-2767 times every week.
From the present observations, the contribution made by the different develop-
mental stages of O. bilobus towards the stable age-distribution has also been
determined. The results are presented in table 5, in which the life-table age-
distribution (L,.) has been worked out with the following formula;
<r-fi
L, = J /A,
9
which, in practice, is given by
j^ *• + (*•"). (3)
It has been observed that, on reaching the stable age-distribution, the egg, larva,
pupa, dormant adult and adult stage of this insect contribute to the extent of
24-95, 68-22, 4-38, M6 and 1-29% respectively.
Acknowledgements
Grateful acknowledgement is expressed to 0r B K Tikader, Director, Zoological
Survey of India, to Prof. T N Ananthakrishnan, FNA, Former Director, Zoological
Survey of India, for their keen interest in the progress of the work, to Dr A K
Das, Officer-in-Charge, Andaman andKcobar Regional Station, Port Blair, ZSI,
for providing all facilities for the work. Appreciations are due to all the
members of the staff of Andaman and Nlcobar Regional Station, ZSI, for their
active assistance. Lastly, thanks are also due to the Department of Science and
Technology, New Delhi, for providing fund to support the project.
References
Atwal A S ani Bains S S 1974 Applied animal ecology (Ludhiana : Kalyani Publishers)
pp. 128-135
Birch L C 1948 The intrinsic rate of natural increase of an insect population ; /. Anim. Ecol.
17 15-26
Howe R W 1953 The rapid determination of intrinsic rate of increase of aninsect population;
Ann. Appl. Biol 40 134-155
Khan T N and Maiti P K 1980 Bionomics of the round head borer, Olenecamptus bilobus
(Fabricius) (Coleoptera : Cerambycidae) ; Proc. Zool Soc. Calcutta (In press)
Laughlin R 1965 Capacity for increase : A useful population statistics; /. Anim. Ecol. 34
77-92
Leslie P H and Ranson R M 1940 Mortality, fertility and rate of natural increase in the vole
(Microtus agrestis) in the laboratory ; «/. Anim. Ecol. 9 27-52
Pearl R and Parker S L 1924 Experimental studies on the duration of life. IX. New life
tables for Drosophila ; Am. Nat. 58 71-82
P.(B)-5
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 3, May 19$2, pp. 259-265.
© Printed in India.
Behavioural responses of the Indian gerbil, Tatem indica to
conspecific sebum odour of the ventral scent marking gland
MOHD. IDRIS and ISHWAR PRAKASH
Coordinating and Monitoring Centre for Rodent Research and Training,
Central Arid Zone Research Institute, Jodhpur 342003, India
MS received 14 December 19S1
Abstract. Behavioural responses of the Indian gerbil, Tatera indica, to conspecific
sebum odour of the male ventral marking scent gland were studied in a glass cage
and a plus maze. Male and female gerbils were attracted towards the strange male
sebum odour though its magnitude was low in females possessing the ventral
marking gland, still lower in the females in which the marking gland was absent.
The diversity in preferential behaviour of female Tatera indica is discussed in rela-
tion to the role of ventral marking behaviour in chemical communication among
rodents. Correlating the results of the experiments with our field observations, it
appears that the function of scent marking in T. indica is more of a ' familiarisation '
nature to label the habitat for its own use in orientation or to signal * home *
to the marking animal.
Keywords. Chemical communication ; familiarisation ; gerbil ; homing ; phago-
stimulant ; scent marking ; Tatera indica.
1. Introduction
The Indian gerbil, Tatera indica, a Turanian element (Prakash 1974), is distri-
buted throughout the Indian sub-continent. The sub-species T. i. indica is one
of the predominant rodents found in the desert region, occupying almost all the
habitat types (Prakash 1975). Whereas a number of gerbil families inhabit a
single burrow in the village complex or around an urban area, they live indi-
vidually or in pairs in open desert grasslands. These two distinct types of Tatera
populations exhibit a difference in the intensity of occurrence of the mid-ventral
scent marking gland in females. In the former population in which social
organisation is intense, 5 % adult females possess it whereas in the scattered popu-
lations it is present in 12% of the females. However the gland occurs in about
89% adult males in both the types of populations. The difference in the frequency
of occurrence of scent-marking gland in female Tatera existing in two types of
social organisation is being reported for the first time among rodents and it makes
the functional role of this gland in chemical communication more intricate as
well as interesting. The results of our experiments to investigate the behavioural
259
260 Mohd. Idrfs and Ishwar Prakash
responses of male and female Indian gerbil, Tatera indica indica towards the
sebum (secretion of the ventral marking scent gland) odour are presented in this
communication.
2. Methods
All the gerbils, T. i. indica were collected from the sandy habitat around Jodhpur
26° 18' N, 73° 01' E). The first experiment was conducted in a glass cage
(90 x 30 x 30cm). 60 gerbils (30 & Avg. body weight 129-53 dz 5- 1 g and
30 $, 110-4 ±. 4- 5 g) were individually released in the cage one by one and were
oriented for 6 days and then exposed to sebum odour of a strange male. Before
releasing the next gerbil, the cage was thoroughly washed and dried. One glass
slide smeared with the sebum of a strange male was placed on one side of the
cage and another clean slide on the other side, following Kumari and Prakash
(1981a), to avoid new object reaction (Mathur and Prakash 1980). The experi-
mental gerbil was released in the middle of the cage and its behaviour (sniffing,
licking, urination, defecation, ventral marking) in relation to the individual stimulus
on both the sides of the cage was observed for 30 min. The number of visits and
duration of every behavioural act in the vicinity of the two slides were recorded
with the help of a stop watch. Observations were made at night under infra-red
light at the maximum activity epoch of the gerbils.
In the second experiment, the behavioural responses of T. indica were observed
in a residential plus maze (Bhardwaj and Prakash 1981), both by ocular obser-
vations and by recording the relative food consumption. 10 male (Av. body
weight 120-7 ± 8-2 g) and 10 female (96-0 ± 7-9 g) were released one by one
in the plus maze and were acclimatized for 6 days in the new environment. There-
after, the gerbils were provided weighed quantity of pearl millet (Pennisetum
typhoides) in arms A and C, drinking water in arm D. The arm B remained
empty. 24-hour consumption of millet in both the food baskets was recorded
for 6 days. Whereafter, a slide carrying sebum smear of a strange male (T. indica)
was placed in the arm in which food consumption was lower (A), and a blank
slide in the other arm (C) near the food container. Food consumption was again
recorded in both the arms for 4 days. Rodents were released in the central
chamber of the plus maze and were free to move and explore any of the arms.
A complete record of their visits and duration to every arm was maintained for
15 min soon after the introduction of the two slides.
3. Results
A comparison of the mean number of visits by gerbils to the sebum odour slide
and clean slide in the glass cage indicates that the frequency of visits to each
section was similar but the duration of visits was significantly more in the side in
which the sebum odour slide was lodged (students t, P < 0- 001, Bailey 1959, table 1).
Male as well as female gerbils were attracted towards the sebum odour of strange
male though its magnitude was lower among females as indicated by their beha-
vioural responses (table 2),
'Behavioural responses of the gerbil, T. indica 261
Table 1. Olfactory response of Tatera indica to conspccific male sebum odour.
No. of visits per Duration of response
Stimulus 30 min (seconds)
Mean ± SE Mean ± SE
Male
Male sebum
27-10±l-85
17-60±0-87***
Blank slide
27-16±l-87
12-41 ±0-64
Female
Male sebum
31-13±l-93
16-70±0'38***
Blank slide
3a-90±l-91
13-72±Q-5Q
Level of significance (Student's t test, Bailey 1959).
***=P<C-Q01.
Male as well as female (possessing the ventral marking scent gland) T. indica
sniffed (P < 0-001), urinated (P < 0-05) significantly more times (table 2) in
presence of another male's sebum odour. Male gerbils significantly ventral marked
(P < 0-001) and licked (P < 0-05) more in the cage side carrying the odour
stimulus. No difference was, however, observed in grooming behaviour. The
females, however, did not differ in their response to strange male sebum odour in
respect of various social acts (table 2).
In the plus maze, after the sebum smeared slide was placed near the food
basket in the arm (A) in which consumption of plain pearl millet was significantly
(P < 0-001) lower than in arm C (table 3), the food consumption by both male
and female Tatera indica in the former increased significantly (P < 0-001). How-
ever, in case of males, in the presence of the two set of slides (columns 3 and 4>
table 3) millet intake by male gerbils declined significantly (P< 0-05) in arm
C but in case of females this difference was not statistically significant which
indicates that the attraction towards strange male sebum by the females is of
lower intensity as compared to that by males.
Both male and female T. indica indicated a similar behavioural pattern, as in
the glass cage, by showing an increased frequency of sniffing and licking (P < 0-01)
in the section of sebum smeared slide (figure 1). The frequency of ventral marking
activity by males also exhibited an enhanced rate (P < 0-01) to wards the sebum
slide but the females did not ventral mark at all because these were the females
which had no ventral scent marking gland (figure 1). A similar pattern of the
duration of various social acts was also observed.
4. Discussion
The ventral scent marking gland is present in both the sexes in the genus Meriones
(Sokolov and Skurat 1966 ; Kumari etal 1981) but in the genus Tatera in which
the occurrence of such a gland was reported earlier (Prakash. and Kumari 1979),
it is present in 89 % males and only in a few females. Results of our experiments
262
Mohd. Idris and Ishwar Frakash
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Behavioural responses of the gerbil, T. indica
263
Table 3. Food consumption by Tatera indica in absence and presence of con-
Specific male sebum odour.
Sex
Mean food consumption g/100 g body weight ± SE
Arm A Arm C
(without any stimulus)
Arm A
(with sebum
odour slide)
Arm C
(with plain slide)
Male
Female
l-68±0-37
1-80±0-31
3-25±0-38
3-75±0-42
3-58iO-45
3-90:tO-36
2'46iO-37
3-28±0*42
Level of significance between
1 and 3 male < O'OOl
1 and 3 female < O'OOl
2 and 4 male Not significant
2 and 4 female Not significant
3 aid 4 male< 0'05
3 ar d 4 female Not significant.
i:
I
i *~
"5 5-
ui
0> 4—
*
1
1
i
1:
1
1
1
\
^ *
AC AC AC AC AC AC
SNIPPING LICKING VENTRAL MARKING
Figure 1. Frequency of various behavioural acts by male and female (not possessing
the ventral scent-marking gland) T. indica in the two arms of plus maze, one
carrying strange male sebum odour (A) and the other without it (C).
clearly indicate that all the three categories of T. indica (males possessing scent
marking gland), females possessing the gland (experiment 1 in glass cage) and females
without them (experiment 2 in plus maze) are attracted towards the sebum odour
of strange male suggesting that it has a bio-chemical communication function in
this genus. However, the frequency and duration of social acts by male and
female T. indica clearly indicate that the magnitude of attraction towards strange
male odour is much lower among females as compared to males. In this prefe-
rential behaviour T. indica is similar to Meriones tristrami (Thiessen et al 1973)
264 Mohd. Idris and Iskwar Prakash
and M. unguiculatus (Thiessen etal 1970). Indian desert gerbil, M. hurrianae
prefers the gland odour of similar sex given the choice of odour of both sexes.
However, in the absence of the same sex odour, both male and female are attracted
towards opposite sex odour (Kumari and Prakash 198 la). In this behaviour the
females of T. indica differ from M. hurrianae though they occur in similar habi"
tats in the Indian desert and are the two most predominant rodent species of the
region.
We have already reported that if the food is impregnated with sebum odour
it functions as phago-stimulant in T. indica (Kumari and Prakash 1979). This
observation is further confirmed by the results of experiment 2 in as much as
that even when the experimental females were those which did not possess the
scent gland, their food consumption increased significantly (P < 0*001) in the
presence of the sebum odour (table 3).
Another interesting observation made is about the significant (P < 0 • 00 1 ;. table 2)
enhancement of ventral marking activity by male and females (possessing the
scent-marking gland) in the presence of the sebum odour of a strange male. In
the plus maze experiments, similar enhancement (P < 0-01) in marking behaviour
of males was observed but this social behaviour in females was entirely absent.
These were the females in which the scent gland was absent and as such the social
act of ventral marking was not expected. What is interesting is that even in the
absence of the gland they were attracted towards male sebum odour (table 2).
The variance in the ventral marking behaviour of the two groups of females but
the similarity of their being attracted towards strange male sebum odour compli-
cate the functional role of ventral scent-marking gland.
A number of workers have explained the functions of ventral scent marking
among rodents. Ewer (1968) stated that an animal's own scent might act to
"increase its confidence" in the environment, whereas Eibl-Eibesfeldt (1953)
and Mykytowycz (1968) conjecture that scent marks provide " homeliness " to
the animals. Other functions designated to scent marking are territorial (Thiessen
1973), individual identification (Daly 1977), recognition of pups (Wallace etal
1973), phago-stimulant (Kumari and Prakash 1979), food reservation (Kumari
and Prakash 1981a) and " advertising ready to mate " stage by estrous females
(Kumari and Prakash 1981b). On the basis of limited observations on T. indica
it is rather difficult to delineate the exact function of ventral marking and the
role of sebum odour. However, our observations (unpublished) in a large
enclosure, the Rattery, under infra-red light show that T. indica ventral mark
their burrow openings quite often, more so before entering a burrow opening.
It was also noted that scent marking is carried out at an enhanced frequency by
the chasing Tatera, soon after the chased one enters a burrow. Tatera also mark
the food containers, the grass clumps and any new object whether it is a stone or
a wooden peg. From these observations, it appears that the function of scent
marking in T. indica is more of a " familiarisation " nature, or to signal * home '
to the marking animal or that of labelling the habitat for an animal's own use in
orientation (Johnson 1973). Whether it plays any territorial role is not clear.
Besides, these animals perform an anal drag behaviour and leave a slightly moist
surface, they possibly mark with urine or with a pheromone contained in it.
It is now known that the urine of Tatera indica has a phago-stimulant property
Behavioural responses of the gerbil, T. in die a 265
(Kutnari and Prakash, unpublished data). These odours may leave sufficient
olfactory cues which might deter other conspecifics away from the occupied
territory. Oar observations suggest that marking behaviour can have a number
of functions and more intensive work, which is in progress, may reveal the secrecy
of scent marking in rodents and its role in the bio-chemical communication.
Acknowledgements
The authors are grateful to Dr H S Mann for encouragement and providing
facilities for the work. Their grateful thanks are due to Miss Saroj Kumari for
helpful suggestions and to our colleagues for support and assistance.
References
Bailey N T J 1959 Statistical methods in biology (London : The English University Press)
Bhardwaj D and Prakash I 1981 Movements of Rattus rat t us in an artificial environment ;
Indian J. Exp. Biol 19 794-796
Daly M 1977 Some experimental tests of the functional significance of scent marking by gerbils
(Meriones unguiculatus) ; /. Camp. PhysioL PsychoL 91 1082-1094
Eibl-Eibesfeldt I 1953 Zur Ethologie des Hamsters (Cricetus cricetus L.) ; Z. Tierpsychol. JO
204-254
Ewer R F 1968 Ethology of mammals (London : Logos Press)
Johnson R P 1973 Scent marking in mammals ; Anim. Behav. 21 521-535
Kumari S, Cowan P and Prakash I 1981 The mid-ventral gland of the Indian desert
gerbil ; Acta Theriol. 26 97-106
Kumari S and Prakash I 1979 Conspecific odour as phagostimulant for Indian gerbil, Tatera
indica Hardwicke ; Indian J. Exp. Biol. 17 981-982
Kumari S and Prakash I 1981a Behavioural responses of Meriones hurrianae (Jordan) in conspecific
sebum odour of ventral sebaceous gland ; Biol. Behav. 6 255-264
Kumari S and Prakash I 1981b Scent marking behaviour of Meriones hurrianae during oestrous ;
Anim. Beliav. 29 1269-71
Mathur R P and Prakash I 1980 New food reaction among desert rodents ; Saugetier Mitteil. 28
28-30
Mykytowycz R 1968 Territorial marking by rabbit, Sci. Am. 218 116-126
Prakash I 1974 The Ecology of vertebrates of the Indian desert. Chapter XIII in Biogeography
and ecology in India (eds.) M S Mani, Dr Junk (The Hague : b.v. Verlag) pp. 369-420
Prakash I 1975 The ecology and zoogeography of mammals. Chapter XIX in Environmental
analysis of the Thar desert (eds.) R K Gupta and Ishwar Prakash (Dehradun : English
Book Depot) 468-480
Prakash I and Kumari S 1979 Occurrence of the ventral marking gland in Indian desert rodents ;
Saugetier. Mitteil. 27 315-316
Sokolov W and Skurat L 1966 A specific mid-ventral gland in gerbils ; Nature (London) 211
544-545
Thiessen D D 1973 Footholds for survival ; Am. Sci. 61 346-351
Thiessen D D, Lindzey G, Blum S L and Wallace PI 970 Social interactions and scent marking
in the Mongolian gerbil (Meriones unguiculatus) ; Anim. Behav. 19 505-513
Thiessen D D, Wallace P and Yahr P 1973 Comparative studies of glandular scent marking
in Meriones tristrami an Israeli gerbil ; Harm. Behav. 4 143-147
Wallace P, Owen K and Thiessen D D 1973 The control and function of maternal scent marking
in the Mongolian gerbil (Meriones unguiculatus) ;• PhysioL Behav. 10 463-466
P.(B)-6
Proc. ladian Acad. Sci. (Anim. Sci.), Vol. 91, Number 3, May 1982, pp< 267^3*
© Printed in India.
Effect of temperature and humidity on the development and
fertility-fecundity of Acrida exaltata Walk*
SHAMSHAD ALI
Section of Entomology, Department of Zoology* Aligarh Muslim University,
Aligarh 202 001, India
MS received 16 October 1980 ; revised 16 January 1982
Abstract The effect of temperature and humidity on Acrida exaltata Walk, has
been studied to have two aspects in relation to (i) its effect on hopper development
and (ii) its effects on the fertility-fecundity. The rate of development was affected
by the moisture present in the environment. No development took place at 0%,
10% and 20% RH. Most desirable range of humidity was between 50-70% RH.
Development took place at 20°, 30°, 35° and 40° C in 92-8, 74-8, 71 -8and 66-6 days
in males and 101-0, 86 '4, 85-6 and 75-2 days in females. Percentage of hoppers
reaching the adult stage, longevity of adults, average number of copulation and average
number of eggpods per female was influenced by the temperature.
Keywords, Relative humidity ; fertility-fecundity ; development ; copulation ;
longevity ; temperature.
1. Introduction
consumption was dependent upon the relative humidity (RH) present in
the atmosphere and water content in the food(Sanger 1973). Water content
of the eggpods directly affects the size of hatchlings in Schistocerca gr£garia$
water loss during the last half of incubation period resulting in smaller hatch,,
lings and excess water uptake in larger hatchlings (Bernays 1972). Shulov (1970)
in Noinadacris septemfdsciata and Locusta migratorfa found that hutiiidity and
temperature affects the development and weight of eggs. Development was
arrested when the required moisture was not available, and resumed on being
provided with moisture. Petty (1974) in Locusta migratoria also found that mois-
ture affects development and hatchling. During developmental period, nymphs
preferred higher relative humidity in the field as observed by Riegert (1959).
Mathee (1954) observed in the case of Locusta pardalina that viability of eggs
was dependent upon the high temperature of the soil. According to Symmons
et al (1974), temperature range between 30°-40° C was favourable for develop-
ment. Abou-Elela and Hilmy (1977), Tutkun (1973) and Qayyum and Atique
(1973) found that temperature directly influences hatching of eggs, rate of develop-
267
P.(B)-7
268 Shamshad Alt
ment, precopulation, preoviposition period and reproduction. Studies
made to note the effect of temperature and humidity on the hoppers development
and fertility-fecundity of Acrida exaltata Walk.
2. Material and methods
2.1. Effect of humidity on hoppers development and fertility-fecundity
Newly hatched hoppers were kept in jars and glass tubes at 90, 80, 70, 50, 20^
10 and 0% humidity at a constant temperature of 37° C for observations on
nymphal duration and fertility- fecundity. Desired relative humidity was
obtained through the solution of potassium hydroxide (100 gm of KOH per
100 gm of water) as given by Buxton and Mellanby (1934) (table 1),
2-2. Effect of temperature on hoppers development and fertility-fecundity
Newly hatched hoppers were kept individually in glass tubes (15 x 3- 8 cm)
covered with muslin cloth for observation on hoppers development at 25°, 30°,
35° and 37° C. Nymphs were provided daily with fresh food twice. Standard
error was worked out and results are shown in table 2.
Newly emerged adults were kept in pairs at temperature of 20°, 25°, 30°, 35°
and 37° C in separate glass jars. Number of copulations and ovjpositions was
observed and number of eggpods per female and longevity of adults were noted.
Results are summarized in table 3.
3. Observations
3.1. Effect of humidity on the hopper development and fertility-fecundity
Rate of development of hoppers was affected by moisture present in the environ-
ment. No development took place at 0%, 10%, and 20% RH (table 4).
The most desirable range of humidity was between 50-70% ,RH.
fable li Relative humidity obtained through KOH with differing specific gravity.
RH gm KOH/lOOgm
% of water Specific gravity
90
15'0
1*115
80
25-0
1-175 '
70
35*0
1*265
50
52-0
1-335
20
81-5 .
1*490
, 10
110-0.
1*570
0
Solid KOH
* »
Effect of temperature and humidity on A. exaltata Walk
Table 2. Effect of temperature on the hoppers development
269
Stage
25° C (days) 30° C (days) 35° C (days) 37° C (days)
I Instar
Mean
SE
14-40
0-81
IS-
C'
60
93
11*20
0/66
13
0
-80
•86
7-80
0-70
9-20
0-58
7-20
0-37
8-QO
0-45
II frmar
Mean
SE
14*80
0'66
17-
0-
80
86
13-60
1-03
12
0
•20
•66
7-60
0-61
7-80
0-66
6-60
0-40
7-20
0-58
III Iitstar
Mean
SE
13-40
0-87
IS-
O-
80
86
10-80
0-S8
11
0
•80
•86
8-40
0-76
10-40
0*68
7-40
0-51
8-80
0-58
IV Instar
Mean
SE
12-80
0-58
IS-
a-
80
58
13-80
0-86
13
0
•60
•si
9-80
0'S8
12-20
0-66
S'60
0-60
10-20
0-58
V Instar
Moan
SE
13-60
0-51
16-
o-
40
•76
12-80
0-58
IS
0
•80
•80
9-20
0-37
11-20
0-37
8-40
a- si
10-20
0-58
VI Instar
Mean
SE
14-80
0'37
17-
0'
20
66
14-20
0-86
17
1
•40
•03
14-20
0-37
16-20
0-66
13-40
0-60
14-40
0-60
VII Instar
Mean
SE
14-20
0-37
17-
o-
60
51
14-20
0-58
17
0
•00
•74
15-40
0- 68
18-60
1-03
14-00
0*55
16-40
1*03
Adult
Mean
SE
92*80
2-73
101-
2-
00
57
74-80
2-17
86
1
•40
•72
71-80
0-98
8S-60
3-02
66-60
1-91
75-20
2-35
Pcrcentago of
hoppers
reached adult
34*20 31-00 51-50 47-20 73-50 68'00 76-00 71-50
SE = Standard error.
tage of hoppers reaching the adult stage was the highest, 77-86 and 81-25 in
males and females respectively at 70% RH, while the lowest, 30-75 and 43-0 in
males and females respectively at 90% RH.
As evident from table 2, it was found that at 70% RH, sexual maturation is
hastened, but the longevity of adults was shorter, 87 and 116 days in males
and females respectively due to the rapid rate of sexual maturation and a high
average number of eggs and eggpods per female. As the humidity increased
above the optimum (70% RH) the longevity of adults and the time required for
sexual mattiratipn increased, but the number of eggs and eggpods per female
decreased until 80% RH. Above 80% RH the length of adult life decreased and
the number of eggpods were few or none at all. The number of eggpods pep
female and the number of eggs per pod show a rapid drop at relative humidities
270 Shamshad All
Table 3. Effect of temperature on fertility -fecundity and longevity of Acrida
exaltata Walk.
Longevity of Average Average Average
adults (days) no. of no. of no. of
Temperature Sex No. of Total Mean ± SE copula- eggpods eggs per
0 C pairs mating tion per per pod
male female
20 Male 5
8 68±2-23 1-6 1
48
Female
99i2*82
25 Male 5
14 72±0-55 2-8 2
59
Female
104±l-57
30 Male 5
21 78.±1'39 4-2 4
76
Female
113±l-93
35 Male 5
26 91±l-56 5-2 5
81
Female
121 ±2-82
37 Male 5
28 87 ±1-59 5-6 7
87
Female
116±2-95
SE = Standard error.
3.2. Effect of temperature on the hoppers development and fertility-fecundity
Temperature has a marked influence on the development of hoppers. Rate of
development increased at the higher temperatures while at low temperature
decreased as is clearly evident from table 2. The total number of mating was
increased with the rise in temperature. As shown in table 3, the frequency of
copulation, number of eggpods per female and number of eggs per pod increased
with the rise in temperature. The temperature range between 30°-37° C was. found
favourable for copulation, oviposition and number of eggpods per female. Pre-^
copulation and pre-oviposition period decreased with rise in temperature.
Average survival of adults at 35° C was higher than at 25° C. Longevity of adults
was 91 and 121 days at 35° C while at 25° C, it was 68 and 99 days -in males
and females respectively.
4. Discussion
Effect of temperature and humidity on hoppers development must be discussed
together, because it is very difficult in experimental worfc to separate the two
factors. On the one hand, the relative humidity of the air varies with tempe-
rature, and on the other, hopper metabolism is possibly more affected by -the
water content of food than by air humidity, both of which may influence the
Effect of temperature and humidity on A. exaltata Walk
Table 4. Effect of humidity on hoppers development.
271
50% RH
70% RH
80% RH
90% RH
I Instar
II Instar
III Instar
IV Instar
V InStar
VI Iitstar
VII Instar
Adult
Percentage
itymphs
reaching
stage
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Mean
SE
Mean
SE
of
adult
(days)
(days)
S-00
0-70
9-80
0-73
8-80 10-40
0-66 0-87
8-40 11-20
0-50 1*06
7-20
0-37
6-60
0-40
7-40
0-51
8-00
0-45
(days)
9-80 12-40
0-73 0-93
(days)
15-20 19-20
0-86 1-28
7-20 11-40 13-20 17-00 20-20
0-58 0-92 1-06 0'70 1-39
8-80 11-20 12-20 16-40 18'60
0-58 0-66 0-66 0-76 1-07
8-40 11-00 8-60 10-20 13-00 13-80 15-60 19'80
0-76 0-89 0-60 0-58 1-14 1-06 1-07 1-06
9-20 11-20 8-40 10-20 12-80 15-60 18'20 22-20
0-37 0-66 0-51 0-58 0'58 0'8l 0'86 1-24
12-20 15-60 13-40 14-40 12-80 15'80 16'80 20'80
0-58 0-92 0-60 0*60 0'86 0*80 0'66 1-42
13-80 15-80 14-00 16-40 14'80 16-20 15'00 18-80
0-66 0-66 0-55 1-03 0-86 0-86 1-04 1-15
69-00 82-00 66-60 75'GO 86*60 91-60 111 -CO 124-60
2-22 2-21 1-91 2-35 2-01 2*71 2-35 2-29
71-50 73-25 77-86 81-25 58-00 61-25 38*75 43-00
SE = Standard error.
quantity of food consumed and, therefore, the rate of growth . Nevertheless, some
evidence of temperature effects should be briefly mentioned. Parker (1930) in
his extensive experiments with several American grasshoppers clearly indicated a
shortening of hopper period and an accelerated rate of development with rising
temperature.
At constant temperature, relative humidity affects the rate of ovarian growth
and percentage of hoppers reaching adult stage. These variations in humidity
at constant temperature suggest that the different optimal relative humidities and
the lower limits are explained by different rates of evaporation (Gunn 1933 ;
Koidsumi 1934) at different temperatures. Zolotarvesky (1933) observed in
Schistocerca gregaria Forsk. that relative humidity plays a very important role
in the embryonic and postembryonic development of locust and grasshopper.
272 Shamshad AH
Table 5. Effect of humidity on fertility-fecundity of Acrida exaltata Walk.
Average Average Average
Humidity No. of Sex Longevity of Total no. of no. of no. of Hatching
% RH pairs adults ± SE mating copula- eggpods eggs per %
turn per per pod
. male female . .
10
5
No pairing and
egg-laying
20
5
No pairing and
egg-laying
50
5
Male £2-4 ±1-36 25 4-8
5-2 76 71-86
Female lll*2Q±0-86
80
90
Male 87; 00 ±1-56 28
Female 116'00±2-95
Male 75 -40 ±1-99 23
Female 105- 20 ±2- 23
Male 67-4Q±l-02 14
Femele 95-40±l-86
5-6
3-9
7-1
2-6
1-5
85
69
51
84-20
58-40
21-90
SE = Standard error.
present findings are contrary to the observations of Husain et al (1941) and
Chauvin (1941), that relative humidity has no effect on the development of
hoppers. However, on examining their results, it was found that they had based
their findings on experiments with only a very small number (often only one)
of hoppers, and without controlling the moisture of the food. In very detailed
and extensive experimental studies on locust by Hamilton (1936, 1950), the effects
of a series of combinations of temperatures and relative humidities were studied
and it was suggested that the duration of adult life was the shortest wheii condi-
tions were optimum for sexual maturation and that the length of life gradually
increased as conditions became less favourable. In the present observations 70%
RH is the optimum for development and sexual maturation. As the relative
humidity increased above or decreased below, the time required to reach sexual
maturity was increased, but the number of eggpods decreased. This shows that
with an increase in the average preoviposition period, an increase in the length
of adult life is observed. Similar observations were found by Symmons et al
(1974) in Schistocerca gregaria, that the temperature range between 30°-40° C was
favourable for development. Abou-Elela and Hilmy (1977) observed in the case
of Acrotylus insubricus that temperature has a direct effect on the hatching.period
and development.
Acknowledgements .
The author is highly indebted to Prof. N H Khan for providing laboratory
facilities. Thanks are also due to the Council of Scientific and Industrial
Research, New Delhi, for financial assistance,
Effect of temperature and humidity on A. exaltata Walk 273
References
Abou-Elela R and Hilmy N 1977 Wirkungenden Fotoperiode und Temperate auf die Entwic-
kenngstadien von Acrotylus insubrucus Scop (Orthoptera : Acrididae) ; Anz. Schadlingsk.
Pftantzenschutz, Umwehschutz 50 25-28
Bernays E A 1972 Some factors affecting size in first instar larvae of Schistocerca gregaria
(Forsk.) ; Acrida 1 189-195
Buxton P A and Mellanby K 1934 The measurement and control of humidity ; Bull. EntomoL
Res. 25 171-175
Chauvin R 1941 Contribution a 1'etude physiologique du criquet pelerin et du determinisme
des phenomenes gregaires ; Ann. Soc. EntomoL Fr. 110 133-272
Gunn D L 1933 The temperature and humidity relations of the cockroach (Blatta orientals)*
I. Desiccation ; /. Exp. 10 274-285
Hamilton A G 1936 The relation of humidity and temperature to the development of three
species of African Locusts, Locusta migratoria migratorioides (R. and F.), Schistocerca
gregaria (Forsk.), Nomadacris septemfastiata (Serv) ; Trans. R. EntomoL Soc. 85 17-60
Hamilton A G 1950 Further studies on the relation of humidity and temperature to the develop-
ment of two species of African locusts Locusta migratoria migratorioides (R. and F.) and
Schistocerca gregaria (Forsk.). Trans. R. EntomoL Soc. 101 1-581
Husain M A, Ahmad T and Mathur C B 1941 Studies on Schistocerca gregaria Forsk— Role
of water in the bionomics of the Desert Locust ; Indian J. Agric. Sci. 10 927-944
Koidsumi K 1934 Experimented studien uber die transpiration und den warmchanhalt be
insekten ; Mem. Fac. Sci. Agric. Toihoku 12 1-179
Mathee J J 1954 The effect of constant high temperature on the embryonic development and
pulsation of the lateral body wall in Locustana pardalina Walk. (Orthoptera : Acrididae) ;
/. Ent. Soc. S. Afr. 17 222-231
Parker J R 1930 Some effects of temperature and moisture upon Melanoplus mexicanus mexi-
canus Sauss and Camnula pellucida Scudd. (Orthoptera) ; Bull. Mont. Agric. Exp. Sta. no.
223 132
Petty G J 1974 Effect of humidity on the hatching of eggs of the brown locust, Locusta parda-
lina Walk. ; Phytophylactica 6 305-306
Qayyum H A and Atique M R 1973 Some ecological studies on Chrotogonus trachypterus
Blanch ; Pakistan J. ZooL 5 75-78
Riegert P W 1959 Humidity reactions of Melanoplus bivittatus Say and Camnula pellucida
Scudd. (Orthoptera : Acrididae) : reactions of normal grasshoppers; Can. EntomoL 91
35-40
Sanger K 1973 Consumption by some field grasshoppers (Orthoptera : Acrididae) depending on
different humidity rates ; Verhand. ZooL Bot. Ges. Wien. 113 81-92
Shulov A 1970 The development of eggs of the red locust, Numadacris septemfasciata Serv. and
the African migratory locust, Locusta migratoria migratorioides R and F and its interruption
under particular conditions of humidity ; Ami Locust Bull. 48 1-26
Symmons P M, Green S M, Robertson R A and Wardhough K G 1974 The production of
distribution maps of the incubation and development periods of the Desert Locust, Schisto-
cerca gregaria Forsk. (Orthoptera: Acrididae); Bull. EntomoL Res. 64443-451
Tutkun F 1973 Investigation on the effect of low temperature on the last immature adult stage
of the Desert Locust, Schistocerca gregaria Forsk. ; Bitki Koruma Bull. 13 181-201
Zolotarvesky B N 1933 Contribution & 6tude biologique du criquet migrateur, Locusta migra-
toria acapito Sauss dans ses foyers permanent ; Ann. Epiphyses 19 47-142
Proc. Indian Acad. Soi. (Anim. Sci.), Vol. 91, Number 3, May 1982, pp. 2?5-282.
© Printed in India.
On some blood flukes (Spirorchiidae : Coeuritrematinae) from
freshwater chelonians in India
V TANDON and N K GUPTA*
Department of Zoology, North-Eastern Hill University, Shillong 793 014, India
* Department of Zoology, Panjab University, Chandigarh 160 01 4, India
MS received 2 June 1981
Abstract. Coeuritrema sutlejemis Mehrotra, 1973 and C. sheilae are described in
detail and their validity is discussed. A key to the species of Coeuritrema is provided
and a few of the diagnostic features of the genus are emended.
C. lyssimus Mehra, 1933 is recorded from a new locality and some variations
from the original description are mentioned.
Keywords. Blood flukes ; chelonians ; Coeuritrema ; Spirorchiidae.
During the period from January 1969 to September 1971, 156 specimens of
freshwater chelonians, namely Kachuga tectum tectum (29), K.t. tentoria (5),
K. sylhetensis (69) and Lissemys punctata punctata (53) from different localities
in Punjab, Haryana and Uttar Pradesh were dissected for the collection of
the digenetic flukes. A thorough examination of heart and blood of the hosts
revealed the presence of many flukes. Of these, detailed accounts of two species
Coeuritrema sutlejemis Mehrotra, 1973 and C. sheilae Mehrotra, 1973 are given
here and their validity is discussed. Earlier, only the diagnostic features of these
species had been given in an abstract form (Mehrotra 1973). Another species,
Coeuritrema lyssimus Mehra, 1933, has also been recorded.
Bouin was used as a fixative for the parasites. The^flattened flukes were
stained with Mayer's carmalum, borax carmine or Ehrlich's haematoxylin. The
last-mentioned stain and eosin were used to stain the serial sections cut at a
thickness of 5//.
Family Spirorchiidae Stunkard, 1921
Subfamily Coeuritrematinae Srivastava, 1960*
Genus Coeuritrema Mehra, 1933
Coeuritrema sutlejensis Mehrotra, 1973 (figures 1-5)
* Yamaguti (1971) has mentioned the subfamily name Coeuritrematinae Dwivedi, 1968. In
fact Srivastava (1960), not Dwivedi, had proposed this subfamily name in view of the priority
of the genus Coeuritrema over Tremarhynchus. Dwivedi (1967) has only referred to Srivastava s
(1960) views.
275
p.mv-8
276 V Tandon and N K Gupta
VESSEKEXI.
_GLCE.
RECSEM.I
-CLSA.
Figires 1-5. Coewtrema
whole
Blood flukes from freshwater chelonians 277
Thirteen specimens of Coeuritrema sutlejensis Mehrotra, 1973 were recovered
from the ventricle of the heart of freshwater chelonians, Kachuga sylhetensis (Jerdon)
collected from the River Sutlej at Ropar (Punjab) and Lissemys punctata punctata
Bonnaterre procured from Lucknow (U,P.) and Sangrur (Punjab). The number
of flukes in one host was one or two.
Description (based on 10 specimens ; all the measurements are in mm) : Body
slightly tapering towards extremities, ending in blunt rounded tips, 1-32-2-15
in length by 0*22-0 -40 in maximum breadth across testes. Tegument smooth,
suckers prominent but with weak musculature; oral sucker terminal, 0-08-
0-12 x 0-06-0-11, ventral sucker situated almost at level of middle of anterior
half of body, 0-09-0-12 x 0-08-0-13, almost equal to oral sucker. Oesophagus
0-16-0 -31 long, irregularly distended and surrounded by gland cells. Intestinal
caeca at first forming broad dilated shoulders and then extending backward as
slender tubes, dilating again in post-testicular region and terminating asymmetri-
cally a little in front of rear end of body.
Excretory vesicle Y-shaped with a short stem ; excretory pore terminal.
Testes enormously developed, tandem, intercaecal, with a wavy contour, anterior
testis 0 • 1 7-0 • 27 x 0 • 1 4-0 • 29 and posterior testis 0-19-0-33 x 0-13-0-26.
Vesicula seminatis externa a little posterior to ventral sucker, thin-walled 0-09-
0-15 X 0-04-0 -08. Cirrus sac almost transversely situated in between vesicula
seminalis externa and anterior testis, 0-04-0-08 x 0-09-0- 15. Genital pore
dorsal, to left of median line, close and external to left intestinal caecum, in
front or at level of anterior border of anterior testis.
Ovary sinistral, intertesticular, close to left intestinal caecum, elongated, 0-06-
0-19 x 0-016-0-05. Receptaculum seminis median, intertesticular. Laurer's
canal present. Uterus short, running along left margin of anterior testis. Eggs
not observed in any specimen. Vitellaria extending laterally from level of
intestinal bifurcation up to close behind caecal termination, coalescing in the
regions just 'in front and behind ventral sucker and also in post-testicular zone ;
in some specimens, however, the vitelline follicles have been found scattered in
the region in front of the intestinal bifurcation. Yolk reservoir dorsal to recep-
taculum seminis.
Remarks : So far, seven species have been assigned to the genus Coeuritrema
Mehra, 1933. These are C. lyssimus Mehra, 1933 from Lissemys punctata in
Allahabad (U.P.) ; C. odhnerensis Mehra, 1933 from the same host and locality ;
C. indlcus (Thapar 1933) Mehra 1934 (syn. Tremarhynchus indicus (Thapar 1933)
from Trionyx gangeticus in Lucknow (U.P.) ; C. yoshldai (Ozaki 1939) Takeuti
1942 (syn. Hapalorhynchus yoshidai Ozaki 1939) from Ocadisinensisin China; C. oca-
diae Takeuti 1942 from Ocadia sinensisin Formosa; C. oschmarini Belous 1963 from
Amy da sinensis from the Khanka lake and the River Mo in the far east of the USSR ;
and C. macrotesticularis Rodhe, Leeet Lim, 1968 from Dogania subplana in Malaya.
C. sutlejensis can be distinguished from C. lyssimus in which the body surface
is covered with conical tubercles, the cirrus sac is flask-shaped and obliquely
placed and the vitellaria are postacetabular in distribution ; and from C. macro-
testicularis which possesses deeply lobed testes, the genital pore more or less
in level with the ventral sucker, and the intestinal caeca showing many undulations
in the posttesticular region,
278 V Tandon and N K Gupta
In the general shape of the body C. oschmarini, C. indicus, C. odhnerensis and
C. ocadiae approach C. sutlejensis hut there are many other differences. C. sutle-
jensis stands apart from C. oschmarini in which the testes are entire and oval,
the ovary is rounded, and the cirrus sac is flask-shaped and obliquely placed;
from C. indicus in which the testes are deeply lobed and the vesicula seminalis lies
behind the cirrus sac ; from C. odhnerensis in which the testes are small and
irregularly lobed and the vesicula seminalis lies opposite to the crescent-shaped
cirrus sac ; and from C. ocadiae in which the testes are small and oval and the
vesicula seminalis lies anterodorsally to the enlongated and conical cirrus sac.
C sutlejensis stands very close to C. yoshidai in having the cirrus sac behind
the vesicula seminalis but the position and the shape of the ovary and the
commencement of the vitellaria are the characters which differentiate the two
species ; in C. yoshidai, the ovary is median and transversely elongated and the
vitellaria commence behind the intestinal bifurcation.
Hosts : Kachuga sylhetensis (Jerdon)
Lissemys punctata punctata Bonnaterre
Location : Heart
Localities : Ropar and Sangrur (Punjab), Lucknow (U.P.)
Coeuritrema sheilae Mehrotra, 1973 (figures 6, 6a)
The material consisted of eight specimens collected from the heart, blood and
teased hepatic tissue of Lissemys punctata punctata Bonnaterre in Rudrapur (U.P.),
Patiala and Sangrur (Punjab) and Kachuga tectum tectum (Gray) in Ropar
(Punjab). The number of specimens in one host was never more than two. Of
the flukes recovered, two were immature and one was distorted.
Description (five specimens measured) : Body elongated, somewhat tapering
towards extremities, 1-61-2- 11 in length and 0-18-0-28 in maximum width across
testicular region. Body surface smooth. Oral sucker 0-06-0 -12 long by 0-05-
0-09 wide. Ventral sucker just behind intestinal bifurcation, 0-11-0-13 x 0-09-
0-16, larger than oral sucker. Oesophagus a wide tube, 0 • 29-0 • 41 long, surrounded
by gland cells. Intestinal caeca slender, bending a little inwards just behind
ventral sucker and again turning outwards and running parallel to body margins,
converging behind posterior testis and continuing as straight tubes, terminating
symmetrically 0-13-0-19 in front of posterior end of body.
Excretory system Y-shaped, pore terminal at rear extremity of body.
Gonads in middle third of body. Testes rounded or irregular, anterior testis
0-12-0-16 X 0-11-0- 13, posterior testis 0-11-0-16 x 0-11-0-14. Vesicula semi-
nalis externa (observed in two specimens only) small, opposite to basal portion
of cirrus sac ; the latter elongated, somewhat sinuous, placed more or less obliquely
or longitudinally between ventral sucker and anterior testis, 0-24-0-28 long by
0-11-0-14 wide across its basal region, enclosing a small vesicula seminalis
interna, pars prostatica and protrusible cirrus. Genital pore a little behind ventral
sucker, sinistral, close to left intestinal caecum, may be inter or extracaecal (since
inward bending of the intestinal caeca has been found to be variable, depending
upon the flattened state of the fluke).
Blood flukes from freshwater
279
.0.5.
Y.R,
.GL.CE.
Figures 6, 6A, 7. See page 282 for caption.
280 V Tandon and N K Gupta
Ovary intertesticular, sinistral, somewhat triangular, base of triangle being
parallel to lateral margin of body and apex directed towards median line, 0-08-
0-12 x 0-08-0-13. Receptaculum seminis median. Uterus containing a single
egg (observed in one specimen only) with, its shell forming polar prolongations.
Egg 0-186 x 0-029 (including length of polar prolongations). Vitellaria beginning
immediately behind ventral sucker and extending up to ends of intestinal caeca
overlapping the latter and filling the entire posttesticular intercaecal space.
Remarks : In shape and disposition of the cirrus sac and also the position of
the vesicula seminalis externa (i.e., opposite to the basal portion of the elongate
cirrus that lies somewhat obliquely), C sheilae shows its closest resemblance to
C. ocadiae Takeuti 1942 and C. odhnerensis Mehra 1933, and differs from all the
other known species of the genus. However, C. ocadiae and C. odhnerensis can
also be differentiated from it because of the oral sucker being larger than the
ventral and the vitellaria extending into the preacetabular zone in them, whereas
in C. sheilae the oral sucker is smaller than the ventral and the viteliaria are
restricted to the postacetabular region of the body.
Hosts : Lissemys punctata punctata Bonnaterre,
Kachuga tectum tectum (Gray)
Location : Heart, blood vessels, liver
Localities : Rudrapur (U.P.), Patiala, Sangrur and Ropar (Punjab)
Coeuritrema lyssimus Mehra, 1933 (figure 7)
Hosts : Lissemys punctata punctata Bonnaterre
Location : Heart
Locality : Rudrapur (U.P.)
Remarks : The present collection consisted of two specimens of Coeuritrema
lyssimus Mehra 1933. Variations from the original description are : smaller
gonads, the genital pore inner to the left intestinal caecum and the presence of
two eggs in the uterus. According to Mehra (1933), the genital pore is external
to the left intestinal caecum and the uterus contains only one egg at a time.
Rudrapur (U.P.) is a new locality record for this species.
In view of the observations on the species described by the authors and also
of the descriptions of C. odhnerensis Mehra 1933 and C. ocadiae Takeuti 1942,
a few generic characters of Coeuritrema as given by Yamaguti (1958, 1971) have
been emended. The emended characters (italicised) are as follows :
Ventral sucker larger or smaller than or 'equal to oral sucker. Vesicular semi-
nalis externa anterior, posterior or opposite to cirrus sac. Parasitic in blood
vessels, liver or heart of freshwater chelonians.
KEY TO THE SPECIES OF THE GENUS COEURITREMA MEHRA, 1933
L Body surface with conical tubercles or papillae.
• • • C. lyssimus Mehra, 1933
Body surface smooth ... 2
2. Cirrus sac somewhat oval, placed transversely to vertical axis of body.
Vesicula seminalis externa in front of cirrus sac ... 3
Cirrus sac elongate, flask-shaped, placed obliquely. Vesicula seminalis
externa behind cirrus sac or opposite to it ... 4
Mood flukes from freshwater cheioniaris
3. Ovary elliptical, median. Vitellaria commencing at a level behind intestinal
bifurcation • • -C. yoshidai (Ozaki 1939) Takeuti 1942.
Ovary elongated, sinistral. Vitellaria commencing at the level behind
intestinal bifurcation •••C. sutlejensis Mehrotra, 1973
4. Vitellaria commencing behind ventral sucker
•••C. sheilae Mehrotra, 1973
Vitellaria commencing in front of ventral sucker, i.e., at bifurcal level ---S
Vitellaria extending throughout the body • • • 7
5. Ventral sucker larger than the oral sucker. Intestinal caeca forming undu-
lations in posttesticular region. Testes large, deeply lobed. Genital pore
at level of ventral sucker • • • C. macrotesticularis Rohde, Lee et Lira, 1968
Ventral sucker smaller than oral sucker. Intestinal caeca straight in
posttesticular region. Testes relatively small, not deeply lobed. Genital
pore quite behind ventral sucker ' • • • 6
6. Ventral sucker close behind intestinal bifurcation, both intestinal caeca
bending inwards behind it. Testes lobed ---C. odhnerensls Mehra 1973
Ventral sucker some distance behind intestinal bifurcation, only left intestinal
caecum bending inwards behind it. Testes entire
• • • C. ocadiae Takeuti 1942
7. Testes oval, entire, ovary rounded. •••C. oschmarini Belous 1963
Testes deeply lobed. Ovary elongated and lobed.
•••C. indicus (Thapar 1933) Mehra 1934.
References
Belous E V 1963 Helminth fauna of Amyda sinensis ; Helminthologia 4 79-88
Dwivedi M P 1967 Contribution to the family Spir orchil dae Stunkard, 1961 (Digenea: Trema-
toda) ; Indian J. Helminthol 9 1-14
Mehra H R 1933 New blood-flukes of the family Spirorchiidae Stunkard, from Indian fresh-
water tortoise with discussion on the systematic position of the genus Coeuritrema n.g
and the relationships of families of blood flukes Part I ; Bull. Acad. Sci. U.P. Allahabad
2 203-222
Mehra H R 1934 New blo.od-flukes of the family Spirorchiidae Stunkard, from Indian fresh
water tortoises with discussion on the synonymity of certain genera and relationships of
the families of blood-flukes. Part II; Bull Acad. Sci. U.P. Allahabad 3 169-196
Mehrotra V 1973 Digenea from some reptilian hosts in India ; Parts I, II ; Proc. 6Qth Indian
Set. Cong. Part IV 46-47
Ozaki Y 1939 A new blood-fluke, Hapalorhynchus yoshidai ; Vol. Jubil. Pro Prof. Yoshida 1
29-35
Rohde K, Lee S K and Lim H W 1968 Ueber drei malayische Trematoden ; Ann. Parasit.
Hum. Comp. 43 33-34
Srivastava H D 1960 Presidential address ; 47th Indian Sci. Cong. Bombay
Stunkard H W 1921 Notes on North American Blood flukes ; Am. Mus. Novit. 12 1-5
Takeuti E 1942 Now blood flukes of the family Spirorchidae from Japanese fresh-water tortoise
and marine turtles ; Jpn. J. Med. Sci. Pt. 6, Bacteriology and Parasit. 2 161-174
Thaper G S 1933 A new blood-fluke from an Indian tortoise, Trionyx gangeticus ; /. Helm.
11 163-16$
Yamaguti S 1958 Systema Helimnthuin. Vol. I. The digenetic trematodes of vertebrates
(Pt. I and II) ; (New York and London : Interscience Publishers)
Yamaguti S 1971 Synopsis of digenetic trematodes of vertebrates I ; (Japan : Keigaku Publishing
Co.) pp. 1-1074
V fandon and N ft Gupta
Figures 6, 6A and 7. 6. Coeuritrema sheilae Mehrotra 1973 (whole mount, ventral
view); 6 A. egg of the same. 7. Coeuritrema lyssimus Mehra, 1933 (whole mount,
ventral view). (CI.SA., cirrussac;EG, egg ;EX.P., excretory pore; EX.VE, excretory
vesicle; G.P., genital pore ; GL.CE., gland cells ; INT.CA., intestinal caeca; L.C.P.,
Lauier's canal pore ; MT, metraterm ; O.S., oral sucker ; OES, oesophagus ;OV,
ovary; REC.SEM., receptaculum seminis; T1? anterior testis ; T2, posterior testis ;
V.S. ventral sucker ; VES.SEM.EXT., vesicula seminalis externa ; VIT, vitellaria ;
Y.R yolk reservoir).
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 3, May 1982, pp. 283-295.
© Printed in India.
Life history and behaviour of the cyst nematode, Heterodem oryzicola
Rao and Jayaprakash, 1978 in Rice (Oryza sativa L.)
A JAYAPRAKASH and Y S RAO
Nematology Section, Central Rice Research Institute, Cuttack 753 006, India
MS received 22 April 19S1
Abstract. The embryonic development of the cyst nematode, Heterodera oryzicola
and its emergence from egg masses was completed within & to 9 days. The emerged
second stage juveniles were attracted to roots of rice within 24 hr and penetrated
the roots within 24 hr. After penetration, the endoparasitic juveniles developed
into males within 14 days and white females within 20 days. Orientation of females .
was equal towards hypocotyl (42%) and root tip (48%) while a few (10%) matured
vertically in thin secondary roots or small rootlets. The sex-ratio between males
and females was about 1 : 4. The virgin females secreted a strong male attractant
and the males migrated towards the females in response to this stimulus and mated
with the females. The eggs were laid in gelatinous matrix secreted by the females
within 22 days and the females turned into brown cysts by 24 days. A single
H. oryzicola female laid on an average 19$ eggs in an egg mass and retained
120 eggs within the body of the brown cyst. None of the females was left unmated
and all of them laid a single egg mass each within 30 days. One life cycle was
completed in 30 days and 12 generations occurred in a year.
Keywords. Heterodera oryzicola ; Oryza sativa ; life history ; behaviour.
1. Introduction
Severe leaf chlorosis, stunting and mortality of rice plants were observed in
upland rice fields of Pattambi and its vicinity in Kerala State. Inoculation to
rice cv. CRM 13-3241 under greenhouse condition proved that it was due to a
new root infesting cyst nematode (Rao and Jayaprakash 1977). The nematode
was subsequently described as Heterodera oryzicola (Rao and Jayaprakash 1978).
Information on the life history and behaviour of this new nematode was essential
for adopting control measures in infested soil. Hence, the present investigation
was taken up.
2* Materials and methods
2-1. Embryonic development
Freshly laid eggs in egg masses attached to the posterior end of mature white
females of H. oryzicola from roots of rice were removed and kept in hanging
283
284 A Jayaprakash and Y S Rao
drops of water on a microslide (Dasgupta and Raski 1968). The eggs were
incubated at 28 ±. 1° C. Cell division, blastulation, development and eclosion
of the juveniles were observed at intervals of 2 hi and recorded from 20 synchro-
nous eggs of each egg mass.
2-2. Post-embryonic development
Plastic pots (6x6 cm) were each filled with 100 g of soil to which 40 ml water
was added and one seed of rice was sown for germination. When sprouts were
10 days old, each was inoculated with 100 second stage juveniles of H . oryzicola.
At intervals of 2 days, roots of 4 sprouts were collected and examined for endo
and semi-endoparasitic stages till brown cyst formation occurred in the roots.
The juveniles and adults were recorded. The first appearance of any juvenile
stage was considered as the result of its growth and moulting and accordingly,
the duration of each stage was computed as the period between the first appearance
of two successive stages.
Seedlings of rice were raised and inoculated with H. oryzicola as above on the
1st day of every month for 12 months. Seedlings were sampled on any day after
the 21st day. The number of 'cysts and. white females per plant root system was
recorded and they ware kept in fresh rice root diffusates for hatching. The dato
of the first juvenile emergence was recorded and used as an indication of comple-
tion of a generation.
2-3. Migration of infective juveniles towards rice roots
Sprouts of rice were grown in petri dishes containing 1% agar media and when
the sprouts were 10 days old, 100 freshly hatched second stage juveniles of
H. oryzicola were released at a distance of 10 mm from the nearest root. A
mechanical barrier (by negative film strip or cover glass or any handy items can
be used to make mechanical barrier) to prevent advancing of roots towards the site
of juvenile release was made. The distance of 10 mm was divided into ten zones
and the number of second stage juveniles present in the zones were recorded
at 6 hr interval till 24 hr.
2-4. Penetration
In 48 plastic micro-pots (2-5 x 2-5 cm) 5 g soil was filled and one seed of rice
was sown. Water was added for germination and when sprouts were 10 days
old, each was inoculated with 100 second stage juvenile of H. oryzicola in 4 repli-
cates. Inoculated plants were equilibrated at 28 ± 1° C and kept under light.
Every 2 hr after inoculation up to 24 hr, four sprouts were sampled for study
of juvenile penetration into roots.
2-5. Mating behaviour
Seeds of rice were germinated individually in pUri plates containing 1 % agar
media and when sprouts were 10 days old, each was inoculated with a single second
stage juvenile of H. oryzicola. When the mature females developed in the roots,
Life history of cyst nematode 285
10 males were released from a distance of 10 mm and the agar media was divided
into 10 zones of 1 mm diameter. " The number of males present in the zones was
recorded every 5 min after rel ase.
2*6. Orientation
The orientation of white females or brown cysts in roots of rice from post-
embryonic development study was recorded.
2-7. Fecundity
Sprouts of rice were grown as in post-embryonic development study and each
was inoculated with 100 second stage juveniles of PL oryzfcola. Sprouts were
sampled every 2 days from the 20th to the 30th day following inoculation for
enumeration of endo and semi-endoparasitic stages and egg masses. Soil was
processed for extraction of second stage juveniles (Whitehead and Hemming
1965).
3. Results and discussion
3-1. Embryonic development
Mature white females laying eggs in gelatinous egg mass were frequent. The eggs
when laid were unicellular. Two-celled stage was observed in 4 hr after oviposi-
tion and multicellular stage during the next 44 hr. Juvenile differentiation occurred
in 88 hr. Fully developed second stage juveniles, folded 3 or 4 times inside the
egg shell, were observed in 187 hr (figure 1). Eclosion occurred in 12 to 24 hr.
The entire developmental duration was found to be 8 to 9 days. The eggs in
egg mass were asynchronous and the second stage juveniles hatched on completion
of the development inside the egg shell, whereas the eggs retained in mature cysts
were synchronous and even after completion of development inside the egg they
remained dormant. Similarly, the embryonic development of H. oryzae in rice
from Ivory coast was reported to have been completed within 6-10 days (Brizuela
and Merny 1964).
3-2. Post-embryonic development
Juveniles of third stage (8) (table 1) first appeared in roots on the 6th day after
penetration indicating that the second stage juveniles had established, developed
and moulted (figure 2B). The third moult occurred on the 10th day in the
male (3) and on the 16th day in female juveniles (3). Third stage cuticle with
the coiled fourth stage juveniles of male (3) (figure 2C) were observed on the
10th day after inoculation. Fourth stage juveniles of female (3) (figure 2D, E)
appeared on the 14th day, while the adult males (3) moved out of the root on the
same and by the 22nd day, the females started laying eggs in gelatinous ovi sac
secreted around the vulva (figure 2F, G). The colour of the females (2) turned
light brown by 24th day and dark as it matured. Second stage juveniles emerged
put of the egg masses by 24th to 31st day (table 2).
286
A Jayaprakash and Y S Rao
4.lhr
20.4 hr
32.8hr
88.lhr 98.9hr
Figure 1. Embryonic development of Heterodera oryzicola.
The computed duration of development was 6 days for the second, 4 days for
the third stage male and 8 days for the third stage female juveniles, while the
fourth stage male and female juveniles require 4 and 8 days respectively to
develop into adults. The sex ratio between males and females was 1 : 4 (table 1).
Life history of cyst nematode
287
Table 1. Post-embryonic development of H. oryzicola in roots of rice. Inoculum
level 100 juveniles/seedling. (Average of 4 replicates).
Juveniles
Adults
Days after
A tj W*
inoculation 11
111 IV Male
Female
Female Male
White
Cyst
with egg
with egg
nnss
mass
2 26
26
4 31
..
31
6 27
8
..
35
8 11
20
31
10 4
21 .. 3
28
12
24 .. 6
. .
30
14
25 3 5 3
36
16
16 10 2 6
. .
34
18
6 21 1 8
. .
36
: ' 20
14 .. 8
10
32
22
2 .. 9
19(5)
30
24
4
26 (10)
2(2)
32
26
6
13(13)
17 (17)
36
28
5
6(6)
21 (21)
32
30
7
4(4)
27 (27)
38
Figures in parenthesis indicate egg masses.
Table 2. Number of generations of H. oryzicola from March 1977 to February
1978 in rice under greenhouse conditions.
Date of
inoculation
of
observation
Number of
cysts/pbnt
root System
D:Ao of
first juvenile
emergence
Gono^ation
1- 3-1977
1- 4-1977
1- 5-1977
1- 6-1977
1- 7-1977
1- 8-1977
1- 9-1977
1-10-1977
l-li-1977
1-42-1977
1- 1-1978
1-2-1978
22- 3-1977
24- 4-19^7
22- 5-1977
26- 6-1977
24- 7-1977
22- 8-1977
24- 9-1977
26-10-1977
28-11-1977
28-12-1977
26- 1-1978
24- 2-1978
15-7
14-1
24-4
28-3
16-6
13-7
22-9
21-2
20-4
12-8
15*9
8-9
24- 3-1977
25- 4-1977
29- 5-1977
24- 6r-1977
26- 7-1977
25- 8-1977
25- 9-1977
28-10-1977
29-11-1977
30-12-1977
31- 1-1978
27- 2-1978
1st
2nd
3rd
4th
5th
6th
.7th
8th
9th
10th
llth
12th
288 A Jayaprakash and Y S Rao
One life cycle of H. oryzicola was completed in 30 days from second stage
juveniles of one generation to succeeding second stage. The life cycle was conti-
nuous and 12 generations were completed in a year under greenhouse condi-
tions (table 2).
Males of H. oryzae developed in 14 days and females in 16 days (Brizuela
and Merny 1964), while H. vigni took 13 days and 17 days respectively in
roots of cowpea (Gupta and Edward 1973). H. graminophilam roots of barn-
yard grass (Birchfleld 1970) and H. zeae in roots of maize at 24-30 °C (Varma
and Yadav 1975) took 20 days for female development, while H. betulae took
52 days at 28 °C (Riggs etal 1969). H. avenae completed one life cycle in 9-14
weeks in roots of wheat (Duggan 1961). Thus, it seems that the duration of
post-embryonic development of Heterodera spp. varied with host plant and
environmental temperature.
H. oryzicola completed 12 generations in a year under greenhouse condi-
tions though H. trifolii and H. cajani were reported to complete 9 generations
in a year (Mulvey 1959 ; Koshy and Swarup 1971).
3-3. Migration of second stage juveniles towards rice roots
In 1 % agar media the second stage juveniles migrated from the point of release
towards the rice roots at 10 mm distance and above within 24 hr (figure 3).
In 6 hr only 10 juveniles were present at 0-2 mm distance from the point of
release, but by 12 hr 64% of the juveniles were present between 0-3 mm to
0-6 mm (2 to 15) ; by 18 hr 84% were present in between 0-5 mm to 0-8 mm
(6 to 15) and by 24 hr 13 juveniles actually reached the roots, while 20 juveniles
were close to the root system at 0-7 to 0-9 mm from the point of release. Hence,
there was a steady and stable attraction of the juveniles towards the host roots.
Similarly juveniles of H. schachtii, H. avenae and G. rostochiensis were also
reported to accumulate around host roots (Baunake 1922 ; Wallace 1958 ; Kuhn
1959 ; Vigtierchio 1961). Some bacteria from the .rhizosphere of sugar-beet
plants were reported to attract H. schachtii juveniles (Bergman and Van Duuren
1959). The present study on H. oryzicola as well as those with H. schachtii
and H. oryzae (Johnson and Viglierchio 1969 ; Reversat 1971) confirmed that
attraction occurs in sterile condition also and hence it may be suggested that the
stimulus for attraction emanated from the host roots.
3-4. Penetration
At constant temperature of 28 ± 1° C, the second stage juveniles of H. oryzicola
penetrated into the roots of rice (table 3). Within 2 hr following inoculation, a
few second stage juveniles (2) commenced penetrating into the epidermis and the
maximum number of juveniles penetrated by 6-10 hr (20-29). With the advance
in time, the juveniles reached the cortex in large numbers (18-35). Within 18 hr
a few juveniles (2) had penetrated the endodermis and pericycle, but by 24 hr
most of the juveniles reached to stele (27) (figure 2A). H. glycines and. H. vigni
also penetrated within 24 hr (Endo 1964; Gupta and Edward 1973), while
ff. zeae penetrated within 48 hr to 72 hr (Varma and Yadav 1975),
Life history of cyst nematode
289
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Life history of cyst mmatode
291
24hr
6 8
Distance travelled (in mm)
10
Figure 3. Migration of second stage juveniles towards rice roots.
Table 3. Penetration by H. oryzicola juveniles into rcots of rice. Age of plant at
inoculation = 10 days. Inoculum level = 100 Second stage juveniles'/Seedling.
Temperature = 28 ±1°C (Average of 4
Time in hr
Number of second stage juveniles' penetrated
Epidermis Cortex Endodf;rmis Total
and
Pericycle
2
2
2
4
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. .
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20
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20
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22
10
22
32
24
2
27
29
3-5. Mating behaviour
In 1 % agar media, the males migrated towards the virgin females of H. oryzicola
from the point of release at a distance of 10 mm within 25 min (figure 4). Most
of the males reached 3 to 6 mm distance from the point of release in 10 min
(1-4), by 15 min in 6 to 9 mm .(1-4) and by 20 min in 8 to 10 mm (1-7). After
25 min, . 8 males had actually reached the females indicating the existence of a
292
A Jayaprakash and Y S Rao
OJ
-Q
iai
5min
/\
10rnin
/ / V/
y* / 7°
25min
(920min
0 2 A 6 8
Distance travelled (in mm )
10
Figure 4. Migration of males towards virgin females of H. oryzicola.
strong male attractant force emanating from virgin females. Mating lasted for
an hour and more than one male took part in it. The gelatinous egg sac present
around the vulva was not observed to interfere in mating process. After the
mating the males moved away and did not respond further to the male attractants.
None of the females subjected to experimentation were left unmated. The secre-
tion of male attractants and migration of males towards female were so far
reported in 10 species of Heterodera and Globodera and most females secreted
more than one attractant and most males responded to more than one (Green
1966, 1967 ; Fox 1967 ; Greet et al 1968 ; Green and Plumb 1970). The male
attractants of G. rostochiensis and H. schachtii were also reported to spread or
diffuse in solution, volatalize in air and accumulate in agar blocks (Green 1967 ;
Greet etal 1968). In a 3mm thick agar block it took more than 15 min at
20° C to diffuse sufficiently to be perceptible to males 5 mm from the females
of G. rostochiensis and H. schachtii (Green 1966).
3 '6. Orientation
In this test, an average of 12-5 females of H. oryzicola in the roots were found
oriented towards hypocotyl, 14-5 towards the root tip and 3 matured perpendi-
cular to the long axis of root in secondary roots or small rootlets with their
head ends embedded inside the root (table 4). Almost all the females ruptured
the cortex to be exposed outside and the egg sacs remained completely outside the
root. Orientation of females of H. schachtii towards hypocotyl and root tip was
equal and some juveniles matured at the root surface of sugar-beet to a varying
degree ranging from completely endoparasitic to nearly completely ectoparasitic.
The juveniles maturing at the root surfaces invariably developed into males
(Steele 1977).
3-7. Fecundity
The mature white females and brown cysts with or without egg sac formed in
root system of 6 rice plants during the 30 days following inoculation with 100
second stage juveniles of H. oryzicola varied from 4 to 31 (table 5). The cumulative
Life history of cyst nematode 293
Table 4. Orientation of H. oryzicola females in roots of rice. Age of Seedling
at inoculation = 10 days. Inoculum level = 100 second stage juveniles/seedling
(Average of 6, replicates).
Mean number of cysts/white females oriented towards
Hypocotyl Root tip Vertically in
secondary roots
12-5 14-5 3-0
Table 5. Oviposltion of H. oryzicola in rice. Inoculum level « 100 second
stage juveniles/seedling.
Days after inoculation
Inoculation 22 24
•KTn
26
28
30
.txo.
WIG
E W/C E
W/C E
WIG E
wic E
1 12
20 8
(8)
21 15
(120)
29 21
(1535)
28 28
(1085)
2 15
15 4
(10)
23 17
(245)
26 22
(1815)
27 27
(957)
3 4
16 8
20 7
(88)
26 26
(2318)
21 21
(1578)
4 12
20 12
(15)
25 18
(214)
25 23
(2275)
'22 22
(3105)
5 11
18 9
26 12
(136)
21 20
(3408)
28 26
(1789)
6 8
21 5
22 19
(286)
27 22
(2557)
31 31
(2035^
Total 62
.. 110 46
(33)
137 88
(1089)
154 134
(13908)
157 155
(10549)
Average No. of
eggs/female 0
38 27
112 98,
,115 195
120 198
Average No. of
egg masses/day 0
0-084
0-044
0-046
0-.024
W = White females or C = Cysts; E = Egg masses.
Figures in parenthesis are hatched second stage juveniles.
294 A Jayaprakash and Y S Rao
number of egg masses (hatched and unhatched) laid per female increased from 33
on 24th day to 155 on the 30th day indicating that all the egg masses were formed
within this period. The average daily opposition rate was 0-084 on 24th day
and after which there was a gradual decline to 0-024 on the 30th day. At 30 days,
the average number of eggs laid in egg mass was 198 and retained in cysts was
120 per single female. The cysts of H. avenae were found to retain over
600 eggs (Anderson 1961) and H. schachtii retained from 10 to over 600
with an average of 286 from 500 Utah specimens. The eggs laid in egg sac of
JET. schachtii, H. glycines, H. trifolii, H. cruciferae and H. caro tae were reported
to be as many as 200, in H. fid about 100 and in H. goettingiana, H. avenae
and some large specimens of H. galeopsidis laid quite a few eggs only
(Thorne 1961).
Acknowledgements
The authors thank Dr H K Pande, for facilities and for a Scholarship to AJ.
The help rendered by Dr J S Prasad is gratefully acknowledged.
References
Andersen S 1961 Resistens mod havreal Heterodera avenae ; Medd. Vet. Hisk. Afd. landbr.
Plkult. Nr. 68 179
Baunacke N 1922 Untersuchungen zur Bialogie und Bekamfung des Rubennematoden, Heterodera
schachtii Schmidt ; Arb. Biol. Reichanst. Lartdu. Fostw. 11 185-288
Bergman B H H and Van Duuren A J 1959 Sugar-beet eelworm and its control. VI. The
Influence of host plant roots and their secretion products on the orientation of Heterodera
schachtii larvae in vitro ; Meded. Inst. Suikerbeit 29 1-24
Birchfteld W 1970 The biology of a new cyst nematode on grasses (Abstract) ; Phytopathology
60 1284-1285
Brizuela R B and Merny G 1964 Biologic tf Heterodera oryzae Luc and Berden, 1961 I. Cycle
du parasite et reactions histologiques de l*hate ; Rev. Path. Veg. Entomol. Agric. 43 43-53
Dasgupta D R ar?d Raski D J 1968 The biology of Rotylenchulus parvus ; Nematologica 14
429-440
Duggan J J 1961 Seasonal variations in the activity of cereal root eelworm (Heterodera major
O. Schmidt, 1930) ; Sd. Proc. R. Dublin Soc. Ser. Bl 21-24
Endo B Y 1964 Penetration and development of Heterodera glycinesin soybean roots and related
anatomical changes ; Phytopathology 54 79-88
Fox J A 1967 Reproductive isolation in the genus Heterodera ; Nematologica 13 143-144
Green C D 1966 Orientation of male Heterodera rostochiensis Woll. and H. schachtii Schm.
to their females ; Ann. Appl. Biol. 58 327-339
Green C D 1967 The attraction of male cyst nematodes by their females ; Nematologica 13
172-173
Green C D and Plumb S C 1970 The interrelationships of some Heterodera spp. indicated by
the specificity of the male attractants emitted by their females ; Nematologica 16 39-46
Greet D N, Green C D and Poulton M E 1968 Extraction, standardization and assessment
of the volatility of the sex attractants of Heterodera rostochiensis Woll. and H. schachtii
Schm. ; Ann. Appl. Biol. 61 511-519
Gupta P and Edward J C 1973 Studies on the biology of Heterodera vigni (Heteroderidae :
Nematoda) I. Life cycle ; Indian J. Nematol. 3 99-108
Johnson R N and Viglierchio D R 1961 The accumulation of plant parasitic nematode larvae
around carbon dioxide and oxygen ; Proc. Helmnthol. Soc. Wash. 28 171-174
Johnson R N and Viglierchio D R 1969 Sugar beet nematode (Heterodera schachtii) reared on
axenic Beta vulgaris root explants I. Selected environmental factors affecting penetration ;
Nematologica 15 129-143
Life history of cyst nematode 295
Koshy P K and Swarup G 1971 On the number of generations of Heterodera cajani, the
pigeon-pea cyst nematode in a year ; Indian J. Nematol. 1 88-90
Kuhn H 1959 Zum problem der wirksfindung phytopathogene nematodan ; Nematologica
4 165-171
Mulvey R H 1959 Susceptibilities of plants to. clover cyst nematode, Heterodera trifolii and
the period required to complete a life cycle ; Nematologica 4 132-135
Rao Y S and Jayaprakash A 1977 Leaf chlorosis in rice due to root infestation by a new
cyst nematode ; Ir.t. Rice Res. News Lett. 2 5
Rao Y S and Jayaprakash A 1978 Heterodera oryzicola n.sp. (Nematoda : Heteroderidae) a
cyst nematode on rice (Oryza sativa L.) from Kerala State, India ; Nematologica 24
341-346
Reversat G 1971 Contribution a? etude dela Biologie d'un nematode phytoparasite, Heterodera
oryzae ; Ph.D. Thesis (Univ. Claude-Bernard de Lyon)
Riggs R D, Hirschmann H and Hamblen M L 1969 Life cycle, host range and reproduction
of Heterodera betulae ; /. Nematol. 1 180-183
Steele A E 1971 Orientation and development of Heterodera schachtii larvae on tomato and
sugar-beet roots ; /. Nematol. 3 424-425
Thome G 1961 Principles of Nematology (New York, Toronto and London : McGraw-Hill)
Varma A C artd Yadav B S 1975 Life history of Heterodera zeae on maize under Udaipur
conditions ; Indian J. Mycol. PI. Path. 5 19
Viglierchio D R 1961 Attraction of parasitic nematodes by plant root emanations ; Phyto-
pathology 51 136-143
Wallace H R 1958 Observations on the emergence from cysts and the orientation of larvae
of three species of the genus Heterodera in the presence of host plant roots ; Nematologica
3 236-243
Whitehead A G and Hemming J R 1965 A comparison of some quantitative methods of extracting
small vermiform oematodes from soil ; Ann. Appl. BioL 55 25-38
Proc. Indian Acad. Sci. (Arum. Sci.)» Vol. 91, Number 3, May 1982, pp.
© Printed in India.
Sediment-ostracode relationship in the Bimili backwater and the
Balacheruvu tidal stream
C ANNAPURNA and D V RAMA SARMA
Zoology Department, Andhra University, Waltair 530 003, India
MS received 11 May 1981
Abstract. Based on the collections of benthic ostracodes during January-December
• • • • 1977 from two selected marginal water bodies, namely Bimili backwater and Bala-
cheruvu tidal stream on the east coast of India, the quantitative variations in the
ostracode fauna have been studied in relation to the sedimentological characteristics
like sand, silt and clay and organic matter content.
Keywords. Marginal water bodies ; sedimentological characteristics ; organic matter
in sediment ; ostracode assemblages.
1* Introduction
Studies on sediment-ostracode relationship are rare and whatever is available are
mainly concerned with the distribution of dead fauna. Moreover, information
on the distribution, sedimentological and ecological relationship of living benthic
ostracodes has been published either in Uxonomic papers or in publications
principally concerned with the ecology of other groups. The studies of Remane
(1933), Klie (1936), Elofson (1941), Smidt (1951), Wieser (1959, 1960), Kornicker
(1964) Kornicker and Wise (1960), Puri etal (1964), Mclntyre (1964), Engel
and Swain (1967), Williams (1969), King and Kornicker (1970), Joy and Clark
(1977) and Athersuch (1979) have shown that the nature of the substratum and
organic matter content play a vital role in controlling the biota in the habitat.
Malkin (1954) and Swain (1955) did not find any pronounced correlation
between the distribution of ostracodes and character of the substratum. Kornicker
fl958) found that the correlation was disappointing in the Bimili area, Great
Bahama Bank, while Benson (1959) found that sediment had a marked influence
on some of biofacies in Estero de Puncta Banda.
In the present investigation an attempt has been made to establish a possible
relationship between the ostracode fauna and the sediments in two selected bodies
of water, Bimili backwater and Balacheruvu tidal stream.
2. Areas of investigation
Bimili backwater: The area covered is an exteBsiveshaUow backwater about
4-5 sq. km towards the north of Bheemumpatnam (Long. 83 28 E, Lat. u
297
P.(B>-41
5,98 C Annapurna and J9 V &ama
54' N). Three nearly equidistant stations (I to III) are located for collection
of samples (figure 1).
Balachemvu tidal stream : This meandering stream opens into Bay of Bengal
15 km (by coast line) south of Visakhapatnam (Long. 83° 15' E; Lat. 17° 39' N).
Three stations (I to III) are located in the course of the stream for the collection
of samples (figure 2>.
3. Material and methods
Collections were made at monthly intervals for one year (JaMaty-December 1977)
at six fixed stations, three in the Bimili backwater and three in the B^cheruvu tidal
stream. For quantification of ostracodes, collections were made using a device
developed by Phleger (1960) and the density of ostracode fauna was expressed
as number per 10 cm2.
To study sediment composition and its organic matter, sediment was collected
by pushing a PVC corer of 4- 5 cm diameter. Sand, silt ^nd clay fractions in
the sediment were estimated by the pipette method of Krunibein an$ Pettijohn
(1938). Organic matter was estimated by the method of Gaudette etal (1974).
4. Results
Seasonal variations in fauna in relation to sedimentological parameters are
shown in figures 3 and 4. In the Bimili backwater, the organic matter CQiitent
ranged from 0-32 to 4-12%. In general, higher values were recorded in July
which marks the end of hot weather season and the establishment of the south-
west monsoon season when drainage from the land was high. In addition, the
contribution of organic matter by the decaying algae which grows densely on
the western margin of the backwater is significantly high. .
In, the Balachemvu stream, the organic matter content ranged from 0-34 to
3-56%. Higher values of organic njatter were observed at station II compared
to the values at stations I and III.
Sediment analyses show that saud was dominant over the silt and clay fractions
at all the stations in the Bimili backwater and the Balachemvu stream. Hence
sediments of Bimili and Balachemvu may be categorised as sandy areas following
the categorisation of Folk (1968).
At station I in the Bimili backwater ostracodes were present in greater numbers
from March through September than during the remainder of the year. Except
for a peak in May, the abundance of ostracodes at station II did not vary
markedly during the year. At station III ostracodes were encountered in consi-
derable numbers in January, ani from May to My.
In the Balachemvu tidal stream ostracodes were encountered in considerable
numbers in January, February and December collections. At station 1^ ostra-
codes were present from August to December in higher numbers qompared to
other months. Except for a peak number in January, the abundance of ostra-
codes at station III did not vary markedly during the year.
At stations L and II of Bimili a^d Balachemvu the ma^im^m in the seasonal
abundance of live ostracodes coincided with the highest organic matter content.
Slight deviation from this trend was seen at station HI in both areas.
Sediment-ostracode relationship
299
JILL
S3'
GOUSTHAN
NATURAL SCALE:1:18,156
27' E
83*
2BE
Figure 1. Location map of Bimili backwater.
10' n' 12' 13* u' . t5* - is' IT' *•
Figure 2. Location m^p of Balacheruvu tidal stream^
300
C Annapurna snd D V Rama Sarma
39nN30«3«<
Sediment-ostracode relationship
301
X U3UVM 3IMV9HO
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nssisnzs*
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302 C Annapufna and D V Rama Sarma
The sand, silt and clay fractions at the six stations during different months,
when viewed in the background of total numbers of ostracodes, clearly indicate
that ostracode abundance increased as the sand and clay content increased and
silt content decreased. At station I in the Balacheruvu stream, the sand content
was below 60% and silt above 10% in April and a fall in the ostracode numbers
coincides.
Relatively higher numbers of ostracodes were encountered at station III of
Bimili and station II of Balacheruvu, compared to the other stations. It is inte-
resting to note that sediments at the above stations hold a, higher sand and clay
fraction and relatively high percentage of organic matter content.
5. Discussion
Throughout the survey conducted in Balacheruvu and Bimili backwater,
samples contained faecal pellets in large quantities which the ostracodes seem to
nibble indicating that the pellets form a sizable source of food. The fact that
faecal pellets serve as the food source for the ostracode fauna is well established
(King and Kornicker 1970).
The ostracode abundance in the areas of study increased with the availability
of organic matter. The ostracode abundance varying with availability of food
was observed by Swain (1955), Engel and Swain (1967) and Joy and Clark (1977).
A close examination of the pattern of distribution of ostracodes in relation to
the sediment composition reveals that ostracodes prefer areas high in sand and
clay fraction rather than silty areas. Thus Balacheruvu and Bimili sustain ostra-
codes in considerable numbers* This observation agrees with those made else-
where in similar localities by Klie (1936), Elofson (1941), Smidt (1951), Benson
(1959), Wieser (1959, 1960), Mclntyre (1964) and Williams (1969).
High density of ostracodes observed in the shallow backwater and the tida
stream is due to the high rate of photosynthesis of diatoms in the sediments.
This observation agrees with those made elsewhere in similar localities by
Hagermann (1967).
The stability structure of the sediment exerts a strong influence on the marine
ostracodes in the selection of a suitable substratum. While the smooth-shelled
forms prefer fine-grained muds, the rough and more ornate ostracodes prefer
coarse or calcareous sediments. Such terms like endopelose (silt and clay
burrowers), epipelose (silt and clay wanderers) and epipsammon (sand surface
crawlers) have been suggested by Remane (1933) and Elofson (1941) for ostra-
code assemblages typical of certain bottom sediments which emphasize the control
of the substrate over the character of associated assemblages.
In the present study smooth-shelled forms like Phlyctenophora occurred in
sand-dominated areas but not in muddy areas. Forms such as Tanella, Loxoconcha,
Paijenborchellina and Atjehella which are sculptured and heavily ornamented were
encountered in considerable abundance in the sandy areas. Palmenella which
is the characteristic genus of station III of Bimili backwater is known to be
epipelitic (Remane 1938).
The foregoing account suggests that substratum plays a major role in the distri-
bution of ostracodes both qualitatively and quantitatively. Regions of sandy
sediments containing high percentage of organic matter content were more densely
populated.
Sediment-ostracode relationship 303
Acknowledgements
The authors thank the Head of the Department for facilities. One of us (CA)
is grateful to the University Grants Commission, New Delhi, for a fellowship-
References
Athersuch J 1979 The ecology and distribution of the ostracodes of Cyprus ; /. Nat. Hist.
13 135-160
Benson R H 1959 Ecology of recent ostracodes of the Todos Santos Bay region, Baja Cali-
fornia, Mexico, Univ. Kansas. Paleont. Contrib. Arthropoda, art p. 1-80
Elofson O 1941 Zur kenntnis der mariner Ostracoden Schwedens, mit besonderer Berucksichti-
gung des Skagerracks ; Uppsala Univ. Zool Bidr. 19 215-534
Engel P L and Swain F M 1967 Environmental relationships of Recent Ostracoda in Mesquite,
Aransas and Copano Bays, Texas Gulf Coast ; Trans. Gulf Coast. Assoc. Geol. Soc. 17
408-427
Folk R L 1968 Petrology of sedimentary rocks (Texas : Hemphill's Austin) p. 170
Gaudette H E, Wilson R F, Lois Toner and David W Folger 1974 An inexpensive titration
method for the determination of organic carbon in Recent sediments ; /. Sed. Petrol. 44
249-253
Hagermann L 1967 Ostracodes of the Tvarminne area, Gulf of Finland; Commentat. Biol. 30
1-12
Joy J A and Clark L D 1977 The distribution, ecology and systematics of the benthic Ostracoda
of Central Arctic Ocean ; Micropaleontology 23 129-154
King C E and Kornicker L S 1970 Ostracoda in Texas Bays and Lagoons : An ecologic study ;
Smith. Contr. Zool 24 92
Klie W 1936 Ostracoden der Familie Cytheridae aus sand und Schell von Helgoland ; Kieler
Meeresforsch 1 49-72
Kornicker L S 1958 Ecology and taxonomy of Recent marine ostracodes in the Bimini area,
Great Bahama Bank, Texas ; Texas Univ. Inst. Mar. Sci. Publ. 5 194-300
Kornicker L S 1964 A seasonal study of living Ostracoda in Texas Bay (Redfish Bay) adjoining
the Gulf of Mexico ; Publ. Staz. Zool. Napoli 33 Suppl 45-60
Kornicker L S and Wise C D 1960 Some environmental boundaries of a marine ostracode ;
Micropaleontology 6 393-393
Krumbein W C and Pettijohn F J 1938 Manual of sedimentary petrography (New York J
Appleton-Century Crofts Inc.) p. 549
Malkin D S 1954 Biostratigraphic study of Miocene Ostracoda of New Jersey, Maryland and
Virginia ; J. Palaeontol. 27 761-799
Mclntyre A D 1964 Meiobenthos of sublittoral muds ; /. Mar. Biol. Ass. U.K. 44 665-674
Phleger F B 1960 Ecology and distribution of Recent Foraminifera (Baltimore : The John's Hopkins
Press) p. 297
Puri H S, Bonaduce G and Malloy J 1964 Ecology of the Gulf of Naples; Publ. Staz. Zool.
Napoli 33 Suppl. 87-199
Remane A 1933 Verteilung und organisation der benthonischen Mikrofauna der Kieler Bucht ;
Wiss. Meeresunters. Kiel. 21 161-221
Smidt E L B 1951 Animal production in the Danish Wadden sea ; Medd. Komn. Dann.
Fiskeri-og. Havunders 11 p. 151
Swain F M 1955 Ostracodes of San Antonio Bay, Texas ; /. Paleontol. 29 561-646
Wieser W 1959 The effect of grain size on the distribution of small invertebrates inhabiting the
beaches of the Puget Sound ; Limnol. Oceanogr. 4 1&1-194
Wieser W 1960 Benthic studies in Buzzards Bay n. The meiofauna ; Limnol. Oceanogr. 5 121-
137
Williams R 1969 Ecology of the ostracoda from selected marine intertidal localities on the
coast of Angelsey. In Taxonomy, morphology and ecology of Recent Ostracoda (cd.) J Neal
(Edinburgh : Oliver and Boyd) 229-327
>roc. Indian Acad. Sci. (Anirn. Sci), Vol. 91, Number 3, May 1982, pp. 305-315.
ID Printed in India.
Effect of DDT on brain neisrosecretory cells of adult
Poekilocems pictus (Ortfaoptera : Acrididae)
OM PRASAD and V K SR1VASTAVA
Department of Zoology, Allahabad University, Allahabad 211002, India
MS received 15 December 1980 ; revised 20 April 1982
Abstract. Neurosecrctory cells occur in groups, medially, dorsally, dorsolatcrally,
laterally and mtdventrally, in the protocerebrum and tritoccrcbium of adult
Poekilocems pictus. Mid- brain is devoid of such cells. On the basis of staining
reactions the NS cells have been differentiated into A and B types. The median
group consists of about 50-55 A and 30 B cells lying on either side of the mid-
line in the parsintercerebralis. The other parts of the protocerebium and irito-
ccrebrum are filled with only B cells. Scant NSM is found in the NS ceils of
freshly moulted adult. Synthetic activity increases with age and after about 5 or
6 days the cells contain deep staining secretory vesicles.
Treatment of 1-6 day old P. pictus with DDT for different periods shewed that
short incubation of 24 hr triggers the synthetic activity of NS ceils, but
prolonged incubation of 72 hr leads to a total depletion of NSM and to disruptive
changes, like undulation of cell wall, cell shrinkage and ultimate cellular dismption.
Keywords. Poekilocems pictus ; effect of DDT ; neurosecretory cells.
1. Introduction
Extensive literature is available on the morpholoy and histology of the neuro-
secretory system, but few workers have studied the changes in these cells induced
under chemical stress (Matsuzawa 1964 ; Masner et al 1970 ; Ghosh et al
1968 • Nanda 1970 ; 1973, 1974 ; Voitkevitch and Leonova 1964).
While studying the effect of tranquillizers at the level of brain nuclcoprotem
in Periplaneta americana, Ghosh et al (1968) reported a patchy condition of
cytoplasm, vacuolation in cell perikarya and undulation of cellular membrane.
Nanda (1973) reported various grades of depletion such as marginal dep etion
and accumulation of neurosecretory material in the neurosecretory cells of
insecticides are known to inteifere with many phyriolo^l
305
306 Cm Prasad and V K Srivastavd
much is known about changes in cytoarchitecture or in the activity of NS cell
especially in relation to the period of exposure to the insecticide and age of the^
insect The present investigation deals with cytomorphological changes caused
by DDT in the neurosecretory cells of brain of adult P. pictus.
2. Materials and methods
Adult males and females P. pictus of known age were used from stock reared in
the laboratory on Aak (Calotropis) leaves, in cages at a temperature of 28 dr 2° C.
A solution of synthetic DDT (0-01%) was obtained by dissolving a concentrated
emulsion (25 B.C.) in acetone, and 0-01 ml of the solution was applied topically
to the body surface just near the wing bases with a microapplicator. Controls
were applied with the solvent acetone alone. For each experimental and control
groups, 16 grasshoppers were used. After 24, 48 and 72 hr of incubation the
grasshoppers were dissected in insects Ringer's solution and their brain fixed in
aqueous Bouin's fluid. Paraffin sections (6 /mi) were cut and stained with Gomori's
chromealum haematoxylin phloxine (CHP), paraldehyde fuchsin (PAF) (Ewen
1962) and Heidenhain's azan stain.
3. Results
Neurosecretory cells occur throughout the protocerebrura. and tritocerebrum in
different locations in the brain of P. pictus with the majority lying in the proto-
cerebral lobes. On the basis of their staining reactions the cells have been
classified into two types, A and B. There are two median groups of about
50-55 A type NS cells in the parsintercerebralis. A cells stain purple with PAF,
dark red with Azan and dark blue with CHP. They measure 0-017 x 0-007 mm,
and their nuclei 0-005 mm in diameter. Occurring in the same group 30 B type
cells are comparatively larger and stain green with PAF, light red with Azan
and red with CHP. They measure 0-06 x 0-02 mm, and their nuclei 0-01 mm
in diameter. Small patches of B cells are also present dorsally, ventrally and
midventraliy in protocerebrutn and midventrally in the tritocerebrum. The mid-
brain is completely devoid of NS cells.
Very little neurosecretory material (NSM) is found in both A and B cells of
freshly moulted adult (figure 1). Gradually the synthetic activity increases and by
the time the grasshoppers mature in 5-6 days, the NS cells exhibit the peak of
synthesis with deeply staining NSM in their perikarya (figure 6).
Neurosecretory cells of one day-old adult P. pictus treated with 0-01% DDT
solution and incubated for 24 hr showed greater synthetic activity. Large quanti-
ties of NSM accumulated in the perikarya along with its simultaneous release.
The release of NSM through the axons was unmistakable, as also slight undulations
of the cell wall (figure 2).
On prolonging the incubation period to 48 hr release of NSM became fester,
leaving small amounts in the perinuclear region. Prolonged incubation also
damaged the body of the NS cells which became polygonal or irregular in shape,
apparently by the contraction of the cell wall and its infolding (figure 3). Disrup-
tion of cell wall at places was also observed (figure 4). Or further prolonging the
DDT and neurosecretion in P. pictus
307
Fig ires 1-2. See page 309 for captions, For abbreviations see page 315.
308
Om Prasad and V K Srivastava
Figures 3-4. See page 309 for captions. For abbreviations see page 315.
£>DT and neuro-secretion in P. plot us
309
Figures 1-6. Po&kilocerus pictus. 1. 3-day old adult showing normal neuro-
secretory cells. 2-5. 1-day old adult treated with 0-01% DDT for 2. 24 hr.
3 4. 48 hr. 5. 72 hr. 6. Under control condition showing peak of synthetic
activity. For abbreviations see page 315.
310
Om Prasad and V K Snvastava
Figures 7-8. See page 312 for captions. For abbreviations see page 315.
DDT and neuro secretion in P. pictus
311
Figures 9-10. See page 312 for captions. For abbreviations see page 315.
Urn /V<M</«/ itnJ J
7 II. /W^fMrmt /**»/«* 7 It *vts*i> ,u
0-01", DDT for 7, $.. 24 in <>. 41 !» Ill, ! ii4
I or ahbrcvii»tii»«^ sec
1$
DDT and neurosecretion in P. pictus 313
incubation period to 72 hr the NS cells became hyperactive in release, draining
off the NSM and becoming vacuolated (figure 5).
Six-day old P. pictus treated with 0-01 % DDT and incubated for 24 hr showed
release of NSM from NS cells. Continuous release of NSM from the cell perikarya
imparted the later a foamy appearance (figure 7). At this stage they stained feebly
with the cell wall showing folds and the cell becoming triangular or polygonal
in shape (figure 8). When the incubation period was prolonged to 48 hr, the
discharge of NSM further increased and extensive damage to the NS cells was
noticed in the form of undulation of cell wall and shrinkage of cell perikarya
(figure 9). After 72 hr of incubation no NSM was noticed in the perikarya but
some could be seen at the axonal endings (figure 11),
4. Discussion
Gundevia (1972) studied the effect of insecticides on the NS cells of insects and
reported that short incubation periods of Dimecron, Diazinon and Dieldrex trig-
gered synthetic activity of NS cells in Hydrophilus olivaceous Fabr. Sabesan and
RamaJingam (1979) also observed increased synthetic activity in the median
neurosecretory cells of endosulphon-treated Odontopus varicornis at the initial
stage of poisoning, resulting in an accumulation of secretory material in the cell
perikarya. In our studies in P. pictus DDT acted in the same way. The initia-
tion of synthesis, gradual acceleration in the pace of synthetic activity and accumu-
lation of secretory material etc., was probably an initial response to the emergency
caused by the action of insecticides. Prolonged incubation, however, resulted
in the discharge of secretory material. It thus seems logical that insecticidal action,
up to a certain level, stimulated the synthesis and storage of secretory products
but later it affected a releasing stimulus. The prolonged incubation also seems
to have an inhibitory effect on synthesis and accumulation leading to large scale
depletion of cellular contents and vacuolation, etc.
According to Wilcoxon and Haitzell (1933), Hartzell (1934), Richard and Cut-
komp (1945) and Roche and Lhoste (1958) the motor neurones in general undergo
vacuolation due to the action of insecticides. Gundevia (1972), Nanda (1974) and
Sabesan and Ramalingam (1979) also made similar observations in H. olivaceous,
P. americana and O. varicornis respectively. As neurosecretory cells are modi-
fied motor neurones, the vacuolation caused by the insecticides can be well
compared to the effect observed by the above mentioned workers.
It is quite obvious from our observations that DDT produced histologically
recognisable degenerative changes in the NS cells. They were in the form of
undulations in the periphery of cell wall, loss in compactness, change in cell shape
and sometimes even the disruption of cell wall. Similar changes have been
noted in the nerve cells of insecticide treated insects by a number of workers.
Wilcoxon and Hartzell (1933) and Hartzell (1934) observed trigrolysis of Nissle
granules and tissue disintegration in the brain nerve cells of Tenebrio molitor and
Melanoplus femur rubrwn after Pyrethrum and Pyrethrin treatment. Chang
(1951) showed destruction of Golgi bodies and their almost complete disappearance
at the time of death in the neurones of DDT-treated P. americana and Apis melli-
fera. Brown (1963) also noted some Abnormalities in the central nervous system
314 Om Prasad and V K Srivastava
of P. americana after treatment with Heptachlore. Various grades of distur-
bances in the compactness of NS elements and undulations in the periphery of
cell wall after insecticidal treatment were reported by Gundevia (1972) and Nanda
(1974) also. Loss in compactness, undulations towards the periphery of cell
wall, tendency of the cells to become polygonal or irregular and the disruption of
cell wall in the present investigation could be due to vigorous release of secretory
material and loss of cohesion amongst the NS cells. The studies of Wilcoxon
and Hartzell (1933), Hartzell (1934) and Chang (1951) revealed that the Golgi
elements of the secretory cells became affected due to the action of insecticide.
It is well-known that the Golgi bodies play a major role in the cellular secretion.
Thus it can be assumed that with shorter incubation periods the Golgi bodies of
secretory cells become activated and with prolonged action of the insecticide they
became exhausted leading to depletion and vacuolation in the cell perikarya.
Regarding the nature of the secretory product thus released Sternberg (1963)
reported that excessive stress on NS cells leads to the discharge of pharmaco-
logically active substances. It has been shown recently that insecticidal treatment
causes release of various neurohormones in insects. Maddrell and Casida (1971)
treated Rhodnius prolixus with 42 different insecticides, of which 18 caused paralysis
and release of the diuretic factor at that time. Maddrell and Reynold (1972)
reported that paralytic dose of insecticides caused the release of plasticizing
hormone also. Granett and Leeling (1972) on the other hand showed the appear-
ance of hyperglycaemic agent in haemolymph, causing the trehalose content to
increase in DDT-treated P. americana. More recently Samaranayaka (1974)
reported the release of adipokinetic and hyperglycaemic hormone also in insecti-
cide-treated Schistocerca gregaria.
Taking these cases as typical ones, it seems likely that it may be a general way
of the action of insecticides in which they provoked a more or less simultaneous
release of several, possibly all of the insect neurohormones. In this way it seems
probable that such a widespread and unbalanced release of neurohormones, the
controlling factors, causes serious damages to the insects and is also responsible
for the lethal effect of insecticides.
Acknowledgements
The authors are thankful to Prof. U S Srivastava, Head, Department of Zoology,
for providing laboratory facilities and to Council of Scientific and Industrial
Research, New Delhi, for awarding a fellowship to VKS.
References
Brown A W A 1963 Chemical injuries in Insect pathology (ed.) E A Steinhaus (New York and
London : Academic Press) pp. 65-131
Chang P I 1951 The action of DDT on the Golgi bodies of insect nervous tissue ; Ann. Ent
Soc. Am. 44 311-326
Ewen A B 1962 Histophysiology of the neurosecrftorysystem and reterocerebral endocrine
glands of the alfalfa plant bug, Adolophocoris lineolatus (Geoze) ; /. Morphol. Ill 255-269
Ghosh J J, Ghosh S, Chanda S, Sikdar K and Bhaduri S 1968 Action of tranquillizer drugs
at the level of brain nucleoprotein ; Sci. Cult. 34 62
Granett J and Leeling N C 1972 A hyperglycaemic agent in the serum of DDT-prostrate
American cockroach PeripJaneta americana ; Ann. EntomoL Soc. Am. 65 299-302
DDT and neurasearetion in P. pictus 315
Gundevia H S 1972 Hormonal studies of insect reproduction : Histological, histochemical and
neuroendocrinological studies in relation to reproduction in Hydrophilus olivaceous Fsbr.
(Hydrophilidae— -Polyphaga, Coleoptera), Ph.D. thesis, Banaras Hindu University
Hartzell A 1934 Histopathology of insect nerve lesion caused by insecticides ; Contrib. Boyce
Thompson Inst. 6 211-223
Maddrell S H P and Casida J E 1971 Mechanism of insecticide induced diuresis in Rhodnius ;
Nature (London) 231 55-56
Maddrell S H P and Reynold S E 1972 Release of hormones in insects after poisoning with
insecticides ; Nature (London) 236 404-406
Masner P, Hoot L, Corrivault G W and Prudhomme J C 1970 Effect of reserpine on the
function of gonad and its neuroendocrine regulation in tenebrionid-beetle ; /. Insect.
PhysioL 16 2327-2344
Matsuzawa T 1964 Quantitative chemical and pathological studies on changes of hypo-
physioadrenal system in response to formalin stress ; in Gunma symposia of endocrinology
Vol I 183-189
Nanda D K 1970 Effect of Diptrex administrator on the cephalic neuroglandular element of
Periplaneta americana ; Indian J. PhysioL Allied Sci. 24 69-19
Nanda D K 1973 Effect of some neuropharmacological drugs on the supraeesophageal neuro-
secretory cells of Periplaneta americana ; Folia BioL 21 329-338
Nanda D K 1974 Impact of insecticides on the brain neuroglandular elements of Periplaneta
americana ; Die Naturwissertschqfteit 61 451-452
Prasad Om and Srivastava V K 1980 Effect of BHCon brain neurosecretory cells of Poekilocerus
pictus (Orthoptera : Acrididae) ; /. Mikrosk Anat. Forsch. Leipzig 2, s. 250-256
Ramade F 1967 Contribution a 1'etude du mode d'action de certains insecticides du synthese
plus particulierement du lindanc et des phenomenon de resistance a ces composes chez
Musca domestica ; Ann. Inst. Nation. Agron. Paris 5 11-228
Richard A G and Cutkomp H 1945 Neuropathology in insects ; J.N.Y. Entom. Soc. 53 313-
353
Roche A and Lhoste J 1958 Action de quelques insecticides sur les corps de Nissl dcs ganglions
thoraciques de Drosophila melanogaster Meig. et de Blaiella gennanica L. ; Bull. Soc.
Entomol. France 63 181-184
Sabesan S and Ramalingam N 1979 Effects of endosulphon on the medial neurosecretory
cells of adult male Odontopus varicornis (Pyrrhocoridae : Hetcroptera) ; Entomon. 4
223-228
Samaranayaka A M 1974 Insecticide induced release of hyperglycaemic and adipokinetic hormone
of Schistocerca gregaria Gen. Comp. Endocrinol. 24 424
Sternburg J 1963 Autointoxication and some stress phenomena ; Annu. Rev. Entomol. 8 19-35
Voitkevitch A A and Leonova L K 1964 Effect of vertebrate hormones on the neurosecretory
system of an insect ; Dokl (Proc.) Acad. Sci. U.S.S.R. 157 526-536
Wilcoxon F and Hartzell A 1 933 Some factors affecting the efficiency of contact insecticides.
III. Further chemical and toxicological studies of pyrethrum ; Contrib. Boyce Thompson
Inst. 5 115-127
Abbreviations : NC, neurosecretory cell ; MNC, median neurosecretory cell ;
DNC, damaged neurosecretory cell ; VNC, vacuolated neurosecretory cell ; NSM
neurosecretory material ; Va, vacuole ; Ax, axon.
£roc. tadiaa Acad. Sci. (Aaim Sci.), Vol. $1, lumber 3, May 1082,
© Printed in India.
Rhythmic oscillations in non-aggressive social behaviour in
Bandicota bengalensis
SHAKUNTHALA SRIDHARA and R V KRISKNAMOORTHY
Departments of Vertebrate Biology and Zoology, University of Agricultural Sciences,
GKVK Campus, Bangalore 560065, India
MS received 10 February 19S2 ; revised 6 May 1982
Abstract. Non-aggressive social behaviour rhythms of Bandicota bengalensis were
studied in the laboratory. The species exhibited 95% rhythmicity for social beha-
viour but the rhythms were uni- or bimodal and were influenced by the number and
sex of interacting con&pecifics. Peaks occurred at 0900 and 1800 hr. Males
were more socially active than females.
Keywords. Bandicota bengalensis ; social behaviour ; rhythms ; unimodal ; bimodal.
1. Introduction
The lesser bandicoot rat, Bandicota bengalensis, is a widely distributed pest both in
agricultural fields as well as in warehouses in India, Nepal, Burma, Thailand,
Sri Lanfca, Indonesia and Vietnam (Barnett and Prafcash 1975). In spite of being
a major rodent pest, its behaviour has not been sufficiently investigated (Spillet
1968; Parracfc and Thomas 1970). Here we describe its non-aggressive social
behaviour rhythms observed under laboratory conditions. Rhythms of aggres-*
sive behaviour have been reported elsewhere (Sridhara and Rrishnamoorthy, in
press).
2. Material and methods
2 . 1 Animals
Bandicota bertgatensis in the weight range of 200-250 g were collected from fields
by digging their burrows. On transport to laboratory the subjects were main-
tained in 35 x 35 x 50 cm galvanized iron mesh cages for 15 days to acclimate
them to the laboratory conditions. During this period they were fed on standard
rat and mouse feed (Hindustan Lever, India). Vitamins through water and fresh
vegetables were made available once a week. The photoperiod was regulated at
12 hr light and 12 hr darkness with the former beginning at 0600 hr. Room
temperature was 25 ± 3° C.
317
318 Shakunthala Sndhara and R V Krishnamoorthy
2.2 Behaviour studies
The non-aggressive social behaviour under different social conditions, such as
confrontation between male-female, male-male, female-female, one male-two
females, one male-three females, two males-one female and three males-one female
was observed, and the rhythmicity for each sex was noted. The parameters of
non-aggressive social behaviour were the frequency of occurrence of several acts
and postures namely attend, approach, nosing, nose-nose, investigate, ano-genital
sniffing, push-past, crawling under/over, huddling, allogrooming and the various
sexual activities like following, attempted mount, mount, intromission, ejacula-
tion, post-copulatory groom and lordosis. The terms and identification of beha-
viour patterns were based on the studies of Grant and Mackintosh (1963), Ewer
(1971), Barnett (1975), Begg and Nelson (1977) and Beach (1976).
For studying behaviour the subjects were transported to a 100 x 50 x 50 cm
observation chamber, made of galvanised iron with glass sides and front. The
roof of the cage was made of wire nr/sh. The sides were of sliding type to faci-
litate easy introduction of animals. The chamber was divisible into two equal
portions by inserting a thin galvanized iron sheet into the slot at the centre of
the roof. The two sexes of different combinations were isolated in these portions
for 5-^6 days prior to the study to habituate them to the test chamber. During
observations on social behaviour, the partition was removed for 10 minutes, the
acts and postures of non-aggressive social behaviour scored for each individual
during the ensuing interaction. Rhythms were established after collecting data
during each hour of the L.l>. cycle over a schedule of seven days. The observa-
tions were made under natural light during day-time and under dim red light
during nights. The light was kept 2 M away from the cages.
2.3. Statistics
The cumulative means of each individual were computed into histograms (figures
1-5). The moan ± S.E. of behaviour scores of the two sexes were subjected to
student t test to establish which of the sex is more active socially in the various
confrontations. For group encounters KrushkaMVallis one way analysis of vari-
ance (Siegel 1956) was carried out to determine the rank, order of sociability
amongst the interacting individuals.
3. Results
Except in females of group encounters all the subjects were socially active through-
out the 24 hr L.I>. cycle. The results presented in figures l-«5 indicate that
B. bertgalertsis exhibits rhythmic social behaviour in most of the encounters. The
seven day schedule of observations indicated a fairly consistent rhythmicity (95%).
The rhythms were unimodal or bimodal depending on the sex and number of
interacting conspecifics. For instance, male one of male-male encounter dis-
played a single peak at 1800 hr (figure 2) whereas the male of one male-three
females group confrontation exhibited two peaks at 0900 and 2100 hr. No defi-
nite peak was seen for the two members of male-female interaction (figure 1), male
Social behaviour rhythms in S. bengalensis
s g s
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320
Shakunthala Sridhara and R V Krishnamaorthy
two of male-male encounter (figure 2), female two of female-female pair (figure 3)
and the male of one male-two females combination (figure 4). However slightly
raised social activities were seen at 1800, 2300-0100, and 0100 hr respectively
for these animals. Amongst females, female one and three of one male- three
females group encounter exhibited a single peak of non-aggressive social behaviour
at 0900 hr (figure 5) while two peaks were seen for female one of female-female
interaction at 1200 and 1800 hr (figure 3), female two of one male-two females
group encounter and one male-three females group confrontation. The former
had peaks of social behaviour at 1200 and 1800 hr (figure 4) and the latter at
0900 and 1800 hr (figure 5). Only one female, female one of male-two females
group displayed three peaks of social behaviour rhythm at 0900, 1800 and
0100 hr (figure 4). Majority of the peaks occurred at 0900 and 1800 hr for both
males and females.
Between the sexes male was more active socially in male- male (P < 0-001,
table 1) and one male-two females encounter (F< 0-001, table 1). In isosexual
pairs one of the pairs was significantly more social (P < 0-001 and P > 0-05,
table 1). The rank order of social behaviour in one male-two females group
encounter was male > female one > female two (#= 7-93, P > 0-05) while
there was no such hierarchy in the one male-three females interaction (H = 3-21,
P < 0-05). However, female one was more sociable than female three (t test
Table 1. Comparison of non-aggressive behaviour scores of male and female
B. bengalensis during different social conditions.
Confrontation
between
Scored by
Mean beb. counts
±SE
Cumulative
score fcr the
combination
Male and female
Male
Female
1393 ±47*
959 ±42
2352
Male and male
Male one
Male two
1024±31*
433±52
1457
Female and female
Female one
Female two
733±49*
504±49
1247
One male and two
females
Male
Female one
Female two
1187 ±98*
41 8 ±26
220 ±21
1819
One male and three
females
Male
Female one
Female two
Female three
190±23
281 ±26**
213±28
137 ±14
821
* Significantly higher social activity score*
** More social than female three.
Social behaviour rhythms in B. bengalensis 321
P< 0-01, table 1). When group encounters involved more than one male,
social behaviour was least but aggression was so violent that all males except
the dominant were found dead much before the 24 hr cycle. The death seemed
to be due to the stress of constant threats and fighting rather than due to injuries
since none of the dead males was fatally wounded.
4. Discussion
Several species of rodents exhibit well-established rhythms for movement outside
their burrows in nature (Marten 1976), activity and movement in cages and maize
(Barnett et al 1975), and for aggressive behaviour (Lerwill 1977). These studies
showed two peaks of activity for rats, one soon after dark period and a second
before dawn with little activity during the day-time. A similar bimodal rhythmi-
city for locomotor activity was observed in B. bengalensis by Parrack (1966).
Agonistic behaviour of a group of lesser bandicoots did not exhibit any rhythms;
however, it tended to be increased between 0200 and 0530 hr (Spillet 1968).
Parrack and Thomas (1970) observed dominant B. bengalensis exhibiting peak
aggressive behaviour at 0600 and 0900 hr. This period coincided with the visit
of subordinate rat to the food platform. The present results also demonstrate
that non-aggressive social behaviour of B. bengalensis also manifests both uni-
and bimodal rhythms depending on the sex and number of conspecifics interacting.
Similar alteration of rhythms in response to social interaction has been demon"
strated for several species of rodents. For instance, Kavanau (1967) observed
synchronization of running between female deer mice, while Calhoun (1975)
noticed alteration of locomotor activity by sexual rhythms in Norway rats. Our
earlier study showed circadian oscillations in the aggressive behaviour of
B. bengalensis consequent on social stress (Sridhara and Krishnamoorthy, in
press). Farr and Andrews (1978) described the phase dissociation of both meta-
bolic and behavioural rhythms of deer mice, Feromyscus maniculatus when crowd-
ing increased social interactions. The phase relationships of subordinate subjects
was prone to be much more unstable which the authors attribute to their avoidance
of more aggressive and dominant cohorts. The fluctuation in the social behaviour
rhythms seen in the present study also could be due to the degree of stress experi-
enced by the subordinate animals during the various social encounters staged.
The authors are grateful to the late Dr K Ramakrishnan and Dr R Narayana
for their encouragement and facilities. Technical assistance of M/s. Govindaraju
and Rajanna, and financial aid of Ford Foundation (Grant No. 660-019),
New Delhi, are acknowledged.
References
Barnett S A 1975 The rat ; A study in Behavior Chicago : The University of Chicago press)
Barnett S A, Cowan P E and Prakash I 1975 Circadian rhythms of movements of the house
rat, Rattus rattus L. ; Indian J. Exp. Biol 13 153-155
Barnett S A and Prakash I 1975 Rodents of economic importance in India (New Delhi : Arnold
Heineman)
Shakunthala Sridhara and R V
raalerat : sci
25 291-327
. 4 127_174
I and Andrews R v
of
a f^e living population of black
«
rats; Anim
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 3, May 1982, pp. 323-328.
© Printed in India.
Toxicity of certain pesticides found in the habitat to the
larvivorous fishes Aplochettus lineatm (Cuv. & Val.) and
Macropodus cupanus (Cuv. & Val.)
SHEILA SUSAN JACOB, N BALAKRISHNAN NAIR and
N K BALASUBRAMANIAN
Department of Aquatic Biology and Fisheries, University of Kerala,
Trivandrum 695 007, India
MS received 12 May 1981 ; revised 22 February 1982
Abstract. Bioassay studies reveal the toxicity levels of pesticides utilised in the area
to the larvivorous fishes Aplocheilus lineatus and Macropodus cupanus. The resis-
tance of both fishes decreases with increase in period of exposure to the pesticides.
Comparing the major groups of synthetic organic pesticides, the chlorinated hydro-*
carbons, here exemplified by DDT, are more toxic to the fishes than ekalux and
malathion, the organophosphates experimented with. The carbamate sevin is the
least toxic. Nevertheless, all the pesticides are 'toxic' to 'very toxic* as defined
by the Joint ICMO/FAO/UNESCO/WHO group of experts, having an acute lethal
threshold of below 1 to 100 mg/1. Af. cupanus is the more resistant of the two fishes,
probably on account of its obligate air-breathing nature, and thus its tendency to
absorb less toxicant across the gills. Contrasting the susceptibility of mosquito
larvae and the fishes studied to the pesticides investigated, the closeness of the LC50
values obtained in A. lineatus to. that recorded in certain species of mosquito larvae
indicates that while M. cupanus could be employed in conjunction with pesticides
for anti-larval work, A. lineatus should not be so utilised.
Keywords. Pesticides ; toxicity ; larvivorous fish ; Aplocheilus lineatus ; Macro-
podus cupanus.
I. Introduction
Larvivorous fishes such as Gambusia ejfinis and Poeciliareticulata, the primary
biological control agents of mosquito larvae, have been extensively employed in
certain regions in mosquito abatement programmes (Mallars and Fowler 1970;
Bay and Self 1972). However, indiscriminate releases of these exotics into the
aquatic environment has resulted in the alteration/eradication of valuable faunal
components of the ecosystem (Myers 1965; Bay 1973; Men on 1977). This has
renewed interest in the biocontrol potential of indigenous larvivorous fishes such
as Aplocheilus lineatus (Cuv. & Val.) and Macropodus cupanus (Cuv. & Val.).
An essential aspect of such assessments is information on the danger levels to the
fishes of pesticide contaminants found in the aquatic ecosystem. This problem
has assumed importance owing to the widespread and indiscriminate permeation
. .' 323
P,(B)-14
324 Sheila Susan Jacob, N Balaknshnan Nair and N K Balasubramanian
of pesticides in the aquatic environment (Muirhead-Thomson 1971 ; Edwards
1977) and the consequent risks to larvivorous fish populations. Such data are
not available, leading to this study.
2. Materials and methods
In the present investigation, pesticides were chosen from each of the major groups
of synthetic pesticide utiliseds in agricultural operations in the area — i.e., DDT
(25 EC; manufactured by Bangalore Pesticides Limited) from the chlorinated
hydrocarbons, malathion (50 EC; manufactured by Bangalore Pesticides Limited)
and ekalux (25 EC; manufactured by Sandoz India Limited) from the organ o-
phosphates, and sevin (50% WP; manufactured by Union Carbide) from the
carbamates and bioassay tests were conducted.
Healthy medium sized A. lineatus (mean standard length 25-40 mm) and
M. cupanus (mean standard length 20-^28 mm) collected from streams and water
bodies in the Trivandrum (Kerala, South India) area were acclimated to labo-
ratory conditions in well water at a temperature of 28 ± 2° C, pH of 7'1 and
Oa at near air saturation. The static test method (Doudoroff et al 1951) was
used to directly estimate the toxicity levels, with certain modifications to guard
against a depletion/alteration in the toxic material, as suggested by Muirhead-
Thomson (1971) and Sprague (1973). Stock solutions of the different pesticides
were diluted to the required parts by weight of active ingredient ( = mg/1) by
standard methods (BusVine 1977). However, since the water volume/weight of
fisi ratios utilised for bioassay tests vary greatly (Rita and Nair 1978), here, on
the basis of preliminary trials, 1*8 gm/1 solution and 1 gm/1 solution were chosen
as an adequate weight/volume ratio in A. lineatus and M. cupanus, respectively.
Bioassays were carried out in 5 logarithmic concentrations. The period of expo-
sure for each bioassay was 48 hr as subsequently the mortality curve flattened;
neither the experimental nor control specimens were fed during this period. The
lethal concentration 50 (LC50) for 24 and 48 hr were calculated for each pesticide
by the probit analysis method. The behavioural responses exhibited by the fishes
during the exposure period were also recorded.
3. Results and discussion
A comparative statement of the results of the probit analysis, specifically regres-
sion equations and the LC50 values including the upper and lower limits (ULC50
and LLC50) has been tabulated for both the 24 and 48 hr period of exposure in
the case of each pesticide in tables 1 and 2.
Considering the physical reactions of the fish to the toxic solutions, in all cases
undulation (mild to pronounced) of the body, increased oscillation of the pec-
toral, pelvic, anal and caudal fins, rapid and irregular movements of the oper-
cular folds, loss of equilibrium (ranging from partial .to complete) and excitation
(mild to pronounced) were noted. At extremely toxic concentrations, the
external body surface showed * burnt' patches.
. Tne lowering in the 48 h LC50 values when compared with the 24 hr ones
suggests the decreasing resistance of the fish with increase in experimental time,
SL finding supported by Cairns and Scheier (1964) and Rita and Nair (1978),
Pesticide toxicity to larvivorous fishes
Table 1. Acute toxicity levels? of selected pesticides in A. lineatus.
325
Pesticide
Period of
exposure
(hrs)
LC50 values
(mg/1)
Regression equation
DDT
24
48
0-
o-
1489±0
1228±0
•0212
•0182
log
log
.y
y
=?9
= 8-
•5405'
0885
•log
•log
x x 100-
x x 100 —
6-1893
3-8103
Ekalux
24
48
o-
o-
1939 ±0-0 247
1699 ±0-0228
log
log
y
y
= 10-2105-log
= 9-6205 -log
x X 100 -
x X 100 -
8-1467
6-8348
Malathion
24
48
i-
a-
15GO±a-
9750 ±0
3050
•2120
log
log
y
y
= 5-
= 6-
0873-
1911-
log A: X 10 -
log x X 10 —
0-3972
1-1228
Sevin
24
48
4-
3-
2070 ±0-
7470 ±0-
3750
3100
log
log
y
y
= 14-3413
= 14-6842
log
log-
—
3-9490
3-4242
Table 2. Acute toxicity levels of selected pesticides in M. ciipanus.
Pesticide
Period of
exposure
I C50 values
(mg/i)
P,egrossion equation
Oirs)
DDT
24
2
•813±Q-
453
log
y
= 8-
6338-
bg^ +
M219
48
2'
•277±0-
310
log
y
= 9-
87^-
log x +
1-4720
Ekalux
24
3-
659±0-
434
log
y
= 11
•454-
log x —
1-4533
48
3
•453iO-
584
log
y
-7-
7358-
log x 4- 0-8363
Malathion
24
4-
962±0-
479
log
y
= 13-2989-log x-
-4-2607
48
4-
•594±0-557
log
y
= 10
•5503
•log x~
-1-9859
Sevin
24
14'
•730±0-
590
log
y
= 35
•2288-logx-
- 36-1552
48
13'
•910±0-
380
log
y
= 44
•0285
•log x -
-45-3320
The higher LC50 values in M. cupanus denote its greater resistance than A. linea-
tus. This may be because the principal route of entry of pesticides for non-feed-
ing fish is through the gills (Johnson 1968) ; M. cupanus, being an obligate air-
breather, naturally tends to absorb less toxicant across the gills. Comparing the
main groups of synthetic organic pesticides, the results of the present study where
DDT (a chlorinated hydrocarbon) is more toxic to the fish than ekalux, mala-
thion (organ ophosphates) and sevin (a carbamate), are in agreement with the
findings of Johnson (1968) and Rita and Nair (1978). However, all pesticides
tested are * toxic ' to ' very toxic ' as defined by the Joint ICMO/FAO/UNESCO/
WHO group of experts (1964) since they have an acute lethal threshold of below
1 to 100 mg/1. A comparison of the acute toxicity levels of the pesticides in
various species of fishes, given in table 3, reveals that wide variations in the
)— i4a
326 Sheila Susan Jacob, N Balakrishnan Nalr and N K Balasubramanjan
Table 3. Comparison of some acute toxicity levels of the pesticides investigated in
different species of fishes.
Pesticide
Species investigated
Period of
exposure
(to)
LC50
(mg/1)
(ppm)
Reference
Lepomis macrochims
96
0-016
Edwards (1977)
Salmo gairdneri
96
0-018
Edwards (1977)
Salvelinus fontinalis
36
0-0323
Hatch (1957)
Carassius auratus
72
0-1
Odum and Summerford (1946)
Carassius auratus
96
0-027
Henderson et al (1959)
DDT
Aplocheilus lineatus
24
0-1489
Present investigation
Aplocheilus lineatus
48
0-1228
Present investigation
Gambusia affinis
24
0-5
Mayhew (1955)
Gambusia affinis
36
0-32
Hatch (1957)
Gambusia affinis
72
0-01
Odum and Summerford (1946)
Macropodus cupanus
24
2-813
Present investigation
Macropodus cupanus
48
2-277
Present investigation
Puntius ticto
24
0-0135
Bhatia (1971)
Puntius ticto
48
0-011
Bhatia (1971)
Puntius ticto
72
0-011
Bhatia (1971)
Puntius ticto
96
0-0074
Bhatia (1971)
Salmo gairdneri
96
0-1
Edwards (1977)
Lepomis macrochirus
96
0-12
Edwards (1977)
Aplocheilus blochii
48
1-3
VCRC Annual Report (1979)
Aplocheilus lineatus
24
1-15
Present investigation
Aplocheilus lineatus
48
0-975
Present investigation
Malathion
Cyprinus carpio
96
4-5
Nishiuchi and Hashimoto
(1967) ^ -
Macropodus cupanus
24
4-962
Present investigation
Macropodus cupanus
48
4-594
Present investigation
Labeo rohita
24
7,- 15
Arora et al (1971)
Labeo rohita
96
5-05
Arora et al (1971)
Pimephales promelas
24
25
Tarzwell (1958)
Pimephales promelas
96
12-5
Henderson et al (1959)
Pimephales promelas
96
22
Tarzwell (1958)
Lepidocephalus thermalis
24
22-69
Rita (1977)
Lepidocephalus thermalis
48
20-61
Rita (1977)
Oncorhynchus kisutch
96
0-7
Macek and McAllister (1970)
Ameiurus mdas
96
0-8
Macek and McAllister (1970)
Fundulus similis
24
1-75
Butler (1963)
Lepomis macrochirus
96
2-0
Henderson et al (1959)
Lepomis macrochirus
96
3-4
Edwards (1977)
Salmo gairdneri
96
•3-5
Edwards (1977)
Sevin
Aplocheilus lineatus
24
4-207
Present investigation
Aplocheilus lineatus
48
3-747
Present investigation
' Mugil cufema
24
4-25
Butler (1963)
Perca flavescens
96
5-6
Macek and McAllister (1970)
Casterosteus aculeatus
.24
6-7
Stewart et al (1967)
Pimephales promelas
96
13
Henderson et al (1959) <
Macropodus cupanus
24
14-73
Present investigation
Macropodus cupanus
48
13-91
Present investigation
Pesticide toxicity to larvivorous fakes 327
pesticide concentrations that produce adverse effects have been recorded, depend*
ing on the species, environmental factors and even biological status and origin
of the test organism. It must however be mentioned that the LC50 values
obtained for A. lineatus exposed to malathion are comparable to those recorded
in the related Aplocheilus blochii (VCRO Annual Report 1979). Again, in the
case of specimens exposed to DDT, M. cupanus is even hardier than the * resis-
tant' mosquito fish G. affinis (Johnson 1968). Again, M. cupanus is the most
resistant of all the species studied to sevin.
It may also be noted that Das and Rajagopalan (1976) working on the suscepti-
bility of mosquito larvae to insecticides found that in Anopheles stephensi, Culex
fatigans, Anopheles culicifdcies and Aedes aegypti, the critical doses of malathion
required were 0-8, 0-064, 0-08 and 0-48 mg/1 respectively. In the case of sevin
it was a uniform 4-0 mg/1. With DDT, the LC50 value was 0-2 mg/1 for A. ste«
pheitsi and C. culicifacies while in the case of Ae. aegypti and C. fatigans it was
0-02 mg/1 and 0-03 mg/1, respectively (VCRC Annual Report 1979). These
values are fairly close to those reported in A. lineatus in the present study.
Therefore, while M. cupanus could be utilised in conjunction with such insecti-
cides for anti-larval work, A. lineatus should not be so used under any circum-
stances.
It has thus been demonstrated that even * safe ' and often minute dosages of
pesticides are highly toxic to fish life, as maybe seen from the LC50 values.
Therefore, studies of this nature are essential as they provide information on the
concentrations of environmental contaminants that cannot be tolerated bySsh
populations and consequently aid not only in the effective control of mosquito
larvae by the fish but also in the protection of the aquatic environment
Acknowledgements
One of the authors (SS J) is grateful to the NCERT and CSIR for the award of
a National Science Talent Search Fellowship and a CSIR Senior Fellowship
respectively, during the tenure of which the work was carried out.
References
Arora H C, Shrivastava S K and Seth A K 1971 Bioassay studies of some commercial organic
insecticides-— Parts I and II ; Indian J. Environ. Health 13 226-233 ; 300-306
Bay E C 1973 Exotic fish introductions for mosquito control : possible and purported conse-
quences; WHOIVBCIWPm
Bay E C and Self L S 1972 Observations on the guppy Poecilia reticulata Peters in Culex
pipiens fatigans breeding sites in Bangkok, Rangoon and Taipei ; Bull World Health Org.
46 407-416
Bhatia H L 1971 Toxicity of some pesticides to Puntius ticto (Hamilton) ; Sci. Cult. 37 54$
Busvine J R 1977 A critical review of the techniques for testing insecticides (London : Common-
wealth Institute of Entomology)
Butler P A 1963 Commercial fishery investigation ; Pesticide-Wildlife Studies U.S. Fish. Wild.
Serv. Cir. 199 5-28
Cairns J and Scheier A 1964 The effect upon the pumpkinseed sunfish Lepomis gibbosus (Linn.)
of chronic exposure to lethal and sublethal concentrations of dieldrin ; Notul. Nat. p. 370
Das P K and Rajagopalan P K 1976 Susceptibility of larvae of Culex fatigans (Wiedmann),
Anopheles stephensi (Liston) and Aedes aeypti (Linn.) to insecticides in Pondicherry ;
Indian J. Med. Res. 70 412-416
328 Sheila Susan Jacob, N Balakrishnan Nair and N K Balasubramanian
Doudoroff P, Anderson B G, Burdick G E, GaltsofF P S, Hart W B, Patrick R, Strong E R, f
Surber E W and Van Horn W M 1951 Bioassay methods for the evaluation of acute toxi- |
city of industrial wastes to fish ; Sewage Ind. Wastes 23 130-139
Edwards C A 1977 Nature and origins of pollution of aquatic systems by pesticides ; In Pesti- f
tides in aquatic environments (cd.) M A Q Khia.(Ncw York : Plenum Press) pp. 11-36 >
Hatch R W 1957 Relative sensitivity of salmonids to DDT ; Prog. Fish-Cult. 19 89-91 -V*
Henderson C, Pickering Q H and Tarzwell C M 1959 Toxicity of organic phosphorous and |
chlorinated hydrocarbon insecticides to fish ; Biol. Prob. Water Poll. Trans. 1959 Seminar
Robt. A. Taft San. Eng. Center Tech. Rep. W. 60-3 76-92
Johnson D W 1968 Pesticides and fish— a review of selected literature ; Trans. Am. Fish. Soc.
97 398-424 . .. -
Joint ICMO/FAO/UNESCO/WHO group of experts 1964 Abstract of the 1st report of the 1st
session on the scientific aspects of marine pollution ; Water Res. 3 995-1005 \
*Macek K J and McAllister W A 1970 Trans. Am. Fish. Soc. 99 20 *
Mallars J L and Fowler J R 1970 Mosquito eating fishes in California (California : Calif.
Mosquito Control Ass. Inc.).
Mayhew J 1955 Toxicity of 7 different insecticides to rainbow trout, Salnio gairdneri Richardson ; ?
Proc. Iowa Acad. Sci. 62 599-606
Menon A G K 1977 Fish and malaria control ; Sci. Cult. 43 110-114
Muirhead-Thomson R C 1971 Pesticides and freshwater fauna (New York : Academic Press)
Myers G S 1965 Gambusia, the fish destroyer ; Trap. Fish. Hobbyist 31-32 54-55
Nishiuchi Y and Hoshimoto Y 1967 Toxicity of pesticide ingredients of some freshwater orga-
nisms ; Botyn. Kagaku. 32 5-11
Odum E P and Summerford W T 1946 Comparative toxicity of DDT and four analogs to gold- ;
fish, Gambusia and Culex larvae ; Science 104 480-482
Rita Kumari S D and Nair N B 1978 Toxicity of some insecticides to Lepidocephalus thermalis
(Cuv. & Val.) ; Proc. Indian Natl. Sci. Acad. 44 122-132
Sprague J B 1973 The ABC's of pollutant bioassay using fish ; Biological methods for the assess- \?
ment of water quality ASTM, STP 528, American Society for Testing and Materials pp. 6-30 — ,i
Stewart N E, Milleran R E and Breese W P. 1967 Acute toxicity of the insecticide sevin and *f
its hydrolytic product 1-naphthol to some marine organisms ; Trans. Am. Fish. Soc. 96
25-30 ':i
Tarzwell C M 1958 The toxicity of some organic insecticides to fishes ; Proc. Conf. S.E. Ass.
Game Fish Com. 12 232-239
Vector Control Research Centre 1979 Annual Report (New Delhi : ICMR) '.!
*Not refered to in the original
Proc. Indian Acad. Sci. (Aaim Sci.), Vol 91, Number 4, July 1982, pp. 329-335.
© Printed in India.
Histochemical studies on non-specific esterases in epididymis
of the bat, Cynoptems sphinx sphinx
L T MOTE and M N NALAVADE*
Department of Zoology, Shivaji University, Kolhapur 416004, Maharashtra, India
MS received 3 September 1981; revised 31 May 1982
Abstract. Non-specific esterases were studied in the epididymis of the bat, C. sphinx
sphinx by employing a-naphthyl acetate and 5-bromomdoxyl acetate as substrates
and eserine sulphate (10"4 M) as an enzyme inhibitor. The enzyme activity in
epididymal cells was diffused cytoplasmic and granular in nature. The granular
activity was eserine resistant. Holocrine cells were also observed in the epididymis
of this bat. Seasonal variations in epididymal esterases are described.
Keywords. Bat; Cynoptems sphinx sphinx; epididymis; principal cells; holocrinc
cells; esterases.
1. Introduction
Several lysosomal acid hydrolases have been studied in the gonads and non-gonadal
sex accessories of the vertebrates. Although esterases have been demonstrated
in the epididymis of rat. (Verne, and Hebfirt 1952 ; . Washstein etal 1961 ; Vogel
1967), mouse (Alien and Slater 1957 ; Kirkeby and Blecher 1978a,b ; Blecher
and Kirkeby 1978 ; Chakraborty etal 1975), marmoset (Miraglia etal 1970),
bull (Erkmann 1971) and man (Malaty and Bourne 1954; Kohl 1968), these'
enzymes have not been studied in bats.
The presence of two types of cells, viz., principal cells and holocrine cells has
been reported in the epididymis of some animals. Esterases have been studied
in the holocrine cells of some animals such as rat (Vogel 1967) and mouse
(Martan and Allen 1964). In this regard nothing is known about the holocrine
cells in the bat epididymis.
The present paper deals with non-specific esterases in epididymis of the bat
C. sphinx sphinx. ...
2. Material and methods
The adult male bats (C. sphinx sphinx) were collected monthly for one year. The
animals were killed by decapitation, the epididymedes were dissected out and
To whom correspondence should be made.
329
33J L T M)te and M N Nalavade
fixed in cold (4°C) Baker's fixative. Following fixation (24 hr) the tissues were
transferred to Holt's gum sucrose (Holt 1959). The sections were cut at 6-8 jum
on a Llpshaw cryostat at — 20° C. Before incubation, the sections were washed
with chilled distilled water.
The following two histochemical techniques were employed for enzyme locali-
zation :
(i) a-naphthyl acetate (aNA) as a substrate with Fast Blue B as a coupler
(Gomori 1952). (ii) 5-Bromoindoxyl acetate (5 BIA) as a substrate with ferri-
ferrocyanide as an oxidizing agent (Holt 1958 ; Holt and Withers 1958).
In both the histochemical techniques, eserine sulphate (10~4M) was used as
an enzyme inhibitor.
3. Observations
3 • 1 . Sex-cycle of the bat
C. sphinx sphinx is a megachiropieran frugivorous bat, the females of which
experience two pregnancies in quick succession. The first pregnancy lasts from
November to March and the second from March-April to July (Mote 1981).
Ranukrishna (1947), Baile (1976) and Pawar (1976) also reported on two sex-
cycles in this species of bat. The sex cycle in male bats may be summarized as :
Sexual quiescence— May to August ; Prebreeding period— September and
October ; First active breeding period— November ; Intervening period— December
and January ; Second active breeding period— February-March ; Postbreeding
period—April
Figures 1-8. (1) Epidtdymis. during sexual quiescence (June) stained with OKA
technique to show diffused cytoplasmic staining in epithelial cells (arrows) CT «
c^ctive tissue x 50. (2) Early prebreedmg period (September) stained with 5
e* N°e
T,e* N°te^USf c^P^mic and granular sining in the prdpal
, hMocrme cells (EC) and fibroblast-like cells in connective tissue (FC) x 15
PrCbreed"1S peri°d ^er s "
nnecv
" PrCbreed"1S peri°d P^er) stained with 5 BIA after
.x
- <^
£±=
(arrows) x 150. CHC) and acr°somes of the sperms
Bat epididymal esterases
331
Figure 1-8.
Sat epididymal est erases 333
3.2. Enzyme localization
The enzyme activity in the epithelial cells appeared as diffused cytoplasmic and
in the form of granules (figure 2). The granular staining in the cells was eserine
resistant (figure 3), whereas the diffused cytoplasmic staining was eserine sensitive.
3-3. Seasonal variations in esterases
The regressed epididymis during the sexual quiescence exhibited weak diffused
cytoplasmic staining in the epithelial cells with aNA (figure 1) and SBIA. The
staining was eserine-sensitive and the holocrine cells could not be distinguished
from the low cuboidal principal cells. With the advent of the prebreeding period
two cell types could be identified with aNAandSBU (figure 2) procedures. The
principal cells exhibited moderate staining which was diffused cytoplasmic and
granular in nature. The holocrine cells also exhibited dual localization of enzyme
activity but the staining was more intense than the principal cells. The granular
staining was eserine-resistant (figure 3). Similar results were also seen during |
the late prebreeding period (figure 4). During this period the spermheads (aero- |
somes) also exhibited eserine-resistant enzyme activity.
During both the active breeding periods the principal cells exhibited weak to
moderate diffused cytoplasmic and granular staining, whereas the holocrine cells
exhibited moderate to intense diffused cytoplasmic and granular enzyme activity
(figure 5). The granular staining in both the cell types was eserine-resistant. The
spermheads in the lumina of the tubules also showed eserine-resistant esterase
activity. The staining intensities in both types of cells were slightly reduced during
the intervening period (figure 6). During the postbreeding period the principal
cells exhibited weak diffused cytoplasmic staining, whereas the staining was intense
and granular in the holocrine cells (figure 7). The granular staining in the holo-
crine cells and the sperm-debris in the lumina of the tubules was eserine-resistant
(figure 8).
4. Discussion
Verne and Hebert (1952) reported that the esterase activity appears in the epididymis
of the rat earlier than in the testes during the development. Malaty and Bourne
(1954) showed that the epididymis of a 12-year boy gives a slight positive esterase
activity. Presence of esterase activity has also been reported in the epididymal
epithelium of several mammals as described in § 1. The present histochemical
results indicate that the epithelial cells in the epididymis of the bat contain non-
specific esterases capable of hydrolyzing aNA and 5 BIA.
In recent years, a dual localization of hydrolytic enzymes in the cells has been
suggested and established. Several hydrolytic enzymes such as /J-glucuronidase,
acid phosphatase etc. have been demonstrated in the endoplasmic reticulum and
lysosomes. In the present investigation also the enzyme activity was seen as
diffused cytoplasmic and granular in nature, the latter being eserine-resistant.
The diffused cytoplasmic activity may be correlated with the esterases in the endo-
plasmic reticulum and the granular activity with the lysosomes. Similar histo-
334 L T Mote and M N Nalavade
chemical studies on the epididymis of man (Malaty and Bourne 1954), ram, rabbit,
rat and hamster (Moniem and Glover 1972) also revealed diffused cytoplasmic
and granular esterase activities.
The present results indicate that the epididymal esterases in bat undergo cyclic
variations according to the sex-cycles. During sexual quiescence the diffused
cytoplasmic esterase activity is weak in the epithelial cells, both diffused cyto-
plasmic and granular activities gradually increase from prebreeding period and
become intense during the first active breeding period. With slight reduction
in the staining during the intervening period, again, the enzyme activities increase
during the second active breeding period. During the po,stbreeding period of
regression the diffused cytoplasmic activity decreases but the granular (lysosomal)
activity remains unchanged. The bioassay studies on total esterase activity and
eserine-resistant esterase activity in the epididymis of this bat (Mote 1981) also
substantiate the cyclic variations.
These results indirectly indicate that the epididymal esterases are dependent
on the testicular hormones. This conclusion is based on some circumstantial
evidences involving castration and hormone replacement studies. Allen and Slater
(1957) observed no change in the aliesterase activity in ciliated cells of lobes 2, 3
and 7 of epididymis but the esterase activity was depressed in all other cells after
castration. They further reported that testosterone prop ionate reversed the effects
of castration. Hunter and Allen (1959) and Allen and Hunter (1960) obtained
seven bands with esterase activity from mouse epididymis by starch-gel electro-
phoresis. The bands were designated as A to G. Castration was found to depress
the activity of A, B and C bands and abolished the activity of D, E and G bands
but increased the activity of F band. Androgen administration reversed these
effects. These studies indicate that the epididymal esterases are dependent on
androgen levels.
Of particular interest is the observation that the holocrine cells are present in
the epididymis of this bat. The literature on holocrine cells was reviewed by
Martan and Allen (1964). Recently, Vibhute (1981) studied epididymal muco-
substances in eleven species of bats and noted the holocrine cells only in Rousettus
leschenaulti and Hipposideros fulvus fulvus. Vogel (1967) reported that 5 min
incubation of rat epididymis with aNA gives an intense tinge in entire epithelium
but when the incubation time is reduced to 2 min only holocrine and basal cells
show enzyme activity. The present investigation also revealed the presence of
non-specific esterases in holocrine cells of the bat epididymis. According to
Martan and Allen (1964) acid phosphatase and aliesterase may be the secretory
products of holocrine cells. They may be liberated into the lumen of the duct
when the cells degenerate and would contribute to the seminal fluid. Such
enzymes are known in the seminal fluid and may play a role in the breakdown of
certain phosphate esters and lipids prior to their metabolism by spermatozoa.
References
-Allen J M and Hunter R L 1960 A histochemical study of enzymes in the epididymis of normal,
castrated and hormone replaced castrated mice separated by zone electrophoresis in starch
gels; /. Histochem. Cytochem. 8 50-57
Bat epldldymal esterases 335
and Slater J J 1957 A chemical and histochemical study of alkaline phosphatase and
epididyinis of normal and castrate mice ; Anat . Rec. 129 255-273
1976 Studies on ft-glucuronidase of Bat in seasonal Breeding Cycle ; Ph.D. Thesis,
Lji University, Kolhapur, India
R and Kirkeby S 1978 Histochemical studies on genetical control of hormonal enzyme
in the mouse I. Non-specific esterase activity and regional histology of the
l<3.*d.ymis; /. Anat. 125 247-265
L"bo.rty J, Budd G C and Nelson L 1975 Fine structural localization of cholinesterases
^productive tract of male white mouse ; Biol. Reprod. 13 397-407
£trij Q 1971 Hlstologische und histochemische untersuchungen zur segmenteinteilung des
"keiihodens vom rind vor und nach der geschlechtsreife ; Cytobiologie 3 37-69
^* G 1952 Microscopic histo chemistry, principles and practice (Chicago : University Press)
J> 1958 General cy to chemical methods (ed.) J F Damelli (New York and London :
Press) pp 375
1959 Factors governing the validity of staining methods for enzymes and their bearing
t the Gomo,ri acid phosphatase technique ; Exp. Cell Res. (Suppl.) 7 1-27
* J and Withers R F J 1958 Studies in enzyme cytochemistry V. An apprasial of indogenic
;a-etions for esterase localization ; Proc. R. Soc. (London) B148 520-531
::r R L and Allen J M 1959 The esterases and phosphatases of the epididymis in normal
and vas efferentiectomized mice using the zymogram technique ; J. Histochem.
. 7 307
S and Blecher S R 1978a Studies on the oxydizing system in Holt's medium for histo-
demonstration of esterase activity ; Acta Histochem. 62 44-56
S and Biccher S R 1978b Histochemical studies on genetical control of hormonal
"xxyrne inducibility in the mouse II. Esterase isozymes of the normal epididymis demon-
x*a.ted by substrate variation and by enzyme inhibitors ; Acta Histochem. Cytochem. 11 30-40
\V 1968 Ltpafuchsin and lysosomes in the human epididymis, fluorescent microscopy and
Istochemical investigations; Histochemie 16 236-286
ty H A and Bourne G H 1954 The distribution of 'simple' esterase in human tissues ;
Ictct Anat. 21 249-258
J and Allen J M 1964 Morphological and cytochemical properties of the holocrine cells
tlie epididymis of the mouse ; /. Histochem. Cytochem. 12 628-639
T, Telles M F Jr and Labao C B A 1970 The male-reproductive system of the common
rtar-nioset (Callithrix jacchus) ; Acta Anat. 76 594-611
ern K A and Glover T D 1972 Comparative histochemical localization of lysosomal
nzymes in mammalian epididymis ; /. Anat. Ill 437-452
L T 1981 Studies on non-specific esterases in gonads and associated reproductive organs
>f some vertebrates ; Ph.D. Thesis, Shivaji University, Kolhapur, India
r IE* B 1976 Studies on muco substances in gonads and associated reproductive organs of Bat ;
*li.lD. Thesis, Shivaji University, Kolhapur, India
a P A 1947 Post-partum oestrous in the short-nosed fruit bat, Cynopterus sphinx
Curr. Sci. 16 186
3 J" and Hubert S 1952 Uactivite esterasique des cellules interstitielles du Eesticule et de 1'
rpidielyme chez le rat ; Ann. Endocrinol. 13 924-930
*ite? H G 1981 Studies on mucosubstances in testes and associated reproductive organs of
•hiroptera ; Ph.D. Thesis, Shivaji University, Kolhapur, India
;1 J" 1967 Zur differenzierung der Esterasen in den epithelzellen des nebenhodens der ratte ;
Acta Histochem. 26 64-72
Astern M, Meisel E and Falcon C 1961 Histochemistry of thiolacetic acid esterase: a
^o-mparison with non-specific esterase with special regard to the effect of fixatives and
L:r*liibitors on localization ; /. Histochem. Cytochem. 9 325-329
Proc. Indian. Acad. Sci. (Anim. Sci.)> Vol. 91, Number 4, July 19S2, pp. 337-348.
© Printed in India.
A study of pupal-adult intermediates produced with juvenoid
treatment of Spodoptera litum Fabr. pupae
U S SRIVASTAVA and S S PRASAD
Department of Zoology, Allahabad University, Allahabad 211002, India
MS received 21 May 1982
Abstract. Pupae of Spodoptera litura were treated topically with a juvenoid, 6, 7-
epoxy-3-ethyl-l (p-ethyl phenoxy)-7 methylnonane. The effects comprised death,
production of pupal-adult intermediates of varying grades and adultoids. The
production of pupal-adult intermediates was studied in relation to age of the pupa
at the time of treatment and the dose of the compound administered. It was found
that up to. the age of 20 hr the degree of morphogcnetic response was directly corre-
lated with the dose of the compound administered and inversely with age, but after
this age, an increase in the quantity of the hormone, beyond the effective dose did
aot further augment the effect.
Keywords. Spodoptera litura ; juveaoid effect ; pupal-adult intermediates.
1. Introduction
Several workers have noted the production of pupal-adult intermediates in endop-
terygote insects by treating pupae with juvenile hormone or its various analogues
(Sarcophaga bullata, Srivastava and Gilbert 1969 ; Bhaskaran 1972 ; Tenebrio
molitor, Critchley and Campion 1971 ; Trogoderma grananum, and Caryedon
gonagra, Metwally and Sehnal 1973 ; Trogoderma granarium, Srivastava and
Srivastava 1974 ; Ceratitis capitata, Daoud and Sehnal 1974 ; Ephestia kuhniella,
Tan 1975 ; Cylas formicarius, Ram etal 1980). In Sarcophaga bullata, Srivastava
and Gilbert (1969) concluded that the thorax is the first to become refractory to
the hormone during pharate adult life, followed by the head and then the abdomen ;
and that the age of pupa is the most important factor which influences production
of intermediates. A dose twice that which is effective in 46 to 50 hr old pupae
is ineffective in pupae which are more than 70 hr old. Similarly, Riddiford and
Ajami (1973) have observed maximum sensitivity to JH in 24-30 hr old pupae
of Manduca sexta and greater morphogenetic response with higher doses of the
juvenoid treatment and Reddy and Krishnakumaran (1973) noted that in Tenebrio
molitor highest morphogenetic response is shown by 18-32 hr old pupae. In the
same insect, however, Socha and Sehnal (1972) observed more or less uniform
morphogenetic response in 0-24 hr old pupae. Further, the intermediates
produced with hormone treatment may differ greatly in respect of their characters,
possessing the pupal and adult characters in varying proportions. For instance,
337
338 U S Srivastava and S S Prasad
S.ma - - C.W ^d *« *r tt. £*?
carefully examined, nor has sufficient attention been paid to the factois which
determine the production of intermediates and their different types.
In the present study, different grades of pupal-adult intermediates were produced
by treating pupae of Spodoptera litura of various ages with different quantities
of a juvenoid and an attempt has been made to relate the age and quantity ot the
compound with the morphogenetic characters of the intermediates.
2. Materials and methods
Larvae of tobacco cutworm Spodoptera litura were 'reared on castor leaves in the
laboratory at 28 ± 2° C and allowed to pupate. " The pupal life at this tempe-
rature was 6-7 days. Pupae of known ages from the stock were placed in cavities
made with plasticine and known quantities of the juvenoid, 6, 7-epoxy-3-cthyi-l
(P-ethyl phenoxy)-7 methylnonane cis/trans mixture (RO-1 0-3 108/0 18 of R. Maag
Ltd.) dissolved in acetone were topically applied to the anterior abdominal region
dorsally with the help of a microapplicator. Controls were similarly treated with
acetone only. After emergence of the controls, the old pupal cuticle of the
unemerged experimental pupae was carefully removed and the specimens studied
for their morphological characters. Specimens which died prior to the completion
of developmental period were disregarded in evaluating the results.
3. Results
Pupae of S. litura (2, 14, 20, 26 and 38 hr old) were treated with five different
quantities, viz. 2, 5, 10, 15 and 25 fig of the juvenoid. The effect comprised
death, failure of emergence and emergence of adultoids. When the pupal cuticle
of the specimens showing failure of emergence was removed, they were all fonnd
to be pupal-adult intermediates of different categories. On the basis of the
combinations of pupal and adult characters, these intermediates were classified
into the following six grades (table 1 and figures 1-6).
Figures 1-6. Pupal— adult intermediates of varying grades of S. litura. 1. Grade
I showing adult external structures with b-furcated proboscis. 2. Grade II showing
anterior abdomen pupal, wings developed but unstretched and proboscis bifurcated.
3. Grade III showing anterior and middle abdomen pupal, wings developed but
unstretched and proboscis bifurcated. 4. Grade IV showing anterior and middle
abdomen pupal wings fused and pigmented and proboscis bifurcated. 5. Grade
V showing pupal abdomen except tip of abdomen is adult-like, wings non-pigrneated
and proboscis bifurcated. 6. Grade VI showing whole body pupal.
Pupae treated with juvenoid
339
Pupae treated with juverioid 34!
Table 1. Different characters of p-a intermediates in relation to different giades;
Grades of p-a intermediates
II III IV V VI
Adult abdomen 4
Part of abdomen adult 4 4-
Adult thorax 4- 4- 4^
Adult head 4-4-4*
Wings unstretched 4- -u -f~
Wings fused and pigmented 4-
Wings non-pigraented/undev loped 4. -f
Proboscis bifurcated 4-4-4-44-
<J ext. gen, deformed ; socii, gnathos absent ; ovip,
fused 4
<J ext. gen. less developed and deformed ; ovip. fused
and reduced 4-4-4*
<J ext, gen. smaller, less developed and deformed,
ovip. absent 4-
Fully pigmented compound eyes 4*
Pigmented and less pigmented eyes 4-4-4-4-
Partly pigmented eyes 4-
Anterior abdomen pupal 4-4-4-4 4-
Middle abdomen pupal 4-4-4-4-
Posteiior abdomen pupal ~*~
Pupal thorax "*"
Pupal head "*"
Grade I : An adultoid with bifurcated proboscis and unstretched wings ;
Eternal genitalia developed but deformed. In males, socii and gnathos not
developed; in females, substitutional ovipositor fused. Eye pigmentation resembles
that of normal compound eye.
Grade II: Anterior abdomen pupal ; wings [developed but unstretched;
proboscis bifurcated. In external genitalia of males, tegumen, uncus, gnathos and
socii not developed while the remaining parts are deformed. Substitutional ovi-
positor in females fused and reduced. Eyes showing a strip-like heavily pigmented
zone towards the proboscis and the remaining weakly pigmented zone. The ratio
of pigmented and weakly pigmented zone is about 1 : 6.
342 U S Srlvastava and S S Prasad
Grade III : Anterior and middle abdomen pupal ; wings developed but
unstretched ; proboscis bifurcated. Condition of external genitalia and eye
pigmentation and ratio between heavily pigmented and weakly pigmented zones are
similar to that of Grade II.
Grade IV : Anterior and middle abdomen pupal ; wings fused and pigmented ;
proboscis bifurcated. Condition of external genitalia and eye pigmentation and
ratio between heavily pigmented and weakly pigmented zones are similar to that
of Grade II.
Grade V: Abdomen pupal but the tip of abdomen is adult like; wings non-
pigmented ; proboscis bifurcated. Male genitalia very small and deformed.
Tegumen, uncus, gnathos and socii of males not developed. Substitutional ovi-
positor in females absent. Eye pigmentation similar 1o that of the eyes of Grade
II, but the pigmented area is relatively larger. Ratio between heavily pigmented
and weakly pigmented zones is about 1 :2.
Grade VI : Whole body pupal. The eye can be differentiated into a strip-like
pigmented zone, about 0-39 mm in width and lying between two unpigmented
zones and the unpigmented zone toward the proboscis about 0-26 mm in width.
Ratio of heavily pigmented and unpigmented areas is about 1 : 2.
It would be seen from table 1 that in this scoring system, a higher grade signi-
fies more pupal and less imaginal characters which means that lesser pupal-adult
transformation has taken place and vice versa a lower grade indicates more
imaginal and less pupal characters, showing relatively greater pupal-adult trans-
formation. It was also observed that by and large a particular grade of pupal-
adult intermediate was produced by the treatment of pupae at a certain age with
a certain amount of the juvenoid (JHA).
3-1. Sensitivity to JHA in relation to age and dose
Table 2 gives the number of pupae of different ages which died, produced p-a
intermediates or gave rise to adultoids as a result of treatment of pupae of diffe-
rent ages with different quantities of the juvenoid. It also indicates the grades
of the intermediates. It would be clear from the table that lethal action was more
pronounced when the treatment was given to early pupa (2 hr old) and as the age
of treatment increased, there was a gradual decrease in the number of pupal deaths
irrespective of the doses tried. All the treated pupae which failed to emerge
were p-a intermediates of varying grades.
Table 2 and figures 7 and 8 give the number and grades of the p-a intermediates
produced by the treatment of pupae of different ages with different quantities of the
compound. It would be observed that with each of the five doses of the juvenoid
tried here, treatment of the youngest pupae (2 hr old) led to maximum morpho-
genetic response, viz., intermediates produced had maximum pupal characters
and as the age of the pupa increased, there was a corresponding decrease in such
response and more adult characters were shown by the intermediates produced.
Treatment of 38 hr old pupae produced very low morphogenetic response and
adult organs had developed although they were often deformed, e.g., the proboscis
was bifurcated and the wings were unstretched (Grade I). In some cases, adultoids
with crumpled wings emerged.
Pupae treated with juvenoid 343
Table 2. Treatment of 2-38 hr old pupae of S. litura with 2-25 fig of the JHA.
Age of
pupae
(in hr)
Dose
(in fig)
No. of
pupae
treated
No.
dead
No. and score of
pupal-adult
intermediates
No. of
adultoids
2
2
25
8
17
V
0
14
2
25
6
19
III
0
20
2
25
2
23
II
0
26
2
25
2
23
II
0
38
2
25
0
21
I
4
2
5
25
11
14
V
0
14
5
25
2
23
IV
0
20
5
25
4
21
III
0
26
5
25
2
23
II
0
38
5
25
0
22
I
3
2
10
25
8
17
VI
0
14
10
25
4
21
IV
0
20
10
25
4
21
III
0
26
10
25
2
23
II
0
38
10
25
2
20
I
3
2
15
25
6
19
VI
0
14
15
25
7
18
IV
0
20
15
25
6
19
IV
0
26
15
25
3
22
II
0
3$
15
25
4
18
I
3
2
25
25
7
18
VI
0
14
25
25
3
22
IV
0
20
25
25
3
22
IV
0
26
25
25
3
22
II
0
38
25
25
3
22
I
0
It may also be noted that there was only a slight increase in morphogenetic
response by the treatment of 2 hr, 14 hr and 20 hr old pupae with increasing
doses of the juvenoid. Treatment of 26 hr and 38 hr old pupae with larger
doses of the compound brought no worthwhile difference. A few adultoids
emerged with the application of 2-15 & to 38 hr old pupa only.
4. Discussion
Williams (1961) described JH as the ' status quo ' hormone and regarded that the
classical status quo effect on the treatment of larva or pupa with JH is mani-
fested in the form of a supernumerary larval or pupal moult. This would
ntesumably happen when the larval-pupal or pupal-adult transformation is fully
arrested by the administration of the hormone before the process of transformation
begins and quantity of hormone necessary to bring about the arrest of transfor-
rnation is available,
344
U S Srivasiava and S S Prasad
e tv
o 2
© 5 JJg
ft lOJLlg
• 25 JU9
Age of Pupa (in hrs )
Figure 7. Different grades of p-a intermediates produced by the treatment of pup?e
of S. litura of different ages with JHA.
V!
.2 IV
ill
Treated pupae:
2 hrs old
14 hri old
eOhrtold
26 hrs tld
38 hrs old
10 t
Doses (in
15
20
Figure 8. Different grades of p-a intermediates produced by treatment of pupae of
S. litura with different quantities of JHA.
It is clear from the present observation that the age of the pupa and dose of
the juvenoid administered are crucial factors which regulate the occurrence and
nature of its morphogenetic response. We shall deal with these two factors sepa-
j?ately with reference to the present observations in Spodoptera
Pupae treated with juvenold 345
4-1. Morphogenetic response in relation to age
Several workers have noted that the age of the pupa is a very important factor
in the production of p-a intermediates and found that treatment of early pupae
with juvenoids is most effective in producing intermediate forms (Srivastava and
Gilbert 1969 in S. bullata ; Critchley and Campion 1971 in T. molitor ; Metwally
and Sehnal 1973 in T. granarium and C. gonagra ; Bagley and Baurnfeind 1972
in P. interpunctella ; Bhatnagar-Thomas 1972 and Srivastava and Srivastava
1974 in T. granarium ; Tan 1975 in E. kuhniella, Srivastava 1980 in several stored
grain insects). In T. molitor, Reddy and Krishnakumaran (1973) noted that the
highest morphogenetic response is shown when 16-32 hr old pupae are adminis-
tered the juvenoid, but in the same insect Socha and Sehnal (1972) noted more
or less uniform morphogenetic response on treatment of 0-24 hr old pupa. In
Manduca sexta, Riddiford and Ajami (1973) have observed maximum sensitivity
to JH in 24-30 hr old pupa. Recently, in pupae of Cylas formicar ius, Ram et al
(1980) have noted juvenilising activity in 61-2% cases when treatment was given
to newly moulted pupae and only in 11*5% when 2-day old pupae were similarly
treated with the same compound.
On the basis of the morphological characters of different categories of p-a
intermediates produced by us in 5. litura, we find that the age of the pupa is
most important with all the doses of the JHA tried. It is clearly established in
this insect that the youngest pupa (2 hr old) shows highest morphogenetic response
and as the age of treatment of pupa increases there is a gradual decline in the
effectivity of the compound.
4-2. Morphogenetic response in relation to dose
Bhatnagar-Thomas (1972) in T. granarium observed that treatment of early pupae
with very low concentrations of JHA produced no visible morphogenetic effect
and normal emergence occurred, whereas with higher concentrations, the number
of insects showing abnormalities increased in more or less direct ratio with the
concentration of JHA. She also noted that in the case of treatment of late pupae,
only higher concentration was effective in producing abnormal forms up to a certain
extent. However, Riddiford and Ajami (1973) in M. sexta and Ram et al (1980)
in C. formicarius have noted that the quantity of compound administered is also
an important factor in producing morphogenetic effect at the time of maximal
sensitivity only alongwith the age of pupa. Slama et al (1974) have noted that
application of different doses of the juvenoids during the critical period evokes the
formation of intermediate forms between the previous and subsequent develop,
mental stages. Our observation on Spodoptera pupae have shown that up to
20 hr there is an increase in morphogenetic response with increasing doses of JHA
but an increase of JHA dose has no effect when older pupae (26 and 38 hr old) are
treated. It has been observed that when 2 hr old pupae were treated with higher
doses (10-25//g) pupal-adult transformation was more or less completely inhibited
and p-a intermediates of grade VI were produced, but with lower doses (2-5 jug),
the differentiation of proboscis, thorax and posterior abdomen was not inhibited
although the external genitalia were deformed (Grade V). When 14 hr old pupae
were treated with the lowest dose (2/^g), the developmeitf of thorax, wings an4
346 U S Srivastava and S S Prasad
posterior abdomen with comparatively less developed and deformed external
genitalia could not be inhibited (Grade III) and with higher doses (5-25 /zg),
differentiation of thorax and posterior abdomen was not inhibited (Grade IV).
Finally when 20 hr old pupae were treated with 2 //g, the differentiation of the
middle abdomen also could not be inhibited (Grade II). In short, the twin factors
of age and quantity of hormone administered together determine the extent of
inhibition of p-a transformation up to the age of about 20 hr but beyond this age
an increase in the dose of juvenoid above the effective quantity does not enhance
the juvenilising effect.
The production of larval-pupal or pupal-adult intermediates of different grades
by JH administration in different quantities and to larvae or pupae of different
ages shows that (i) the critical time for the onset of the transformation of the
different parts or organs of the body of an insect is different and (ii) similarly the
critical concentration of the hormone required to inhibit such transformation is
also different. Slama etal (1974) and Willis (1974) had also shown earlier that
the critical periods for different organs and cells in a larva or pupa occur at
different times. In S. bullata, Srivastava and Gilbert (1969) noted that the thorax
is the first to become refractory to hormone during pharate adult life followed
by the head and then the abdomen. Gilbert and Schneiderman (1960) in
A. polyphemus and Metwally and Sehnal (1973) in T. granarium and C. gonagra
found that external genitalia and the abdomen as a whole were the first sensitive
parts, then the thoracic structures and finally the appendages and mouth parts.
In many species of endopterygotes, Slama (1971) had noted that the epidermal
cells of the wing lobes, thoracic and head appendages, and external genitalia lose
their sensitivity to juvenoids sooner than the epidermal cells of abdomen. In
stored grain insects, Srivastava (1980) noted that the order of decreasing sensitivity
was abdomen > thorax > head. In Spodoptera pupae, the sequence of trans-
formation of different body regions on the basis of different grades of pupal-
adult intermediates can be described as follows : anterior abdomen > middle
abdomen > posterior abdomen, external genitalia and wings > head and thorax.
Bowers and Williams (1964), Krishnakumaran et al (1967) and Riddiford and
Ajami (1973) believe that in Manduca sexta, maximal sensitivity to exogenous
JH occurs at the beginning of epidermal retraction which is immediately followed
by DNA synthesis, prerequisite to the formation of adult cuticle. Thus, according
to this view, in order to be effective an increased titre of JH/JHA should be available
in the insect system and it should start action before the process of cellular differen-
tiation is set into motion, probably at a time when the gene sets are being
reprogrammed for a new developmental cycle.
Metwally and Sehnal (1973) have observed that even the most severely affected
specimens of Trogoderma resulting from pupal treatment, displayed slightly pig-
mented eyes, outlines of segmentation of appendages, and most important of
these, the absence of pupal hair. The maximally affected individuals of Caryedon
also resembled normal pupae except that their eyes and appendages showed adult-
like differentiation. In the course of our observations published elsewhere, we
have noted that the pigmentation of the developing compound eyes as seen in
these intermediates, is not normal. In fact, the pigmented part comprises an area
where an excessive deposition of pigmented material in the cuticle occurs
Pupae treated with juvenoid 347
in this area or areas, ommatidial differentiation and development are greatly
inhibited. Such pigmentation, therefore, does not indicate normal or near normal
development of the ommatidia in comparison to the unpigmented region which
is assumed to indicate lack of ommatidial development and from a study of the
eyes of different grades of p-a intermediates it becomes clear that the extent of
abnormal (i.e. heavy) pigmentation and ratio of heavily pigmented and un-
pigmented/weakly pigmented zones increases, while the ommatidial differentiation
decreases with increasing score of p-a intermediates.
The present work indicates that in the case of 5. litura, the order of critical
periods for the metamorphic transformation of several parts or organs can be
separately worked out if similar but a more intensive study was carried with the
pupal period relatively prolonged, say by rearing the pupae at lower temperature.
Acknowledgements
Financial assistance from CSIR in the form of fellowship to SSP is gratefully
acknowledged. Thanks are due to R. Maag Ltd., Basel, Switzerland, for the
gift of the compound.
References
Bagley R W and Bauernfeind J C 1972 in Insect juvenile Hormones, (ed) J J Menn and
M Beraza (New York : Academic Press)
Bhaskaran G 1972 Inhibition of irniginal differentiation in Sarcophaga bullata by juvenile
hormone ; /. Exp. Zool 182 127-142
Bhatnagar-Thomas P L 1972 Laboratory evaluation of synthetic juvenile hormone analogue for
the control of Trogoderma granarium Everts ; Indian J. Entomol. 34 87-93
Bowers B and Williams C M 1964 Physiology of insect diapause~XII. DNA synthesis during
the metamorphosis of Cecropia silkworm ; Biol Bull. (Woods Hole Mass.) 126 205-219
Critchley B R and Campion D G 1971 Effect of synthetic juvenile hormone and a juvenile
hormone analogue, methyl farnesoate dihydrochloride, on pupal development of the yellow
mealworm Tenebrio molitor L ; Bull. Entomol. Res. 61 293-297
Daoud D S and Sehnal F 1974 Effects of juvenoids on the Mediterranean fruit fly, Ceratitis
capitata (Wied.) (Diptera, Tephritidac) ; Bull. Entomol Res. 64 643-651
Gilbert L I and Schneiderman H A 1960 The development of a bioassay for the juvenile
hormone of insects ; Trans. Am. Microsc. Soc. 79 38-67
Krishnakumaran A, Berry S J, Oberlander H and Schneiderman H A 1967 Nucleic acid
synthesis during insect development-II. Control of DNA synthesis in the Cecropia silk-
worm and other Saturniid moths ; /. Insect Physiol. 13 1-57
Metwally M M and Sehnal F 1973 Effects of juvenile hormone analogues on the metamorphosis
of beetles Trogoderma granarium and Caryedon gonagra ; Biol Bull. (Woods Hole Mass.)
144 368-382
Ram G M, Rao B K, Thakur S S and Rao P N 1980 Effectivity of a synthetic juvenile hormone
on the pupae of sweet potato weevil, Cylas formicarius F. (Coleoptera : Curculionidae) ;
Proc. Indian Acad. Parasitol. 1 30-34
Reddy G and Krishnakumaran A 1973 Changes in the morpho.genetic response of Tenebrio
molitor pupae to juvenile hormone in relation to age ; /. Insect Physiol. 19 773-780
Riddiford L M and Ajami A M 1973 Juvenile hormone : its assay and effects on pupae of
Manduca sexta ; /. Insect Physiol 19 749-762
Slama K 1971 Insect juvenile hormone analogues ; Annu. Rev. Biochem. 49 1079-1102
Slama K, Romanuk M and Sorm F 1974 Insect hormones and bioanalogues (Wien— New
Yoik : Springer Verlag)
348 V S Srivastava and S S Prasad
Socha R and Sehnal F 1972 Inhibition of adult development in Tenebrlo molitor by insect
hormones and antibiotics ; /. Insect Physiol. 18 317-337
Srivastava R C 1980 Effect of certain juvenile hormone analogues on the biology of certain
stored grain insects ; D.Phil. Thesis, University of Allahabad, Allahabad.
Srivastava U S and Gilbert L I 1969 The influence of juvenile hormone on the metamorphosis
of Sarcophaga bullata; J. Insect Physiol. 15 177-189
Srivastava U S and Srivastava R C 1974 Effects of juvenile hormone analogue on certain
aspects of the biology of Trogoderma granarium Everts ; Proc. Natl. Acad. Set. 44 99-120
Tan K H 1975 Effect of a synthetic juvenile hormone and some analogues on Ephestia spp,
(Lepidoptera— Phycitidae) ; Ann. AppL Biol 80 137-146
Williams C M 1961 The juvenile hormone II. Its role in the endocrine control of molting,
pupation and adult development in the Cecropia silkworm ; Biol. Bull. ( Woods Hole Mass,)
121 572-585
Willis J H 1974 Morphogenetic action of insect hormones; Amu. Rev. Entomol. 19 97-115
Piroc. Indian Acad. Sci. (Aitim. Scl), Vol. 91, Number 4, July 1982, pp. 349-355,
© Panted in India.
A comparative study on certain biochemical aspects of red and
white myotomal muscles of the black skipjack tuna, Euthynnus affinis
Cantor
N GOPINATHAN PILLAI and K M ALEXANDER*
Department of Zoology, University of Kerala, Kariyavattom695581,Trivandnim,
India.
; MS received 23 May 19S1 ; revised 21 May 1982
Abstract. The biochemical assay of certain metabolites of the red and white myo-
tomal muscles of the tuiia, Euthynnus affinis Cantor has been carried out. The
metabolites exhibited a marked variation in their distribution pattern in red and
white muscles. The narrow red fibres are characterised by higher levels of lipid,
glycogea, myoglobin and SDH while the broader white fibres had lessor amount of
the above metabolites. The distribution of metabolites-— the myoglobin and SDH,
revealed a gradientfrom the superficial towards the inner layers of the red myotomal
muscle in both the pectoral and middle regions. The physiological relevance of
these biochemical variations in diverse 'regions of the red and white muscle is
discussed. ' '•:"•• :
Keywords. Skipjack. tuna.; red and: wfcite muscles; prof eib ; fuel reserves-;
rnyoglobin ; st>H, Ettthynnus: affinis. *
1. Introduction
Tuna are actively swimming, commercially important teleosts, exhibiting unique
adaptation for maintaining a higher, body temperature than the surrounding ambient
medium. Similar to certain other teleosts, tuna also possess two types of myo
tomal muscles —the red and white, with the red muscle lying near the spine and
Constituting about 5 to 20% of the total body weight (Modigh and Tota 1975).
Morphological and biochemical investigations on red and white muscles of fishes
have elaborated the functional differences between them. (Love 1970). Generally the
red fibres are adapted for long-term cruising movements, utilizing lipid as the
main .source of .energy and the white fibres for short-term activity metabolising
glycogen as the chief fuel (George 1962 ; Black et al 1962 ; Bilinisky 1963 ;
Bone 1966 ; Love 1970). However, relatively very little information is available
regarding the physiology of these muscles and their nutritional significance
in the black skijyack tuna, Euthynnus. .qffinfa. . . •• . .... ./.:
\Accor4ingly a study has beep, undertaken to -elaborate, the comparative bio-
chemical aspects ,of these red.aud white muS©leS;ih Euthynnus affinis ^^ Cantor. ; ... .
made. -.V.^"...> .:.,^... .-. >•,-.:.".; ^<.i. t.^:.^-..:
349
350 ff Gopinathan Filial and J£ M Alexander
2. Materials and methods
Investigations were carried out on tuna weighing 2 to 3 kg having a range of
35 to 55 cm total body length. The fishes were collected from boats immediately
on landing at the Shankumugham Beach at Trivandrum and were transported
immediately in refrigerated containers to the laboratory. The muscle samples
were excised from the superficial, middle and inner most layers from three
different regions of the body, viz, the pectoral, middle and the caudal regions
(single sample) for biochemical assay by employing standard -analytical techniques;
The following methods were employed for the estimation of lipid, glycogen, total
protein, myoglobin and SDH.
(a) Lipid Nayeemunisa and Rao (1972) <
(b) Glycogen Anthrone Reagent technique of Seifter et al (1950)
(c) Protein Wong's microkjeldhal method (1923)
(d) Myoglobin Tappan and Raynaferjee (1957)
(e) SDH Kun and Abood (1949) using the tetrazolium salt as
the electron acceptor.
The optical density, of the aliquots obtained was measured in a photoelectric
colorimeter (Bausch and Lomb, Spectronic 20).
3. Results
The data on biochemical aspects of these muscles are shown in table 1. Regarding
moisture content, the pectoral and middle regions of the red muscle showed only
a very slight variation between the superficial layer and the inner layer (Pectoral
PS-69-68 % ; PM-70-71 %. and PI-7Q.-97% and .middle . MS^7Q- 09% ; MM-
69-88% and MI-69-60%) with the caudal region exhibiting a moisture level of
69-84%. The white muscle exhibited relatively higher percentage of moisture
72-57%.
Comparatively, the protein levels of the red muscle did not reveal any variation
with the middle layer of the pectoral region having the maximum amount of protein
(20- 08 %). The inner layer of both pectoral and middle regions exhibited slightly
lower values (19-23% and 19-65%). The caudal region had 19-91%. The
white muscle exhibited a relatively higher protein level (22-48%).,
The maximal amount of lipid has been recorded from the middle layers of
pectoral and inner layer of middle -regions (17-02% and 14-98%). The super-
ficial layers of these regions showed slightly lower values of lipid (15-07% and
13-26%). Interestingly enough both these regions exhibited a gradient in distri-
bution of lipid with the maximum being at the inner and minimum in the super-
ficial. The caudal region had a lower level .(12*42%) of lipid. Regarding tfc£
white muscle the lipid. concentration, .was very much lower than that of the red
muscle ($-.96%). . ... .......
The middle layers of pectoral and middle, regions exhibited maximal quanta of
glycogen (PM«300- 95 and MM-^365-85 /jg/lOQ-mg) with a minimal amount at the
inner layer (PW230-99 and MI~-262*63 //g/100mg). The superficial layers
showed 233-38 and 284-49 /Kg/100 mg of glycogen in the pectofal a^d
regions respectively. A higher amount qf £lycQge&. {$&-£l /€/I(>6
Myotomal muscles of E. ajftnis Cantor
351
352 N Gopinathatt Pillai and.K M Alexander
discernible at the caudal region. As for the white muscle the glycogen concen-
tration was significantly very much lower (60-15/*g/100 mg).
The myoglobin levels of both pectoral and middle regions exhibited a marked
gradient from the superficial to inner layers (PS— 8-6293 ; PM— 12-0627 ; PI—
13-3008 and MS— 8-1042; MM— 14-5783 and MI— 15-8273 mg/g wet tissue.
The caudal region had 13-4902 mg/gm of myoglobin. Relatively, the white
muscle revealed only a very much lower value for myoglobin (2 -4635 mg/g).
The concentration of SDH at the pectoral and middle regions exhibited an
increasing gradient from superfici alto wards the inner layers— viz., (Pectoral ; PS —
42-85 ; PM— 57-46 ; and PI-59-19 and middle ; MS— 45-67 ; MM— 62-49 and
MI— 65-68). The SDH level in the caudal region was relatively higher (68-41)
while the concentration in the white muscle was very much lower (27-87 //gm —
formazan/min/g).
4. Discussion .
The moisture concentration revealed only a very narrow range of variation in
the red muscle samples from different regions of the tuna fish. Nevertheless a
comparatively higher percentage has been noted in the white muscle: Alexander
(1955), Love (1970) and Chinnamma (1975) had reported a higher value of
moisture content in white muscles of fishes.
Studies on the protein content of Euthymus affinis did not reveal any significant
variation in the red muscle samples. However, protein level was comparatively
higher in the white muscle. This is similar to those of the ayian muscles such as
p.geon pectoralis imiscle'.as reported by Pishawikar (1961). It has been suggested
that the higher total protein content in the white muscle is due to structural proteins*
such as actin and myosin and the higher water soluble protein in the red fibers is
due to their higher enzyme concentration (George and Berger in Ayian myology
1966). Further, lower levels of total protein had been reported in the dark muscles
of Sardinia, melanosticta (Fujikawa andNaganuma 1936) ; Scomber ycombrus and
Thunnus thynnus (Braekkan 1959) which are also in conformity with the values
observed In the dark muscles of Euthynnus affinis.
Regarding fuel reserves, data indicate that the red muscle fibres have a higher
fuel reserve with a preferential dependence on lipid. Lipid constitutes an impor-
tant source of fuel reserve for muscle contraction ; the metabolism of which yield
sufficient ATP (West etal 1956). In tuna, the relatively highu? level of lipid in
the pectoral region may be due to the continuous activity 6f the pectoral fins.
Farther, it is also possible that the red muscle fibres are capable of providing the
requisite amount of energy for the slow and sustained contraction for the long
term swimming activity of the fish by the aerobic oxidation of the lipids. The
relatively lower level of lipid in the white muscle maybe due to their non-involve-
ment in slow and sustained activity and are mainly meant for fast spurts of move*
ment using mamly glycogen as the fuel. The breakdown of lipid is most evident
in those fish which migrate without feeding (Bilinisky 1963). It has been reported
that in the trout (Salmo gairdnerii) the ability of dark muscles to oxidise fatty
acid was much greater than that of the ordinary muscles by the presence of an
enzyme system in the red muscle (Bilinisky 1963). The observations of Drummond
Myotomal muscles of E. affinis Cantor 353
and Black (1960) had revealed that fat metabolism provides the energy for
sustained swimming in the up stream migration of salmonids. Further, studies
of Braekkan (1959) in Clupea harengus ; Gadus virens, Salmo salar ; Alexander
(1955) in Scatophagus argus and Labeo rohita; George (1962) in Rastrelliger
:kanagurta ; Zama (1963) in Thynwts orientalis etc., had revealed a much higher
lipid concentration in the red muscles of these fish.
Glycogen is one of the major fuel reserves of the muscle. Studies on Euthynnus
affinis have revealed a higher concentration of glycogen in the red muscle almost
ranging over two to three times than that of white muscle. In fishes usually the
white muscle produces much of the energy for sudden bursts of activity by
anaerobic metabolism (Rayner and Keenan 1967). Apart from this, certain functions
have been attributed to the red muscles in fish myotome. Among these, Arloing
.and Lavocat (1875) have suggested that the two types of fibres— the red and
white were active during different phases of swimming. George (1962) had
reported in the teleost, Rastrellinger kanagurta, the red muscle was adapted for
continuous and slow contractions while the white fibres effecting quick and fast
contraction. Further, the observations of George (1962) ; George and Bokdawala
(1964) ; Bone (1966) ; Love (1970) corroborate the view that the red muscle facili-
tates continuous muscular activity of the animal.
Comparatively a lower concentration of glycogen was recorded in the tuna white
muscle. Bokdawala and George (1967) had suggested that the probable deple-
tion of glycogen in the white muscle may be due to the fact that it might have been
used up since the white muscle fibres are involved in quick and sudden movement
during capture by utilizing the energy derived from the breakdown of glycogen
Studies by Driedzic and Hochachka (1976) in carp muscle had revealed an increase
in glycolytic intermediates during activity. Thus in tuna, Euthynnus ajfinis, the
.^comparatively higher levels of lipid and glycogen in the red muscle indicates the
.continuous -and higher rate of utilization of these fuels for the active swimming
habits of this teleost.
The characteristic red colour of the tuna red muscle is due to the preponderance
of myoglobin. It plays a salient role in the transport and storage of oxygen in
the nxuscle (Lawrie 1952) and has the capacity for rapid oxygenation and deoxy-
genation. Thus in tuna, the higher myoglobin levels in the red muscle facilitate
a better diffusion of oxygen into the red muscle and function as a store house of
oxygen for the aerobic oxidation. Further, in red muscle the main energy source
.is lipid and it can be metabolised aerobically which warrants a sufficient supply
of oxygen. Observations of Modigh and Tota (1975) in Thunnus thynnus revealed
that mitochondria from deep red muscle consume more than thrice as much
oxygen as those from white muscle when the complete electron transport chain
is in dperation. Moreover, in Euthynnus affinis, the .higher level of myoglobin
in the inner layers of pectoral and middle red muscle regions, wherein the arteries
and veins are highly concentrated may possibly have specific role in the produc-
tion and maintenance of slightly higher body temperature together with the
<6 rete mirabili ", which plays a prominent role in these parts of the? muscle
^(Carey 1973). - . . . •
The data obtained on SDH (succinic dehydrogenase), the prime mover of
oxidation in the metabolic process going on in a muscle, indicate that its levels are
much higher in the red muscle with an increasing gradient from the superficial to
354 N Gopinathan Pilldi and K M Alexander
the inner layers in both pectoral as well as the middle regions. However, the
white muscle exhibited a significantly lower SDH level It is well-known that
the level of SDH in different layers of the muscle can be correlated to their oxidative
capacity. Further, it provides an indication of the mitochondrial intensity
Hence the higher levels of SDH in the red muscle fibres of the tuna fish reflect its
higher oxidative capacity. Similar data have been reported by Talesara aiid
Narang (1979) in- mammalian and avian muscles. In fact the relatively higher
metabolic demands of red muscle warrant a higher SDH concentration in corre-
lation with the increased myoglobin content.
In consensus, the significant variation discernible in the biochemical para-
meters, especially fuel reserves,m yoglobin and SDH in red muscles are in accord
with the specific functional requirements of these red muscles viz., the substained
muscular activity, production of increased metabolic energy for maintaining a
higher body temperature than the ambient medium.
Acknowledgement
NGP is indebted to the University of Kerala and University Grants Commission
for providing financial assistance in the form of fellowship.
References
Alexander K M 1955 A compraisoa of the gross chemical composition of red and white muscles
in the two fishes Scatophagus argus and Labeo rohita ; /. Anim. Morphol.Physiol. 1 58-61
Arloing S and Lavocat A 1S75 Recterches Sur I'. Anatomic et al physiologic des muscles
stress piles et foices ; Msm. Acad. Sci. Inscript. Toulouse T. 7 177-197
Bllinisky E 1963 Utilization Of lipid by fish. 1. Fatty acid oxidation by tissue slices frofti dark
and white muscles of Rainbow trout Saimo gairdmrrt ) ; 'Can. J. Biochem. PhysioL 41
107-112
Black E C, Connor A R, Lam K C and Chia W G 1962 Changes in glycogen, pyruvate and
lactate in rainbow trout (Saimo gairdneril) during and following muscular activity ; /. Fish.
Res. Board Can. 19 409-436
Bokdawala F D and George J C 1967 A quantitative study of fat, glycogen,lipase and succinic
dehydrogenase in fish muscle ; /. Anim. Morphol. PhysioL 14 223-230
Bone Q 1966 On the function of the two types of myotomal muscle fibre in elasmobranch fish;
/. Mor. Biol. Ass. U.K. 46 321-349
Braekkan O R 1956 Function of red muscle in fish; Nature (London) 178 747-748
Braekkan O R 1959 A comparative study of vitamin in the trunk muscles of fishes ; Fisk. Dir.
Skr. Ser. Teknol. Und. Kol. 3 42
Carey F G 1973 Fishes with warm bodies ; Set, Am. 228 36-50
Chirkaamma George 1975 Biochemical differences between the red and white meat of tuna
(Katsuwonus pelamis) and changes in quality during freezing and storage ; Fish. Technol.
1270-74
Driedzic W R and Hochachka P W 1976 Control of energy metabolism in carp white muscle ;
Am. J. PhysioL 230 579-582
Drummond G I and Black E C 1960 Comparative physiology fuel of muscle contraction ;
Annu. Rev. PhysioL 22 169-190
Fujikawa K and Naganuma H 1936 Chemical composition of sardine, Sardinia melanosticta
(C and V) from Tyosen. 1. Comparative study on dark muscle and white muscle ; Bull.
Jpn. Soc Sci. Fish. 5 95-102
George J C 1962 A histophysiological study of the red and white muscles of the mackerel;
Am. MM. Nat. 68 487-494
Myotomal muscles of E. affinis Cantor 355
George J C and Berger A C 1966 Avian myology (New York : Academic Press)
George J C and Bokdawala F D 1964 Cellular organization and fat utilization in fish, muscle •
/. Anim. Morphol. PhysioL 11 124-132
Kun E and Abood L G 1949 Colorimetric estimation of succinic dehydrogenase by Triphenyl
tetrazolium chloride ; Science 109 144-146
Lawrie R A 1952 Biochemical difference between red and white muscle ; Nature 170 122
Love R M 1970 The chemical biology of fishes (London and New York : Academic Press)
Modigh M and Tota B 1975 M'tochondrial respiration in the ventricular myocardum and in
the white and deep red myotomal muscles of the juvenile tuna fish (Thunnus thynnus L.) ;
Acta PhysioL Scand. 93 289-294
Nayeemunisa and Rao K P 1972 Effects of thermal acclimation in thelipid metabolism in the
earthworm ; Lampito mauritii^A method for lipid estimation ; Comp. Biochem. PhysioL
B42 167-173
PIshawikar S D 1961 A study of the phosphates, phosphatases and certain inorganic ions in the
pectoralis muscle of some birds with special reference to that of the pigeon. Doctoral Thesis
M.S. University of Baroda, Baroda, India
Rayner M D and Keenan M J 1967 Role of red and white muscles in the swimming of skip-
jack tuna ; Nature (London) 214 392-393
Seifter S, Dayton S, Novic B and Muntwyler E 1950 The estimation of glycogcn with the
Anthrone reagent; Arch. Biochem. 25 191-200
Talesara C L and Vasdev Narang 1979 A comparative study of myofibrillar ATPase (/w-ATPase)
and succinic dehydrogenase (SDH) activities in certain specialised muscles of rat (Rattus
norwegicus)fnom various representative regions; Indian J. Exp. Biol. 17 219-221
Tappan DU and Raynaferjee B 1957 Tissue pigment manifestations of adaptation to h;gh alti-
tudes ; Am. J. PhysioL 190 99-103
West E S, Todd W R, Manson H S and Bruggen J T V 1956 A text book of Biochemistry.
(New York: Macmillan Co.)
Wong 1923 Estimation of proteins in blood plasma ; /. Biol. Chem. 55 427
Zama K 1963 Studies on the phospholipids of aquatic animals; Mem. Fac. Fish. Hokkaido
Univ. 11 pp 73
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 4, July 1982, pp. 357-360.
© Printed in India.
Orcadian basis for the photoperiodic response in the male
blackheaded bunting (Emberiza melanocephala)
VINOD KUMAR and P D TEWARY*
Department of Zoology, Banaras Hindu University, Varanasi 221 005, India
MS received 7 November 1981 ; revised 19 May 1982
Abstract. Short day (6 hr light in a 24 hr cycle (LD 6 : 18)) inhibits growth and
development of the testes in male blackheaded buntings, whereas the same (6 hr)
nonstimulatory photoperlods in a 36 hr cycle (LD 6 : 30) induce complete testicular
recrudescence and development. In another experiment of 24 hr cycles, using the
same (6 hr) main photoperiod, testes were stimulated when the dark period was
interrupted by light at 12 to 13 hr after the onset of basic photoperiod (LDLD 6:6:
1 : 11). The results appear to conform to the tenets of the external coincidence
model.
Keywords. Blackheaded bunting ; photoperiod ; circadian ; rhythm ; light ; dark
cycle ; external coincidence model.
1. Introduction
Since the pioneer studies of Hamner (1963) on house finches (Carpodacus mexi-
canus), the nature of the photoperiodic response mechanism(s) has been experi-
mentally investigated in many photoperiodic birds (see reviews, Farner and Lewis
1971 ; Follett 1973 ; Farner 1975 ; Farner etal 1977 ; Turek 1978). The results
from these experiments agree with the classical Biinning hypothesis which mentions
an endogenous circadian rhythm of sensitivity to light as the physiological basis
for photoperiodism (Biinning 1973). The validity of a circadian basis for photo-
periodic time measurement in birds is generally tested by resonance and night-
interruption experiments (see reviews, Follett 1973 ; Farner 1975 ; Turek 1978),
Here, we report the results of night-interruption experiments designed to test the
influence of an endogenous circadian rhythm in the photoperiodic time measure-
ment of blackheaded buntings.
1. Materials and methods
Wild adult male blackheaded buntings (Emberiza melanocephala) were acclimatized
to laboratory conditions for a fortnight. These acclimated birds were pretreated
* To whom correspondence should be made.
357
358
Vinod &umar and P D Tewary
for 8 weeks with short days (LD 8 : 16) ensuring that they were photosensitive at
the time of exposure to different light regimes. Three groups (numbered I, II
and III) of birds then were marked individually and held under different
programmed photoperiods (LD 6 : 18, LD 6 : 30 and LDLD 6:6:1:11, respec-
tively) for a fixed period (see table 1) inside light-boxes. Food and water were
freely available. The birds were lit by fluorescent tubes at an intensity of about
400 lux at perch level. The first experimental photophase was in phase with the
pretreatment schedule and commenced at 06-00 hr. The birds were laparotomized
at the beginning and end of experiments, and only during the main light phase of
the cycle. Testicular growth was assessed as combined testicular weight in situ
and by comparing with the standard set of gonads of known weights. The error
by this method is about (±) 20 %. The data from one bird of group III that
died during the course of study were not included in our statistical analysis. The
data were analysed using student's ' t ' test.
3. Results and discussion
The results are presented in table 1. The birds either of group I (LD 6 : 18) or
of II (LD 6 : 30) received equal photoperiods (6 hr) per cycle but only the birds of
the latter group responded. Since the extended period of darkness could appear
to initiate the gonadal recrudescence, in a separate experiment, buntings were held
in constant darkness (DD) for 100 days and found not to respond (unpublished
results). Further, the birds of group III (LDLD 6:6:1:11) also responded although
the total amount of darkness which these birds received per cycle (17 hr)
was even less than the amount which birds of group I were experiencing (18 hr).
The duration of light also cannot be a factor in initiation of the testicular growth
in the buntings, since the total amount of light per cycle given in all the experi-
ments (6 hr or 7 hr) was much shorter than the photoperiodic threshold for the
species which lies at 11 to 12 hr light per day (Kumar and Tewary 1982). Further,
it is to be noted that a light regimen consisting of 8 hr photoperiod (LD 8 : 16)
Table 1. The gonadal responses of Emberiza melanocephala exposed to 3 different
light regimes.
Group
Light regime
(light : dark)
Light
cycle
(in hr)
Combined testicular weight (mg)
treatment
(in weeks)
Initial
Final
I
LD 6 : 18
24
5
9-5il-68(6)
7-00±0-63(<5)
II
LD 6 : 30
36
5
8-0±0-00(7)
285 -71 ±21 -03 (7)
ni
LDLD 6 :6 :1 : 11
24
6
7-5±0'50(6)
216-00±22-49(5)
Value in parenthesis gives the number of individuals
Time measurement in buntings 359
for 6 months could not induce the testes of blackheaded buntings (Tewary and
Kumar 1982). :
It appears that neither the amount of light or dark nor the ratio of light to
dark is the determining factor in stimulating the gonadal growth and develop-
ment in blackheaded buntings. Our data agree with those obtained with
similar experiments on other known photoperiodic birds (Hamner 1963, 1964 ;
Follett 1973 ; Farner 1975 ; Tewary and Kumar 1981a, b ; Chandola et al 1976).
Such results may best be interpreted on the basis of an endogenous circadian
rhythm involvement in the * photosensitivity ' of the hypothalamo/hypophyseal/
gonadal system (Follett 1973 ; Farner 1975 ; Turek 1978). According to the
external coincidence model, first developed by Biinning (1973), a photoperiodic
induction occurs if and only if photophase coincides (repeatedly, daily or other-
wise) with the photosensitive phase ( = photoinducible phase, subjective night)
of the circadian rhythm. In the present experiments presumably the birds of group
II received 6 hr light at alternate cycles, and of group III were receiving 1 hr
light period daily in the photosensitive phase and a response was obtained in both
the groups. In contrast, the birds of group I were receiving light periods (6 hr)
daily only in the photoinsensitive phase (= non-photoinducible phase, subjective
day), and hence photostimulation failed to occur.
Acknowledgement
Financial assistance from the University Grants Commission, New Delhi, is grate-
fully acknowledged. The results were presented at the third Conference of Indian
Society for Chronobiology : A joint meeting with the International Society for
Chionobiology, Varanasi, 1979.
References
Biinning E 1973 The physiological dock. 3rd (ed.) (New York, Heidelberg, Berlin : Springer-
Verlag)
Chandola A, Singh R and Thapliyal J P 1976 Evidence for a circadian oscillation in the gonadal
response of the tropical weaver bird (Ploceus philippinus) to programmed photoperiods ;
Chronobiologia 3 219-227
Farner D S 1975 Photoperiodic controls in the secretion of gonadotropins in birds ; Am. Zool.
15 117-135
Farner D S, Donham R S, Lewis R A, Mattocks P W, Darden T R and Smith J P 1977
The circadian component in the photoperiodic mechanism of the house sparrow, Passer
domesticus ; Physiol Zool 50 247-268
Farner D S and Lewis R A 1971 Photoperiodism and reproductive cycles in birds ; in Photo-
physiology (ed.)} A. C. Giese (New York and London : Academic Press) Vol. 6 pp 325-370
Follett B K 1973 Circadian rhythms and photoperiodic time measurement in birds ; /. Reprod.
Fertil 19 5-13
Hamner W M 1963 Diurnal rhythm and photoperiodism in testicular recrudescence of the
house finch ; Science 142 1294-1295
Hamner W M 1964 Circadian control of photoperiodism in the house finch demonstrated by
interrupted-night experiments ; Nature (London) 203 1400-1401
Kumar V and Tewary P D 19S2 Response to experimental photoperiods by a migratory bunting?
gmberiza melanocephala ; Ibis (accepted).
360 Vtnod Kumar and P D Tewary
Tewary P D and Kumar 19Sla Involvement of circadian rhythm in photoperiodic response in
the male common Indian rosefinch Carpodacus erythrinus ; Indian J. Exp. BioL 19 77-79
Tewary P D and Kumar V 1981b Circadian periodicity and the initiation of gonadal growth in
the male blackheaded bunting (Emberiza melanocephala) ; /. Comp. PhysioL 144 201-203
Tewary P D and Kumar V 19S2 Photoperiodic response of a subtropical migratory finch, the
blackheaded bunting (Emberiza melanocephala) ; Condor 84 168-171
Turek F W 1978 Diurnal rhythms and the seasonal reproductive cycle in birds; In Environmental
endocrinology \ (ed.) I. Assenmacher and D S Faraer (Berlin, Heidelberg, New York: Springer-
Verlag) pp 144-152
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 4, July 1982, pp. 361-366.
© Printed in India.
Steroid metabolism in target related to nuptial plumage
production in the Baya weaver bird
V C KOTAK and G K MENON*
Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388 120,
India
* Department of Zoology, Faculty of Science, MS University of Baroda,
Baroda 390 002, India
MS received 5 January 1982 ; revised 21 June 1982
Abstract. To elucidate the possible utilization of gonadal sex hormones by the
nuptial plumage producing skin, histochemical localization of 3)?-, 3a- and 17P-
hydroxysteroid dehydrogenases was carried out in the skin from crown region
(characterized by bright yellow plumage) and ventrum, and testes of the Baya weaver
bird Ploceus philippinus (L) during the breeding phase. Results indicate higher
activity of 17/?-hydroxysteroid dehydrogenase in the crown skin, when testosterone
was used as substrate. Possibly, skin from the crown region actively metabolizes
androgens and this in turn is correlated to the production of nuptial plumage.
Keywords. Baya weaver bird ; histochemistry ; steroid dehydrogenases ; skin.
1. Introduction
It is a generally accepted concept that the accessory sex organs and secondary
sexual characters of vertebrates are under the control of sex hormones. Sexual
dimorphism of birds could be genetically determined (as in house sparrow) or
hormone mediated. As in human skin cytosol (Mowszowicz et al 1981), androgen
receptors have been reported in the uropygial glands of male ducks (Daniel et al
1977) which would be quite typical of an androgen target site. Interestingly, an
extragonadal direct effect of luteinizing hormone (LH) on the seasonal plumage
changes has been proposed in the orange weaver finch Euplectes frandscanus
(Witchi 1950) and Indian weaver bird Ploceus philippinus (L) (Thapliyal and
Tewary 1961, 1963 ; Thapliyal and Saxena 1961). It would be noteworthy to find
out the advantage of gonadotrophic control over gonadal steroids in the formation
of nuptial plumage in the weaver birds. The present work was aimed at finding
out whether the crown skin producing bright yellow nuptial plumage of the Baya
weaver bird is capable of utilizing gonadal hormones.
361
362 V C Rotak and G K Menon
2. Material and methods
Adult male Baya weaver birds Ploceus philippinus (L), were shot down in Vidya-
nagar University Campus during their breeding phase (August/September). Part
of the defeathered skin from the crown region and ventrum (ventral normal
colour skin) was fixed on the AO cryostat chuck maintained at - 20°C. Testes
of the same birds were also fixed in the cryostat. Sections (12 /mi thick) were
cut on the microtome and sections were processed for the demonstration of 3/J-
hydroxysteroid-dehydrogenase (3/J-HSDH ; Wattenberg 1958) ; 3a-hydroxysteroid-
dehydrogenase (3a-HSDH ; Balough 1966) and 17^-hydroxysteroid-dehydrogenase
(17/3-HSDH ; Kellogg and Glenner 1960). The pHfor 3a-HSDH incubation medium
was maintained at 7-7 (Ambadkar and Kotak 1978). The control sections in all
cases were incubated in media devoid of hormones.
3. Results and discussion
The enzyme intensities have been graded as under :
— Nil, -h minimum, + + moderate, -f + + maximum.
In all the three enzymes studied, epidermis revealed more activity than the dermis,
both in, crown as well in ventrum.
3-1. 3jS-HSDH (pr-egnenolone)
Activity in the ventrum was nil ( — , figure 5) and in the crown skin, moderate
(+"+, figure 2). Probably, interconnexions involving proandrogens may occur
in the crown skin. Testes exhibited high intensity (+ + +, figure 6) being more
or less uniform in the Ley dig cells and the seminiferous epithelium. Possibly, the
Sertoli cells play an important role in andrbgen synthesis as the interstitial cells.
It is now widely accepted that the Sertoli cells produce steroids that may influence
spermatogenesis (Bentley 1976). This aspect in the Baya weaver bird, however,
would demand more extensive seasonal investigations, particularly prior to, during
and post-reproductive phases vis-a-vis lipid cycle in the testicular compartments.
3-2. 3a-HSDH (androsteroney
Pattern of localization was more or less same as in case of 17 j8-HSDH (testo-
sterone) but the intensity was weak (+, figure 3). To a lesser extent, interconver-
sions between /^5-3 hydroxysteroids and A4 3-ketosteroids may occur in the
crown skin (e.g. androsterone to testosterone). Testes and ventrum showed
practically no activity. .
3-3. 17j8-HSDH (testosterone and estradiol)
Both, the ventrum and crown skin, showed minimum (+) activity with estradiol
as substrate. The female sex hormone does not seem to be metabolized by the
integumentary regions. As against this, the enzyme legalization with testosterone
Steroid de'iydrogenases in Say a skin
363
364
V C Kotak and G K Menon
%
Figures 4-6. 4 and 5. Ventrum (< », rest is pith tissue) reveals feeble lift-
HSDH (testosterone) and B^-HSDH (pregnenalone) localization (x 100). 6. T.S.
of testis shows high S^-HSDH (pregnenolone) in the seminiferous epithelium ( X 50).
Steroid dehydrogenases in Bay a shin 36$
revealed moderate to high activity (++, 4- + -h) in the crown skin (figure 1) and
minimum (+) in the ventrum (figure 4). There is thus good likelihood of 17-OH-
steroid to 17-ketosteroid (androgens) turnover in the skin from the crown region
(e.g., testosterone to androstenedione or vice versa), whereas the ventrum appears
to be hypo-sensitive in this regard. Testes exhibited moderate enzyme locali-
zation, once again, the seminiferous epithelium and interstitial cells displayed no
visible distinction at this time of the year.
In the South African weaver finches, the yellow and black breeding plumage
is believed to be under the control of LH (and not androgens) since castration
has no effect on plumage change. Also, administration of anterior pituitary
extracts to females or non-breeding males, castrate or intact, is followed by the
appearance of dark feathers (cited from Turner and Bagnara 1976). However,
Ralph et al (1965) have demonstrated that there is no direct action of LH on
changes in feather colour in these birds. Besides, in castrated birds, augmented
output of adrenocortico androgens cannot be ruled out. That avian skin is capable
of sex steroid hormone interconversions can be seen from the study on Sebright
cocks (George et al 1981). These workers have suggested conversion of testosterone
to estradiol by skin which is responsible for the typical female feathering trait of
males. Other target sites too possess such property; in drakes for example,
testosterone is rapidly converted to dihydrotestosterone (DHT) in target organs
(Horst and Paulke 1977). Our present report does not permit a conclusive
comment on the involvement of androgens in nuptial plumage production. How-
ever, it is apparent that the target skin (nuptial plumage producing crown region)
is endowed with relatively greater ability for steroid hormone metabolism than the
non-target areas.
Acknowledgements
Thanks are due to Prof. J J Shah, Head of the department of Biosciences, S P
University, Vallabh Vidyanagar, for facilities.
References
Ambadkar P M and Kotak V C 197$ Histochemical observations on 3 a- and 17jJ-hydroxysteroid
dehydrogenases in extra gonadal tissues of feral blue rock pigeon Columba livia G. ; Indian
J. Exp. Biol. 16 298-301
Balough K 1966 Histochemical demonstration on 3a-hydroxysteroid dehydrogenase activity;
/. Histochem. Cytochem. 14 77-83
Bentley P J 1976 Hormones in reproduction in Comparative vertebrate endocrinology (New York :
Cambridge University Press) pp 332
Daniel J V, Vignon F, Assenmacher I and Rochefart H 1977 Steroids 30 703
George F W, Noble J F and Wilson J D 1981 Female feathering in Sebright cocks is due to
conversion of testosterone to estradiol in skin ; Science 213 557-595
Horst H J and Paulke E 1977 Comparative study of androgen uptake and metabolism in
domestic and wild mallard drakes (Anas platyrhynchos L.) ; Gen. Comp. Endocrinol. 32
138-145
Kellogg D A and Glenner G G 1960 Histochemical localization of human term placental 17;?-
Estradiol dehydrogenase reaction ; Nature (JLondon) 187 763
V C Kotak and G K Menon
Mowszowicz I, Riahi M, Wright F, Bouchard P, Kuttenn F and Mauvais-Jarvis P 1981
Androgen receptor in human skin cytosol ; /. CHn. EndocrinoL Metab. 52 338-344
Ralph C L, Hall P F and Grinwich D L 1965 Failure to demonstrate a direct action of
luteinizing hormone (LH or ICSH) on regenerating feathers in African weaver birds ; Am.
ZooL 5 212
Thapliyal J P and Saxena R N 1961 Naturmssenschaften 24 751-742
Thapliyal J P and Tewary P D 1961 Science 134 738 cited from : Reproduction in Indian birds
by J P Thapliyal in Pavo 1978 16 151-161
Thapliyal J P and Tewary P D 1963 Naturwissenschaften 15 529-531
Wattenberg L W 1958 Microscopic histochemical demonstration of 3^-ol-dehydrogenase reaction ;
/. Histochem. Cytochem. 6 225-232
Witschi E 1950 Zur biologischen charakterisierung der gonadotropen hormone ; Naturwissenschaften
37 81
Proc. Indian Acad. ScL (Anirri. Sci.), Vol. 9J, Number 4, July lH2> pp. 367-371
© Printed in India.
Sex pheromone ID a stomatopod crustacean Squilla holoschista
M DEECARAMAN* and T SUBRAMONIAM**
Department of Zoology, Sri Theagaraya College, Madras 600021, India
* Department of Zoology, University of Madras, Madras 600005, India
*
**
MS received 25 January 1982 ; revised 29 May 1982
Abstract The stomatopods are well-known for their aggressive and agonistic
encounters. The males are normally aggressive ; the females too in the non-
reproductive condition show such a behaviour with males. In S. holoschista
mating is frequent as well as repetitive. The present paper explains whether there
is any involvement of sex pheromone. The sex pheromones are considered to be
present in ovaries, cement glands as well as oviducal extractions. These substances-
were tested for their pheromonal activity. The results indicate that there may not be
such attraction as evidenced by the lack of mating gestures from the isolated males
in the presence of these substances. It is therefore suggested that the mating in the
stomatopod, S. holoschista is indiscriminate. The physiological effect of such a
repeated and indiscriminate mating on the female is discussed.
Keywords* Pheromone ; natural sex attractants ; Squilla ; premating gestures.
1» introduction
The accumulation of evidences drawn from insects led to the introduction of
pheromone concept (Karlson and Liischer 1959). Recently sex attractants in the
form of pheromones have been found to exist in several crustaceans (Dahl 1975).
However due to lack of proper controlling methods, the mere existence of phero-
monos in Crustacea is questioned (Dunham 1978).
In decapod crustaceans the behavioural movements may be due to chemical
Or visual stimuli (Salmon 1965, 1971 ; McLeese 1971 ; Ryan 1966 ; Teytaud
1971). Initial behavioural contact between male and female is followed by mutual
exchanges of communicating signals. This helps in transmitting information of
species, identification of sex and reproductive drive from one animal to another
(Haztett 1975). Alteration in the agonistic behaviour has been shown to result
in the pair of reproductive male and female (Hazlett and Winn 1962 ; Nolan
and Salmon 1970). The act of copulation in marine Crustacea varies from one
species to another. In general, mating occurs in the freshly moult condition.
But there are exceptions to this rule (Hartnoll 1969). In lobsters and anomuran
species mating occurs normally between a fresh moult female and an intermoult
male (Berry 1970 ; Hazlett 1970, 1972). In some hermit crabs, Hazlett (1972)
367
P rm— <?
368 M Deecaramart and T Subramoniam
observed frequent mating and copulation was prolonged in hard-shelled crabs.
Dingle and Caldwell (1972) have observed in a stomatopod, Gonodactylus
breedini that mating is not preceded by moulting.
In spite of the elaborate mating processes reported in some decapod crustaceans,
not much is known on the pheromonal attraction among the males. Rittredge
etal (1971) have pointed out that " the closest parallel to insect pheromone commu-
nication observed in marine organisms are the sex pheromones of marine
Crustacea. " The available information on crustacean sex pheromone indicates
that their behavioural assays accepted as admissible evidence for sex pheromone
are as follows : (i) chemokinetic reactions, (ii) chemotaxic reactions, (iii) releaser
reactions (Dunham 1978). The presence of non-diffusible stimulating substances
have been found by Carlisle (1959) and Forster (1951) in Pandalus borealis and
in Leander serratus respectively. They have found that the stimulant is not
restricted to any particular part of the body ; instead even antennal contact seems
to be sufficient for exciting the male. Ryan (1966) reported on the water soluble
sex pheromone released through the urine of Portunus sanguinolentus during pre-
moult stage. Atema and Engstrom (1971) and McLeese (1971) have also demon-
strated the existence of water-soluble sex pheromone released by moulted mature
female lobsters. Kamaiguchi (1972) has shown in Palemon paucidens that such
sex attractant^is released from the sternal glands during prepaturial moult. Sex
pheromonal activity of the moulting hormone (crustecdysone) itself has been
indicated by Kittredge etal (1971). However Atema and Gagosian (1973) found
no evidence for the pheromonal activity for ecdysone or its analogue in the mature
males. Perhaps the occurrence of sex pheromone in more crustacean species
should be demonstrated in order to draw conclusions on its physiological speci-
ficity on the males. It is of interest to note in this connection that in a male crab
Emerita asiatica mating occurs indiscriminately without the involvement of any
sex pheromone (Subramoniam 1979). The aim of the present paper is to find out
whether the extracts of various female reproductive organs as well as the " female
water " possess pheromonal activity on the mature males kept in isolation in the
laboratory.
2. Material and methods
Squilla holoschista (Woodmason) used in the present study were collected from
the Madras coast and maintained in the laboratory in glass aquaria containing sea
water. Water was changed and sufficiently aerated every day. The animals were
fed regularly with fresh muscles of fish and prawn.
Behavioural sequences were observed in the glass tanks. Before the experi-
ments commenced the matured males and females were fed ad libitum and trans-
ferred into a tank of dimensions 60 X 25 X 31 cm, with sufficient sea water.
Before the experiment began the males were fully fed. Then the aqueous extracts
of ovary (0-5g of ovary in 1 ml of filtered sea water), cement glands (0*25g
of cement glands in 1 ml of sea water) and the oviduct (from three animals with
0-5 ml of sea water) were tried on the males when it comes to a motionless state.
This was repeated many times in order to find out the changes if any in the beha-
vioural patterns towards the premating gestures or agitated or searching beha-
Sex pheromone in stomatopod
Table 1. Behavioural sequences
369
Normal behaviour— Male
(In isolation within a period
of 30 min)
I. Mating— Male and female
(Deecaraman and Subramoniam 19&la)
(Generally at evening hrs — diffused
light)
Aggressive males
(Frequently exhibits)
1 . Antennule flicking
2. Spreading of the
raptorial meri at narrow
angle
3. Telson thrust
4. Forward and backward
movements
5. Motionless
6. Cleaning the cephalo-
thorax with telson spines
7. Coiling by bringing the
telson close to the
cephalic region
1. Antennule flicking— male and
female
2. Contact— male initiates
3. Male spreads the raptorial meii
female remains motionless
4. Male holds the female by
cephalothoracic appendages,
grasps and tilts the female
5. Male erects the intromittent
organs and moves towards
the female
6. Male exhibits thrusting move-
merts— Female orientates
towards the male
7. Release of male by the female
—strikes (indicates to some
extent aggressive behaviour)
II. Repeated mating
1. Both mile and female involve
2. Some behavioural movements
repeated as in column 1.
1. Antennule flicking
2. Meri spread out
widely
3. Strikes the oppo-
nents
4. Chase
Female
1. During non-
receptive condi-
tion
2. Strikes the male
at the end of
copulation
vioutal patterns as reported by Ryan (1966), Atema and Engstrom (1971) and
Kamiguchi (1972).
Similarly the effect of " female water " in changing the behavioural pattern
of male was also tested. The femaJe water was obtained by keeping a mature
female in a glass tank for six hrs. This female water was tested on males kept
in isolation in a glass tank. The behaviour of the males after the addition of
female water is compared with the normal mating patterns (Deecaraman and
Subramoniam 1981b). .
3. Results
In S. holoschista normal mating behaviour (Deecaraman and Subramoniam
1981b) could be easily differentiated from the aggressive encounters. The male
usually exhibits aggressive behaviour. However, the female also exhibits th$
370 M Deecaraman and T Subramontam
same when it is not in the receptive state. This aggressive behaviour by females
is also exhibited at the end of copulatory sequences.
The males when introduced into the trough start flicking the antennules in all
directions. This movement may last for few sec. Subsequently, the animals
spread the raptorial meri on both sides and withdrew them immediately. Then
the males move backward using the telson spines and the walking legs. Some-
times, the animals remain motionless up to 10 min, but keep the antennules and
the pleopods in motion. Following this the males exhibit forward and backward
movements using the thoracic and abdominal appendages, with telson spines
planted on the substratum. Frequently the animals clean the maxillipedes with
telson spines and also demonstrate " coiling " by bringing the telson and the head
close together. These movements may last from a few sec. to some min.
3-1. Experiments with ovary, oviduct and cement glands extracts
A mature male was introduced into the tank and its normal behavioural pattern
was observed. When the animal comes to a motionless state at one end, the
aqueous extract of ovary was introduced at the other end opposite to the animal
drop by drop. In the beginning the animal shows a positive response by moving
towards the point of application of the ovarian extract ; however, it immediately
retreats to its original place without showing any behavioural pattern positive
to premating gestures. Repeated application of the ovarian extract failed to show
any effect on eliciting the premating gestures. Similarly, the application of
oviducal and cement glands extracts did not have any effect on the males (table 2).
3 • 2. The female water
To test the effects of female water on the male behavioural pattern the mature
males were introduced in the tank. Even here the males failed to elicit any posi-
tive behaviour for attraction.
All these preliminary experiments suggest that there may not be any specific
stimulation of the male by the female by way of any pheromonal substances
(table 2). It is therefore suggested that mating in the stomatopod under labo-
ratory conditions occurs indiscriminately.
4. Discussion
Many available evidences in invertebrates clearly suggest the involvement of
pheromone and one such phenomenon is the settlement of marine larvae of
gregarious organisms (Dahl 1975; Crisp 1974). Another phenomenon of sex
pheromone is that of "epidemic spawning" (Gaits off 1938, 1940; MacGinite
and MacGinite 1949).
Various stomatopod species are known to exhibit agonistic and aggressive beha-
viour in natural copulation (Dingle and Caldwell 1969, 1976; Caldwell and Dingle
1976). Malacostracam, especially the brachyuran crabs, have been shown to
exhibit prolonged premating gestures before the external pairing (Hazelet 1975).
Maay others have attempted to explain this phenomenon by way of p
Sex pheromone in stomatopod
Table 2. Behavioural sequences— Male (within a period of 30 min)
371
I. Ovary
II. Oviduct
1. Normal behaviour
2. Motionless
3. Application of ovary extract
4. Antennule flicking
5. Advances towards the point of application
6. Retreats to the normal position immediately
7. No premating gestures or searching behaviour or agitated movements
8. Restore to the normal behaviour
1. Normal behaviour
2. Motionless
3. Application of oviduct extract
4. Antennule flicking
5. Avoids the point of application
6. No premating gestures or search behaviour
7. Restore to the normal behaviour
I [I. Cement glands
1. Normal behaviour
2. Mptionless
3. Application of cement glands extract
4. Antennule flicking
5. No premating gestures or searching behaviour or agitated movements
6. Restore normal behaviour
"Female water "
1. Antennule flicking
2. Motionless
3. No premating gestures or searching behaviour or agitated movements
4. Normal behaviour.
attraction (Ryan 1966; McLeese 1971; Kittredge etal 1971). Virtually nothing
is known about the origin of pl^romone i$ the aquatic invertebrates (Dunham
1978). Recently, JCittredge etal 1972 and Kittredge and Tak&haahi (1972) have
reported that the crustecdyzone or the related compound acts as sex pheromone
in some decapod crabs, however Atema and Gagosion (1973) have reported nega-
tive results to the sex pheromone response to any one of these? compounds in
Homams americamis. In Portuws sanguinofentus Christofibrson (1970) has
reported that the sex pheromone is of 1000 or less of molecular weight.
372 M Deecaraman and T Subramoniam
A recent study on the mating behaviour of sand crab E. asiatica has shown that
the tiny males may not be attracted to the female by any sex pheromone as the
attachment of the males to the females occur long before the actual deposition
of spermatophore and the attachment could also occur at any time of moult
cycle (Subramoniam 1977). It was also reported that the mating at least in this
crab is indiscriminate and that there may not be any pheromonal attraction
involved in it (Subramoniam 1979). It was also suggested that a pheromone may
not work in an environment of rapid water movements such as intertidal region
inhabited by E. asiatica.
The present results have not provided any evidence in support of a sex phero-
monal attraction in S. holoschista.
Acknowledgement
The authors thank Prof. K Ramalingam, Prof. T K Sudhindran, Prof.
S Augustine Chellappa, and Mrs. D Jayalakshmi for provision, facilities and
encouragement. One of us (MD) gratefully acknowledge the award of
fellowship of U G C
References
Atema J and Eflgstrom D G 1971 Sex pheromone in the lobster Homams americanus • Nature
(London) 232 261-263
Atema J and Gagosian R B 1973 Behavioural responses of male lobsters to ecdysones • Mar
Behav. Physiol. 2 15-20
Berry P F 1970 Mating behaviour oviposition and fertilisation in the spiny lobster Panulims
homarus (Linnaeus) ; Invest. Rep. Ocean Res. Inst. S. Afr. 24 1-16
Caldwell R L and Dingle H 1976 Variation of agonistic behaviour between populations of
the stomatopod Haptosquilla glyptocercus ; Evolution 31 220-223
Carlisle D B 1959 On the sexual biology of Pandalus borealis. The termination of the male phase*
/. Mar. Biol. Ass. U.K. 38 381-395
Christofferson J P 1970 An electrophysiological and chemical investigation of the female sex
pheromone of the crab Portunus sanguinokntus (Herbst.) ; Ph.D. Thesis, University of
Hawaii, Hawaii
Crisp D J 1974 Studies of barnacle hatching substance ; Comp. Biochem. Physiol. 30 1037-1048
Dahl E 1975 Pheromones in aquatic invertebrates, Bio signals Kungl. Fysiografiska Sallaskapet
of Lund, Sweden
Deecaraman M and Subramoniam T 1981a Repeated mating and its effect on the female reproduc-
tive physiology with special reference to the fate of male accessory sex gland secretion ;
Proc. First All India Symp. Inv. Repro. Madras
Deecaraman M and Subramoniam T 19&lb On the question of sex pheromonal attraction in
the crustacean stomatopod Sqidtta holoschista ; Abstr. IQth Ann. Conf. Etho. Soc. Bangalore
Dingle H and Caldwell R L 1969 The aggressive and territorial behaviour of the mantis shrimp,
Gonodactylus breedini Manning (Crustacea : Stomatopoda) ; Behaviour 33 115-136
Dingle H and Caldwell R L 1972 Reproductive and maternal behaviour of the mantis shrimp
Gonodactylus breedini Manning (Crustacea : Stomatopoda) ; Biol. Bull 142 417-426
Dingle H and Caldwell R L 1975 Distribution abundance and intraspecific agonistic behaviour
of two mudflat stomatopods ; Oecologia 20 167-178
Dunham P J 197S Sex pheromones in Crustacea ; BioL Rev. 53 555-583
Forster G R 1951 The biology of the common prawn Leander serratus Peimat ; /. Mar. Biol
Ass, U<K. 30 333-36Q
Sex pheramone in stomatopod 373
Galtsaff P S 1938 Physiology of reproduction of Ostrea virginica II. Stimulation of spawning
in the female oyster ; Biol. Bull 75 286-307
Galtsoff P S 1940 Physiology of reproduction of Ostrea virginica. III. Stimulation of spawning
in the male oyster ; Biol. Bull 78 117-135
Hartuoll R G 1969 Mating in Branchyura ; Cmstaceana 16 161-181
Hazlett B A 1970 Tactile stimuli in the social behaviour of Pagurus bernhardus (Decapoda,
Paguridae) ; Behaviour 36 20-48
Hazlett B A 1972 Shell fighting and sexual behaviour in the hermit crab genera Pagurites
and Caldnus with comments on Pagures; Bull. Mar. Sci. 22 806-823
Hazlett B A 1975 Ethological analysis of reproductive behaviour in marine Crustacea ; PubL
Staz. Zool Napoli. 39 671-695
Hazlett B A and Winn H E 1962 Sound production and associated behaviour of Bermuda
crustaceans. (Panulims, Gonodactylus, Alpheus and Synalpheus) ; Cmstaceana 4 25-38
Kamiguchi Y 1972 Mating behaviour in the freshwater prawn, Palaemon paucidens. A study
of the sex pheromone and its effect on male ; /. Fac. Sci. Hokkaido Univ. 18 347-355
Karlson P and Luscher M 1959 "Pheromones" : a new term for a class of biologically active
substances ; Nature (London) 183 55-56
Kittredge J R, Terry M and Takahashi F T 1971 Sex pheromone activity of the moulting
hormone, crustecdysone, on male crabs (Pachytr.apsus crassipes* Cancer antennarius,
C. anthonyi); Fish. Bull. 69 337-343
Kittredge J S and Takahashi F T 1972 The evolution of sex pheromone communication in
the Arthropoda ; /. Theo. Biol. 35 467-471
MacGinitie G E and MacGinitie N 1949 Natural history of marine animals. (New York, London
and Toronto : McGraw-Hill Book Co.) 1-473
McLeese D W 1971 Detection of dissolved substances by the American lobster (Homarus
americanus) and olfactory attraction between lobsters ; /. Fish. Res. Canada 27 1372-1378
Nolan B A and Salmon L 1970 The behaviour and ecology of Snapping shrimp (Crustacea :
Alpheus heterochelis and Alpheus nomanni) ; Form Funct. 2 289-335
Ryan E P 1966 Pheromone : Evidence in a Decapod crustacean ; Science 151 340-341
Salmon M 1965 Waving display and sound production in the courtship behaviour of Uca
pugilator with comparison to U. minax and U. pugnax ; Zoologica 50 123-150
Salmon M 1971 Signal characteristics and acoustic detection by the fiddler crabs, Uca rapax
and Uca pugilator ; Physiol Zool. 44 210-224
Subramoniam T 1977 Some aspects of sexual biology of a crab Emerita asiatica Milne Edwards;
Mar. Biol. 43 369-378
Subramoniam T 1979 Heterosexual raping in the mole crab Emerita asiatica \ Int. J. Invertebr.
Reprod. 1 197-199
Teytaud A R 1971 The laboratory studies of sex recognition in the blue crab Cattinectus
sapidus Rathbun; Sea Grant Tech. Bull. 15 1-63
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 4, July 1982, pp. 375-380
© Printed in India.
A new species of Argulus Muller (Crustacea: Branchiura), with a
note on the distribution of different species of Argulus in India
P NATARAJAN
Fisheries College, Tamil Nadu Agricultural University, Tuticorin 62S003, India
MS received 26 February 1981
Abstract. This paper describes a new species of Argulus, Argulus mangalorensis
collected from the estuarine stretch of Nethravathy river of Manga lore, S. India.
The distribution of different species of Argulus reported from India is also indicated-
Key words. Argulus mangalorensis, tt.sp. description, distribution.
1. Introduction
Two specimens of Argulus obtained from the plankton samples from Nethravarthy
estuary of Mangalore, are found to belong to a new species which is described
here. Generally argulids are known to parasitize marine and freshwater fish.
The present report records the occurrence of Argulus in the estuarine habitats
as well. Both the specimens were gravid females and appear to have left their
hosts for egg laying. The species of Argulus known so far from India are
A, indicus Weber, A. giganteus Ramakrishna, A. bengalensls Ramakrishna,
A, siamensis Wilson, A. siamensis peninsularis Ramakrishna, A. puthenveliensis
Ramakrishna, A. siamensis sub sp. Sundari Bai, A. fluviatilis Thomas and Devaraj
A. cauveriensis Thomas and Devaraj, A. japonicus Thiele and A. quadristriatus
Devaraj and Ameer Hamsa.
2. Descriptions
Argulus mangalorensis sp. nov. (figures 1-11)
Material : Two gravid females were obtained from the plankton samples from
Nethravarthy estuary on 3 April 1979. The holotype, a female measuring 8mm
long, will be deposited in the Indian Museum, Calcutta.
\dult female : Body (figures 1, 2) 8 mm long, carapace longer than wide 7-5 x
5- 6 mm, anterolateral sinuses distinct, cephalic area 2mm wide, convex above
and spined ventrally, lateral lobes of carapace 5 mm long, rounded behind, spined
3n anteroventral surface, dorso-median sinus moderately deep, reaching to the
level of anterior end of fourth thoracic segment. .
The dorsomedial pair of longitudinal ribs of carapace convergent in the middle,
curve outward beyond paired eyes anteriorly and below sucker posteriorly, posterior
375
376
P Natarajan
pieces parallel, end near transverse groove of cephalic region. Each dorsomedial
rib bears a pair of sutures at about its middle region, a pair of longitudinal sutures
arise from below compound eyes, run sideways and proceed backwards and join
the transverse groove of cephalic region. Secondary sutures arise from the triangular
sutures, extend backward, parallel to the lateral lobes of carapace and reach
almost at the level of base of fourth thoracic segment, and are connected with
each other by a transverse groove. None of the sutures are marked by any
pigments or coloured stripes.
Abdomen is 2-6 x 3- Omm, truncate anteriorly, posterior lobes subacute, sinus
deep, narrow anteriorly, and broad posteriorly. Caudal ratni small, each with
three terminal subequal setae.
1 mm
Figure 1
A new species of Argulus
377
The anterior respiratory areas oval, equal in size, found near lateral margins
of carapace between the level of base of suckers and maxillipeds. Posterior
respiratory areas elongated, kidney-shaped, found between the level of origin of
first thoracic segment and base of fourth thoracic segment.
Basal segment of first antenna with a strong medial outcurved spine, next
segment with a stout spine and antennular spine strongly curved. Slender terminal
segment of antenna with three minute spines and setae distally (figures 3, 4).
Second antenna (figure 5) four segmented, basal segment broad with a stout spine
at base, six setae on dorsal and four on ventral margins. Second segment elon-
gated with four setae of which two are on dorsal margin, remaining two at
distal seta. Fourth segment small, club-shaped with three small equal apical
mm
Figure 2
378
P Natarajan
0.5 mm
Figures 3-11
Figures 1-11. Argulm mangalorensis sp. nov. 1. dorsal view ; 2. ventral view ;
3. first and second antenna ; 4. distal end of first antenna enlarged ; 5. second
antenna enlarged ; 6. maxilliped ; 7. basal segment of maxilliped enlarged ;
8. distal segment of maxilliped enlarged ; 9. stylet ; 10. single rib with plates ;.
11. fourth leg.
spines. Postantennal spine very stout, maxilliped (Fig 6) five segmented, basal
segment with three nearly equal stout posteromedial spines and a large ova!
spinous pad which carries nine setae along its setae. Second and third segments
are provided with rectangular pads which carry scale-like spines and a row of
A new species of Argulus 379
Table 1. Distribution of Species and sub-species of Argulus from India.
Parasite
Hast
Locality
Author and Year
A.
siamensis
Not known
Harischandrapur, ^
W. Bengal
Ophiocephalus
Champahati,
punctatus
W. Bengal
Labeo rohita
Siripur, Bihar
1 Ramakrishna 1951
Not known
Mahanamia River,
Base of Himalayas
Murrel
Saurashtra
A.
indicus
Ophiocephalus
Champahati,
Ramakrishna 1951
punctatus
W. Bengal
A.
giganteus
Not known
Not known
Ramakrishna 1951
Tetradon oblongus
Bombay
Rangnekar 1957
A.
bengalensis
Not known
Harischandrapur ,
Ramakrishna 1951
W. Bengal
A.
siamensis sub sp.
Not known
Rajahmundry
Ramakrishna 1951
peninsularis
Ambassis ranga
Rajahmundry
Malaviya 1955
A.
puthenveliensis
Not known
Not known
Ramakrishna 1962
Esomus danrica >
Puntius vittatus
Macropodus eupanus
S Kerala
Thomas 1961
Panchax panchax
f
blochii /
A.
siamensis sub sp.
Lebistes reticulatus
Hasaragatta,
Sundari Bai 1973
Bangalore
A.
fluviatilis
Not known
Hoginekal,
Thomas and Devarai
Tamil Nadu
1975
A.
cauveriensis
Not known
Hoginekal,
Thomas and Devaraj
Tamil Nadu
1975
A.
japonicus
Labeo fimbriatus
Sathanur fish farm,
Prabhavathy and
Catla catla
Tamil Nadu
Sreenivasan 1976
Cyprinus. carpio
A.
quadristriatus
Psammoperca
Palk Bay, Maudapam
Devaraj and Ameer
waigiensis
Hamsa 1977
similar spines on the margin of the third segment. Fourth segment which is
smaller than the third, also carries spines. Fifth segment small with a blunt lobular
distal end and two dissimilar claws on inner margin.
Paired lateral eyes conspicuous, located at base of antennal spine, median eye
well developed, proboscis midventral, in between suckers. Distal half of proboscis
expanded, anterior part narrow, terminating in a stylet (figure 9). Sucker 0-6 mm
(inside diameter), composed of 115-118 ribs of 38 to 40 imbricated plates each
(figure 10).
Distal ends of rami of third and fourth legs reach a little beyond carapace.
Flagella of swimming legs absent, basal lobe of fourth leg boot-shaped, carries
setae on ventral margin, basal segment of basipod with nine and distal segment
with two plumose setae (figure 11). The thoracic segments and basipods with
spines ventrally. Uterine eggs are four to five sided, arranged, in honey-comb
pattern.
380 P Natarajan
Colour .: Body greenish yellow, papillae algal green, thoracic segments and legs
straw yellowish and uterine eggs dull brown.
3. Discussion
In the arrangement of respiratory areas, A. mangalorensis agrees with 22 species of
Argulus in Wilson's (^944) report. A. kusafugu and A. scutiformis from Japanese
fishes (Yamaguti and Yamasu 1959), A. indicus and A. giganteus from India
(Ramakrishna 1951), A. japonicus from pond fishes of Tamil Nadu (Prabhavathy
and Sreenivasan 1976) and A. quadristriatus from a marine fish (Devaraj and
Ameer Hamsa 1977). In the arrangement of the respiratory areas as well as the
suction cup being composed exlcusively of imbricated plates, A. mangalorensis
is similar to A. melanosticus, A. pugettensis, A.niger, A.floridensis&nd A. giganteus
and A. quadristriatus. However, the present species is distinct from the others by
the following characteristics-— (1) 115-118 number of ribs in each suction cup ;
(2) 38-40 imbricated plates in each rib ; (3) three spines and three setae at the
distal end of first antenna ; (4) absence of flagella on any of the swimming, legs.
The distribution of Argulus spp. in India is given in table 1.
Acknowledgements
The author wishes to acknowledge his gratitude to Dr. N. Krishna Pillai, Professor,
University of Kerala, Trivandrum, for his help in identification of the species. He
also thanks. Prof. N. Balakrishnan Nair, University of Kerala, Trivandrum and
Dr. M Devaraj, Professor, Central Institute of Fisheries Education, Bombay for
critical reading of the manuscript.
References
Devaraj M and Amser Hamsa K M S 1977 A new species of Argutus (Branchiura) from a
marine fish> Psammoperca waigiensis (Cuvier) ; Cmstaceana 32 129-134
Malaviya R B 1955 Parasitism of Ambassis ranga H.B. by Argulus siamensis sub sp. peninsularis
Ramakrishna ; Curr. Sci. 24 275
Prabhavathy G and Sreenivasan A 1976 Occurrence of Argulus japonicus in brood fish ponds
in Tamil Nadu ; /. Inland Fish. Soc. India 8 131-133
Ramakrishna G 1951 Notes on the Indian species of the genus Argulus Muller (Crustacea:
Copepoda) parasitic on fishes ; Rec. Indian Mus. 49 207-216
Ramakrishna G 1962 On a new species of Argulus Muller (Crustacea : Copepoda) from Kerala ;
Proc. All India Congr. Zool I 178-179
Ranganekar M P 1957 Copepod parasite of the families Arguiidae, Caligidae, Dichelesthidae
and Lernaeopodidae ; /. Univ. Bombay 26 8-20
Sundari Bai A 1973 The occurrence of Branchiura parasite Argulus sp. (Argulidea : Arguiidae)
on the carnivorous fish. Lebistes reticulatus (Peters) In Mysore State ; Curr. Res. 2 79-75
Thomas M M 1961 Observations on the habits and post-embryonic development of a para-
sitic brahchiuran Argulus puthenveliensis Ramakrishna ; /. Mar. Biol. Ass. India 3 75-86
' Thomas M M and. Devaraj M 1975 Two new species at Argulus Muller (Crustacea : Branchiura)
from River Cauvery with a key to Indian species ; Indian J. Fish. 22 215-220
Wilson C B 1944 Parasitic Copepoda in the United States National Museum ; Proc. U.S. Nat.
Mus. 94 529-582
Yamaguti S and Yamasu T 1959 On two species of Argulus (Branchiura : Crustacea) from
Japanese fishes ; Biol. J. Okayama Univ. 5 167-175
Proc. Indian Acad. Sci. (Auim. Sci.), Vol. 91, Number 4, July 19S2, pp. 381-389.
©Printed in India.
The effect of cephalic transection on the micromorphological
changes in the ventral nerve cord-neurosecretory system of
earthworm, MetapMre peguana (Rosa, 1890) during anterior
regeneration
D K NANDA and P S CHAUDHURI
Department of Zoology, Calcutta University, 35, Ballygunge Circular Road,
Calcutta 700 019, India
MS received 11 September 19S1 ; revised 4 June 1982
Abstract. Transection of anterior 5 segments in Metaphire peguana engenders
characteristic changes in the functional activity of the ventral nerve cord-neura-
secretory system in the event of cephalic regeneration. Of the two types of neuro-
secretory cells, the moderately stained cells remain more susceptible when the cell
structure, location of nucleus, amount of secretory inclusions and their transportation
to the zone of accumulation are considered. Overall engorgement of neurosecretory
substances refrained from axonal transport, moderate axonal flow coupled with
slight depletion and finally acute depletion at 24, 4$ and 72 hr after amputation
respectively are some of the notable features registered in course of this investi-
gation. Disarray in the sequential changes involved in the secretory dynamics of
neurosecretory cells, as well as extent of NSM accumulation both within and outer
periphery of the ganglia provide evidence for the utilisation of material through
repaired vascular systems during regenerative proliferations cf anterior segments.
Keywords. Metaphire peguana', neurosecretory cells; NSM; regeneration; secretory
dynamics.
1. Introduction
The importance of the central nervous system in the phenomenon of oligochaete
regeneration has been elucidated by several classical investigators (Morgan 1902 ;
Avel 1929 ; Sayles 1940). Harms (1948) experimentally established the indis-
pensability of the brain for regenerative growth of the anterior segments in
Lumbricus terrestris. Later Herlant-Meewis (1964) refuted the solitary role of
the cerebral ganglia and advocated the involvement of the ganglionic comple-
ments of the ventral nerve cord in both anterior and posterior regeneration in
Eisenia foetida. These observations have been substantiated by Farber (1965)
who reiterated that neurosecretion of the ventral nerve cord has a profound role
in the segmental regeneration of L. terrestris. In her detailed analysis, Herlant-
Meewis (1972) opined that C3 cells of each segmental ganglion exhibit spectacular
cytological response to the loss of anterior segments. Synchronous release and
synthesis of neurosecretory material (NSM) in the ganglia immediately proximal
381
382 D K Nanda and P S Chaudhuri
to the level of amputation of either anterior or posterior segments in E. foetida
have been recorded by Marcel (1973) who also concluded that neurosecretory
system promotes some aspects of regeneration.
The present investigation deals with the extent of histomorphic changes in the
neurosecretory system of the ventral ganglionic complements following anterior
amputation. An attempt has also been made to assess sequential reactive res-
ponse in the ganglia concerned.
2. Materials and methods
Full grown clitellate earthworms, Metaphire peguana (average length 120 mm)
were collected from the neighbourhood of Calcutta and acclimated for one week
in the laboratory at room temperature 29° C and RH 78 %. Amputation of the
first five anterior segments by a sterilized paragon knife was made in the group
comprising fifteen worms which were kept in a petridish containing 1-5 inch
thick bed of moisturized soil. Ganglionic complements from anterior, middle.
and posterior regions (each region containing 40 segments) of the remaining nerve
cord were fixed in Bouin's fluid after 24, 48 and 72 hr of amputation. Identical
sets of ganglionic complements were dissected out from unoperated earthworms
which, however, served as controls. Sections (7 /^m thick) were stained with both
Gomori's chromealum-haematoxylin phloxin (Bargmann 1949) and simplified
aldehyde fuchsin (Cameron and Steele 1959) staining techniques following acid
permanganate oxidation.
3. Observations
3-1. Control
A majority of the neurosecretory cells of the ventral nerve cord are in various
phases of secretion (figure la) which can be determined on the basis of the staining
intensities in descending order to locate the concentration of secretory material.
Relatively small deep stained cells do not exhibit detectable cytoplasmic inclusions
and usually possess more or less homogeneously stained cytoplasm (figure Ib).
Large moderately stained cells, however, possess variable amount of secretory
inclusions apart from clarity in their cytoarchitecture (Nanda and Chaudhuri 1981).
Some of the moderately stained cells exhibit axonal transport of secretory material
and their subsequent discharge. Evidence for rich NSM accumulation both at
the margin of the neuropile, as well as, the outer periphery of the ganglia as such
are not seldom.
3-2. Experimental
Appearance of regeneration blastema is first noticed within 24-48 hr after cephalic
transection (Nanda and Chaudhuri 1982). Completion in the formation of a
full fledged anterior segment, however, is accomplished around 72 hr after ampu-
tation. Such operation renders multiple cytomorphic alterations that arc
Ventral nerve cord-neurosecretory system
^ ^ ^ Jm fLA
383
Figure 1. (a) Control section of the ventral nerve cord of Metaphire peguana
showing CHP-positive neurosecretory cells with various phases of secretory activity
( x 1500). (b) Control section showing AF-positive cytoplasm of deep and mode-
rately stained cells. Note homogeneously stained cytoplasm of deep stained cell
( X1500).
Figure 2. Experimental: Section showing typical "shrunken condition" of CHP-
positive moderately stained cells in the ventral nerve cord following anterior tran-
section (x 1500).
384
D K Nanda and P S Chaudhuri
Figure 3. Experimental : (24 hr after anterior transection.) (a) Section showing
trend in the massive accumulation of AF-positive m?terial in the neuro-secretory
perikarya ( x 1500). (b) Sectio-n showing same moderately stained cells with secretion
in the form of aggregates and axon oriented nuclei. Note discrete accumulation
of cap-positive material at the margin of neuropile ( x 1500).
Ventral nerve cord-neurosecretory system
385
Figure 4. Experimental : (48 hr after anterior transect! on), (a) Section showing
CHP-po$itive moderately stained cells with cytoplasmic vacuoles in the perikarya
(X1500). (b) Section showing AF-positive cells discharging their secretory material
through ' axo-n bundle'. Note axon oriented nuclei ( X 1500). (c) Section showing
accumulation of AF-positive secretory colloids at the peripheral margin of the ganglion
(X 1500).
386
D K Nanda and P S Chaudhuri
Figure 5. Experimental : (72 hr after anterior transection.) (a) Section showing
sudden drop in staining intensity of AF-positive secretory neurones. Note contrasting
staining feature of the neuropile due to accumulation of secretory material (X 1500).
(b) Section showing cap-positive neurosecretory cells with cytoplasmic vacuoles
Note ramification of intraganglionic blood vessels endowed with secretory inclusions.
(X1500),
Ventral nerve cafd-rteurasecretory system 387
more apparent in case of moderately stained cells. These cells in contrast with the
deep stained cells also reveal conspicuous " shrunken conditions " of the cell
body (?) which is pronounced up to 48 hr of experimentation (figure 2).
3-3. 24 hr after amputation
Most of the neurosecretory cells irrespective of their types especially of the anterior
region of the ventral nervecord show intense accumulation of secretory inclusions
in their perikarya (figure 3a). These inclusions may exist in the form of close
aggregates so as to render the cytoplasm coarse in appearance. This condition
is rather predominant in moderately stained cells which have aggregates mostly
concentrated at the anterior half of the perikarya. Nuclei, however, are often
observed more towards the axon hillock region. The margin of the neuropile
remains sprinkled with secretory inclusions (figure 3b).
3-4. 48 hr after amputation
Despite deep stainability in the majority of the neurosecretory cells in the ventral
nerve cord a few cells are in a state of depletion and vacuoles in the perikarya
are not scarce (figures 2 and 4a). Majority of the cells bear axon oriented nuclei
with brilliant phloxinophilic nucleoli and exhibit axonal transport (figure 4b).
In contrast, the neuropile falls short of NSM while enhanced accumulation of AF-
positive material is obvious at the outer periphery of the ganglia (figure 4c).
3-5. 72 hr after amputation
The general trend for the deep stainability of cells demonstrating discrete secretory
inclusions as found above suddenly declines (figure 5a). A few deeply stained cells
still exist but they do not demonstrate coarse cytoplasm. On the other hand,
they. remain homogeneously stained and are comparable to those of the control.
Moderately stained cells are very clear and they exhibit intense vacuolation in their
perikarya which often are devoid of any cytoplasmic inclusions. Occasional
axonal transport throughout ganglionic complements of the ventral nerve cord
may be noticed. Incidentally, the margin of the neuropile, as well as the intra-
ganglionic blood vessels show secretory inclusions but the peripheral region of
the ganglia demonstrate very little NSM (figure 5b).
4. Discussion
Cephalic transection on the rest of the ganglia of the ventral nerve cord in
M. peguana has elicited altered secretory activity in the neurosecretory cells
especially when the position of the nuclei, the concentration of cytoplasmic
inclusions and the rate of axonal migration of NSM are considered. Such oscil-
lation in the functional activity may have correlation with their spectacular * hyper-
activity' (Herlant-Meewis 1964). Changes in the neurosecretory cells are most
conspicuous 24 hr after amputation, close to the level of transection than at
other regions. But thereafter almost uniform changes are noticed throughout
the nervecord at 48 and 72 hr after amputation. The reason is not clear and
388 D K Nanda and P S Chaudhuri
may have bearing in relation to the intensity of stress in course of segmental proli-
feration. Causes for the shrunken conditions of some neurosecretory cells in
general and moderately stained cells in particular are not understood but involve-
ment of " generalised stress action " as reiterated by Farber (1965) could be the
reasons. Further, the disarray in sequential changes in the neurosecretory peri-
karya following transection of ventral nervecord of M. peguana incur disruption
in neurohormonal balance so as to trigger temporary accumulation of secretory
substances in all the neurosecretory cells (Herlant-Meewis 1964). In fact, tempo-
rary cessation of neurosecretory transport has some bearing in the event of resti-
tution and subsequent blastema formation. Participation of the NSCS to discharge
their elaboration either partially or indiscriminatelxinto the just repaired circula-
tory system at late post-amputation periods, i.e. at 48 and 72 hr provides clue
for their indispensability in the management of restoration of lost part or " replace-
ment of element " during regeneration (Herlant-Meewis 1962 ; Dey and Nanda
1979). Indeed, increment in the number of moderately stained cells with specta-
cular intracellular changes at 72 hr post anterior transection period in contrast
to accelerated rate of axonal transport and initiation in the transformation of the
moderately stained cells at 48 hr post-amputation period seem to indicate func-
tional change over in the secretory dynamics of the deep and moderately stained
cells. Relatively rich accumulation of NSM around the periphery of the neuropile,
at the initial stage of experimentation and subsequent exhaustion of the same
at 48 hr followed by massive accumulation at 72 hr of post-amputation tend to
indicate fluctuation in the secretory rhythm pertaining to increased axonal flow.
In consequence, rapid disposal of NSM in the intraganglionic capillaries to miti-
gate restorative response and thereafter resumption to near-normal condition
ensure.' Adverse physiological stress condition in the form of injury possibly
release cellular products that act as an adjunct to stimulate the neurosecretory
neurones of the ventral nerve cord for the production of "regeneration
promoting hormones" leading to segment proliferation (Hoar 1975). Besides
these, non-existence of discrete non-neural endocrine gland, as well as distinct
neurohaemal organ in oligochaetes in general and M. peguana in particular,
it is reasonable to assume that nervous system as a whole plays a "versatile" role
to meet altered physiological eventualities.
Acknowledgements
The authors are grateful to Mr E G Easton, Annelida Section, British Museum
(Natural History), for identification of the specimen. Thanks are also due to
Mr Ashim Bej and other research workers for their active co-operation during
the course of this investigation.
References
Avel M 1929 Rechejrclies experimentales stir les caract&res sexuals somatiques des Lumbriciens ;
Bull. Biol France et Belg. 63 149-318
Bargmann W 1949 Ober die neurosekretorische Verkaiipfung Von Hypothalamus und Neqrp-
hypaphyse ; Z. Zellforsch. 34 610-634
Ventral nerve COY d-neuro secretory system 389
Cameron M L and Steele J E 1959 Simplified aldehyde fuchsia staining of neurosecretory cells ;
Stain Tech. 34 265-266
Dey M and Nanda D K 1979 Effect of posterior transaction on the brain neurosecretory perikarya
of Pheretima posthuma ; ZooL Beitr. 25 199-204
Farber P A 1965 The histological relationship between neurosecretory activity and anterior
regeneration in Lwnbricus terrestris ; Anat. Rec. 151 348 (Abs.)
Harms J W 1948 t)ber ein inkretorisches Cerebralorgan bei Lumbriciden sowie Beschreibuns
eines verwandten Organs bei drei neuen Lycastis-Arten ; Arch. Entw. -Meek. 143 332-346
Hertent-Meewis H 1962 Neurosecretory phenomena during regeneration of nervous centres in
Eisenia foetida ; Mem. Soc. EndocrinoL 12 267-274
Herlant-Meewis H 1964 Regeneration in Annelids ; Adv. Morphog. 4 155-216
Herlant-Meewis H 1972 Le role du systeme nerveus dans la cicatrisation chez Eisenia foetida
(abst.) ; Gen. Camp. EndocrinoL 18 596
Hoar W S 1975 General and Comparative Physiology (New Delhi : Prentice Hall of India
Private Limited) pp 752-753
Marcel R 1973 Cycle secretoires de cellules de la chaine nerveuse au course de la regeneration
chez Eisenia foetida Sav. f. typica (Annelide, Oligochaete) ; Gen. Comp. EndocrinoL 21
30-44
Morgan T H 1902 Experimental studies of the internal factors of regeneration in the earthworm;
Arch. Entw. -Mech. Org. 14 562-591
Nanda D K and Chaudhuri P S 1981 Studies on the cytomorphology of the ventral nervecord
of the earthworm, Pheretima posthuma with special reference to neurosecretion ; /. ZooL
Soc. India (in press)
Nauda D K and Chaudhuri P S 1982 Regeneration of the neurosecretory system of the nerve
ring in earthworm, Metaphire peguana ; Acta Biol. Cracov. (in press)
Sayles L P 1940 Buds induced by implants of the anterior nerve cord and neighbouring tissues
inserted at various levels in Clymenella torquata ; Biol. Bull. 78 298-311
Free. Indian Acad. Sci. (Anira. Sci.), Vol. 91, Number 4, July 1982, pp. 391-395.
© Printed in India.
Studies on preference of Callosobruchus maculatus Fabricius to some
high yielding varieties of arhar (Cajanus cajan L.)
SATYA VIR
Central Arid Zone Research Institute, Jodhpur 342 003, India
MS received 31 August 1981
Abstract. The oviposition response and development of Callosobruchus maculatus
Fabricius were studied on 14 high yielding varieties of arhar. There was signi-
ficant difference among the varieties in the amount of food consumed per grub.
The average development period was not dependent on the amount of food consumed.
The development of grub was also not better on the grain preferred by the beetle
for oviposition. There was significant difference among the varieties in the loss of
100 seed weight. Average weight of female was more than the male developed on
all varieties. On the basis of food consumed per grub and loss of 100 seed weight
as a combined criterion, the varieties are grouped into least susceptible, intermediate
in susceptibility and the most susceptible varieties.
Keywords. Varietal preference ; Callosobruchus maculatus.
1. Introduction
Storage of pulse seeds is a problem owing to the severe damage caused by the
pulse beetle, Callosobruchus maculatus Fabricius. The damage is sometimes so
serious that whole of the seed material is eaten and only thick seed coat with
empty cavities are left behind. Gokhale (1973), Wadnerker et a! (1978) and Dabi
et al (1979) assessed the relative susceptibility of some varieties of different pulses
to C. maculatus. Attempts have also been made to investigate the cause of diffe-
rential response of different pulses on various life processes of this beetle (Girish
et al 1974), But the available literature reveals that practically no attention has
been paid towards the susceptibility of high yielding varieties of arhar under culti-
vation to C. maculatus. The present investigation was therefore undertaken.
2. Materials and methods
Fourteen varieties of arhar (Cajanus cajan L.) were obtained from the Chief
Scientist, Dry Farming, Central Arid Zone Research Institute, Jodhpur. Healthy
and uncontaminated seeds were sterilized and the moisture contents of seeds were
maintained between 12-5 to 13-0%. 100 seeds of each variety were weighed and
kept in plastic vial (5 X 4 x 3cm). The experiment was replicated five times.
391
392 Satya Vir
Four pairs (4 males -I- 4 females) of newly emerged adults from uniparental culture
were introduced into each vial except the fifth replication, which was kept wihout
beetle as control for each variety. After 10 days the beetles were removed and
the number of eggs laid on each variety was counted. All the experiments were
carried out in an incubator at a constant temperature of 28 ± 2° C and humidity
50-60% r.h.
Commencing from the 20th day of the experiment, the newly emerged beetles
were counted daily till the emergence of last adult. After each observation the
emerged beetles were removed to prevent further breeding. The weight of seeds
and adults were recorded separately with a single pan electric balance (with 0- 1 mg
precision). The average development period and percentage emergence of adults
was calculated. All the data were statistically analysed. The correlation coeffi-
cient (r) was calculated between the various life processes of the beetle and physical
characters of seed to establish possible relationship between them.
3. Results and discussion
The results (table 1) reveal that all the varieties of arhar were utilized by the beetle
for egg laying. The response of opposition however varied significantly. Varieties
4-84, 4-64, BS. 1, K-28 and T-7 (with average of 238 -25 to 273-25 eggs) showed
preference for oviposition as compared to variety T-17 (with average of 170-00
eggs). There was no significant difference in the rest of the varieties where the
average number of eggs laid varied from 199* 50 to 229-75. The minimum number
of eggs laid per seed was 1 • 70. The correlation coefficient (r) between the average
number of eggs laid and the seed characters, viz., seed weight, seed volume and
colour of seed was not significant (table 2). Further, the texture of seed cannot
be taken as a criterion for the preference for oviposition as the texture was smooth
in all the varieties tested.
The average food consumed per grub is a good criterion for the assessment
of relative susceptibility of different varieties (Regupathy and Rathinaswamy 1970;
Dabi etal 1979). There was significant difference among the varieties in the
amount of food consumed per grub (table 1). Varieties HP (WP)-15, T-17, K-16,
B.S. 1, T.T. 4 and 4-64 were least susceptible to C. maculatm (with 30-69 to
34*39 mg of food consumption per grub) than the other varieties. The corre-
lation coefficient (r) between the amount of food consumed per grub and the seed
characters, viz, seed weight, seed volume and colour of seed was not significant
(table 2). Similar observations were reported in the experiment with Calloso-
bruchus chinensis reared on different varieties of pigeonpea (Regupathy and Rathina-
swamy 1970) and with C. maculatus reared on different varieties of cowpea (Dabi
etal 1979). Apparently some factor other than seed characters governs the
mechanism of resistance in pulse seed to the attack of pulse beetle.
The average development period was found to vary significantly which ranged
from 27-82 to 34-71 days (table 1). Coefficient of correlation (r) between the
amount of food consumed per grub and the average development period was not
significant (table 2). The study thus reveals that the development period of the
grub is not dependent on the amount of food consumed. Further, the develop-
ment of grub was also not better on the grain which were preferred by the beetle
for oviposition (table 1). Thus the preference for oviposition is not an indication
Studies on preference of Callosobruchiis maculate*
393
XJ
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in
3 fa O
CU o
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-HoO
i
41
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394 Satya Vi*
Table 2. Coefficient of correlation (r) betwesn physical characters of seed and
life processes of the beetle, L
Average
weight of
100 seeds'
Average
i umber of
seeds/10 ml
volume
Colour of
seed
Average
development
period
Average number of eggs laid
0-137
-0-165
0-210
Level of significance
NS
NS
NS
Average food consumed/grub
0-588
-0-628
-0-125
0-168
Level of significance
NS
NS
NS
NS
NS =Not significant.
of suitability for development. These observations are in accordance with the
findings of Girish et al (1974) and Singh et al (1977), The loss of 100 seed weight
varied from 2-765 to 3-638 g. Varieties Basant, PS-41, T-7, T.T.6, T.T.-5, 4-84
showed significantly greater loss in seed weight as compared to HP (WP)-15, T.T.I 7,
K-16, B.S.I and K-28 at C.D. value of 0-05%. The percentage emergence of
adults on varieties Basant and PS-41 was significantly more than of the other
varieties. Average weight of female was more than the male emerged on all
the varieties tested in the present investigation. Similar tendency was observed
earlier by Howe and Currie (1964) and Gokhale (1973).
From the overall results on the basis of average food consumed per grub of
emerged beetles and loss of 100 seed weight as a combined criterion, varieties
HP (WP) -15, T-17, K-16 and B.S. 1 proved to be the least susceptible whereas
T.T.6, T-7, PS-41 and Basant as the most susceptible varieties. The varieties T.T.4,
4-64, K-28, K-23, T.T.2 and 4-84 are intermediate in susceptibility and none of
the varieties was found immune to the attack of C. maculatus.
Acknowledgements
The authors are grateful to Dr H S Mann, Director and Dr K A Shankar-
narayan, Division of Plant Studies, Central Arid Zone Research Institute,
Jodhpur, for providing necessary facilities to carry out the investigation.
References
Dabi R K, Gupta H C and Sharma S K 1979 Relative susceptibility of some cowpea varieties
to pulse beetle Callosobmchus maculatus Fabricius ; Indian J. Agric. Sci. 49 48-50
Girish G K, Singh Karan and Krishnamurthy K 1974 Studies on the oviposition and deve-
lopment of Cattosobruchus maculatus Fab. on various stored pulses ; Bull. Grain Tech.
12113-116
Studies on preference of Callosobruchus maculatus 395
Gokhale V G 1973 Development compatibility of several pulses in the Bruchidae. 1. Growth,
and development of Callosobruchus maculatus Fabricius ; Bull. Grain Tech. 11 28-31
Howe R W and Currie J E 1964 Some laboratory observations on the rate of development,
mortality and oviposition of several species of Bruchidae breeding ; Bull. EntomoL Res.
55 437-477
Regupathy A and Rathinaswamy R 1970 Studies on comparative susceptibility of seeds of
certain red gram [Cajanus cajan (L.)l Mill sp. varieties to pulse beetle, Callosobruchus
chinemis (L.) (Bruchidae ; Coleoptera) ; Mad. Agric. J. 57 106-109
Singh Suchwant, Odak S C and Singh Zile 1977 Studies on the preference of pulse beetle
(Callosobruchus chinensis Linn.) for different hosts ; Bull. Grain Tech. 15 20-26
Wadnerkar D W, Kaunsale P P and Pawar V M 1978 Studies on preference of pulse beetle
Callosobruchus maculatus Fab. to some varieties of arhar and gram ; Bull. Grain Tech. 16
122-124
ftroc. Indian Acad. Sci. (Aaim. Slci.), Vol. 91, Nfumbcr 4, July 1982, pp. 397-406.
© Printed in India.
Three new species of haematozoans from freshwater
teleosts (pisces)
B D JOSHI
Department of Zoology, Kumaun- University, Campus Almora, Almora 263 60 1 ,
India
Present address; Department of Zoology, Gurukul Kangari Vishwavidyalaya,
Hardwar 249 404 (UP)
MS received 23 June 19SO ; revised 2 June 1982
Abstract. Two new species of haematozoans, Trypanosoma aori (sp. nav.) and
Trypanoplasma mysti (sp. nov.), were found harbouring the blood plasma of fresh-
water teleasts, Mystus aor, while Trypanoplasma atti (sp. nov.), was found in the
plasma of another cat fish Wallago attu. The two hosts are new records for these
parasites. All three species of the parasites described here showed characteristic
polymorphism.
Keywords. Haematozoara ; Trypanosoma ; Trypanoplasm* ; blood; polymorphism.
1. Introduction
In the recent past quite a few new species of piscine haemoflageliate parasites have
been described from various freshwater teleosts of India (Ray Chaudhuri and
Misra 1973 ; Misra etal 1973 ; Tandon and Joshi 1973 ; Pandey and Pandey
1974 ; Mandal 1975, 1977, 1978, 1979 and Joshi 1976, 1978), besides the earlier
reports (Lingard 1904, Demello and Valles 1936, Qadri 1955, 1962 and Hasan
and Qasim 1962). In a recent paper Joshi (1979) reported occurrence of trypano-
somes in thirteen species of freshwater teleosts of Lucknow. This paper describes
three new species of haematozoan parasites from two freshwater teleosts viz.
Mystus aor and Wallago attu.
2. Material and methods
Live specimens of M. aor and W. attu were obtained from river Gomati, trans-
ported to the laboratory, given rest for 12-14 hr in a large glass aquarium under
laboratory conditions and then studied the blood smears, stained with
Leishman and Wrights stains following the usual methods described earlier
(Tandon and Joshi 1973 and Joshi 1978). Camera Lucida drawings were made
of the parasites found in blood slides with precise details and measurements
taken.
3. Observations
Histomorphological and morphometric studies made on the species of Trypano
soma and Trypanoplasma revealed the following characterisitics, and accordingly
397
398
B jD foshi
with the help of existing literature three new species of haemoflagellates/haemato-
zoans are described here :
Parasite : Trypanosoma aori (sp. nov.)
Host : Mystus aor.
Location : Plasma of the host fish.
Locality : River Gomati, Lucknow.
Diagnosis and descriptions : (figures 1-6) table 1.
Body : Parasites were short (figures 1-3), elongated and partly stumpy. Out
of many forms seen in three stained preparations, some typically large elongated
forms were also found (figures 4-6). These forms mainly had both ends blunt
or rounded, while few forms had pointed or beak shaped extremities.
Figures 7 and 13 are those of the RBC's of the host fishes to give a comparative
idea of the blood cell size and the parasite.
Figures 1-14. 1-3. Small sized forms of T. aori (sp. nov.), 4-6. Large sized
farm of T. aori. 8, 9, and 10, T. mysti (sp. nov.) 7. RBC of M. aor, the host
fish. 11, 12 and 14. Polymorphic forms of T. atti (sp. nov.). 13. RBC of
W, atttt, the host fish. e
New species of kaematozoans 399
Cytoplasm : It stained bluish purple, with fine azurophilic dusty granules and
granulation appeared denser in the smaller forms, than in the larger ones,
which also showed vacuoles. Myonemes were not seen in either of the forms.
Nucleus : It was distinct in all the forms, situated towards either of the two
extremities in smaller forms (figures 1-3) or almost in the centre (figures 4-6)
In most of the forms, it was rounded or oval, with a distinct karyosome. Karyo-
some contained, more dense and hyperbasophilic contents than the surrounding
karyoplasm.
Kinetoplast : It was present almost at the posterior terminal end and was
rounded or slightly elongated.
Flagellum : It always arises from the inner end of the kinetoplast, runs towards
the anterior end boardering the undulating membrane before being free at the
anterior extremity. In most cases it took light basophilic stain. The free flagel-
lum showed much variation in size.
Undulating membrane : This structure was conspicuously present in all elon-
gated forms and was well differentiated from the body.
Remark : A distinct polymorphism existed, with high parasitemia and low
instance of infection.
Parasite : Trypanoplasma mysti (sp. nov.)
Host : Mystus aor.
Location : Plasma of the host fish.
Locality : River Gomati, Lucknow.
Diagnosis and description : (figures 8, 9 and 10) table 2.
Body : The? trypanoplasmid forms were stoutly elongated and irregularly curved
(figures 8, 9 and 10). All forms showed conspicuous body width (table 1). Both
ends of the parasite were blunt and wide.
Cytoplasm : The cytoplasm is densely packed with fine to coarse granules.
It took deep bluish black stain. Vacuoles were frequently present and at places
were dense, surrounded by cytoplasmic granules (figure 10).
Nucleus : There occurred marked variation in the shape, size and position of
the nucleus of this new species of Trypanoplasma. In few forms it was mid-
anterior (figure 8) and parallel to kinetoplast, while in others it was situated
at either of the two ends (figures 9, 10), being quite away from the kinetoplast.
The nucleus always showed a distinct and deeply stained karyosome, which occu-
pied much space within the nucleus. Nuclear chromatin around the karyosome
was thinly scattered. The karyosome always took a deep bluish black stain,
whereas the nuclear chromatin was purple blue. The nuclear shape varied from
ovoid (figure 8), reniform. (figure 9) to drop shaped (figure 10).
Kinetoplast : Like that of the nucleus, it also revealed conspicuous variations in
the shape, size and position. It was rod shaped in few forms (figures 8, 9) and
rounded (figure 10) in others. It is situated either at extreme terminal end
P.(B)-9
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New species of haematozoans 403
(figure 9) or nearly towards the central periphery of the cell. It alway? stained
deep bluish black.
Flagellum : All the forms possessed two free flagella, which arise from the two
ends of the same kinetoplast. Anterior flagellum was usually larger than the
posterior (table 2). The posterior flagellum either runs through the cytosome
•(figure 8) or boarders the undulating membrane (figure 9), before being freed at
the post extremity.
Undulating membrane : It was not seen in few forms (figure 8), but distinctly
present in others (figures 9, 10).
Remark : This newly described species of Trypanoplasma aori was found
harbouring the same specimen of the host. species, M. aor, to which the T. aori
(sp. nov.) harboured. The host species thus showed a multispecies parasitemia
of high intensity. Only one specimen of the host fish, out of 40 observed was
found to be parasitized by these haematozoans (Joshi 1979).
Parasite : Trypanoplasma atti (sp. nov.)
Host : Wallago attu
Location : Plasma of the host fish
Locality : River Gomati, Lucknow.
Diagnosis and descriptions : (figures 11, 12 and 14).
Body : The unicellular body showed marked variations, with characteristic
undulations (figure 14), while few others were ovo-triangular (figures 11, 12)>
with irregular shapes, hence conspicuous differences were noted in body size
(table 2).
Cytoplasm : It was homogeneous and densely filled with coarse cytoplasmic
granules. The cytoplasmic contents took most hyperbasophilic stain than in
any other form described. Vacuoles were present in almost all forms, besides
myonemic striations were also seen in few forms (figure 12).
Nucleus : It was short, cylindrical or rod like (figures 11, 12), reniform or ovoid
(figure 14). Karyosome was not seen in any form of this species. Nuclear chroma-
tin loosely distributed within the karyoplasm which stained deep purple blue
(figures 11, 12, 14) to light reddish purple.
Kinetoplast : It was larger in size than in T. mysti (sp. nov.) described above
(table 2), and was usually situated towards either of the extremities within the cell
(figures 11, 14). It always stained bluish black.
Flagellum : One anterior and one posterior flagella were always present in all
forms. In few forms anterior flagellum was longer (figures 11, 12) while in others,
posterior (figure 14). In some forms both the flagella free abruptly, after traversing
through the cytosome (figures 11 and 14), while in others it borders the outer
margin of the cell 01? the undulating membrane (figure 12) and then frees.
Undulating membrane : It was present in few forms (figure 14) and not distinct
in others (figures 11, 12).
404 B D Joshi
Remark : Two specimens of the host fish out of 65 observed were found
harbouring this paiasite.
4. Discussion
Despite the fact that the above description is characteristic to the ne\r species of
the haematozoans described, the problem of new speciation remains complicated
for the haemoflagellates from fish, as also encountered earlier by various authors
(Baker 1960 ; Becker 1970 ; Putz 1972 ; Joshi 1978 and Mandal 1979). The
problem becomes more complicated when a particular species of these haematozoan
parasites show a great degree of polymorphism (Laired 1948, 1951 ; Tandon and
Joshi 1973 and Joshi 1978). Besides, experimental studies have also revealed
that many of these haematozoans are euryhostpitalic (Becker 1977). Further*
recently Froes et al (1978, 1979) and Grogl et al (1980) have described six and
one new species of the trypanosomes, respectively, from seven new host fish. They
have also used the same criteria of species specificity and varying morphometric
characteristics in all cases to create new speciation.
In the present case T. aori (sp. nov.) is not only different in having a new host
fish, hitherto not described, but also in various cytomorphological and meristic
characteristics. A high degree of polymorphism was also evident. The morpho-
metric differences from the twentytwo species of the trypanosomes described
earlier from the Indian freshwater teleosts viz, four species by Qadri (1955, 1962),
one by Hasan and Qasim (1962), one by Misra etal (1973), two by Tandon and
Joshi (1973), two by Ray-Chaudhury and Misra (1973), one by Pandey and
Pandey (1964), seven by Mandal (1975, 1977, 1978 and 1979) and four by Joshi
(1976 and 1978). The new species, T. aori is also conspicuously different from
many other forms described from various freshwater host species by Button et al
(1906), Hoare (1932), Baker (1960), Smirnova (1970) and Abolarin (1970). The
degree of polymorphism encountered here is well comparable with those described
by Laired (1952) for several species of trypanosomes.
Table 1 provides a comparative account of meristic morphological characteristics
of eight species of trypanosome described from the siluroid hosts inhabiting the
freshwater realms of the Indian subcontinent. Interestingly, when linear over-
laps of all these species including the new species described here, are compared
(as suggested by Mayr 1969), then at least 10-30% of all the measurements given
for a species are overlapped by one or the others. However, despite these facts,
the present new species T. aori, described here, have two major differences than
the others described earlier. These are (i) small forms are most charac-
teristic in appearance (acquiring a twisted grub-like structure) and (ii) these forms
have usually maximum body width at the centre of the nucleus. Besides, the
flagellar length, post nuclear distance, presence of the karyosome within the
nucleus and comparatively blunt posterior extremity are its species characteristics.
Two new species of trypanoplasma viz., T. mysti and T. atti, differed not only
from C. indica (Mandal 1979), described from Af. vittatus, but also from related
species like C. borsott (Laveran and Mesnil 1901), C. salmositica (Katz 1951)
and C cataractae (Putz 1972), in almost all morphometric and cytomorphic charac-
ter. It is important to mention that till recently the diflagellate haemotozoans
New species of haematozoans 405
harbouring in the blood stream of the fishes were described under the genus
Cryptobia (viz., Katz 1951) and since recently it has been resolved thai all the
diflagellate haematozoans be described under the generic name of Trypanoplasma,
as pointed out by Woo (1979).
Acknowledgements
The author is indebted to Dr R S Tandon, Department of Zoology, University of
Lucknow, in whose laboratory part of this work was done, and to UGC for
financial assistance (vide grant No. UGC 10671).
References
Abolarin M O 1970 A note on the trypanasomes of the African freshwater fishes and some
comments on the possible relationship between taxonomy and pathology in trypanosomes;
Bull. Epizoot. Dis. Afr. IS 221-228
Baker J R 1960 Trypanosomss and dactylosomes from the blood of freshwater fish in East
Afric? ; Parasitol. 5 515-526
Becket C D 1970 Haematozoa of fishes, with emphasis on North American Records. Spl.
Publ. No, 50 82-100 American Fish Society. Washington, D. C.
Becker C D 1977 Flagellate parasites offish ; Parasitic Protozoa (ed) JP Kreier Academic Press
pp 357-416
De-Mello I F and Valles C F 1936 On the Trypanosoma found in the blood of the Indian
freshwater fish Clarias batrachus (Linn.) ; Proc. Indian Acad. Sci. B38 120-124
Dutton J E, Tood J L and Tobey E N 1906 Concerning haemoflagellatcs of an African fish
i| Clarias angelensis; J. Med. Res. 15 491-494
Froes O M, Fortes E, Lima D F and Lrite V R V 1978 Trypanosomes (Protozoa, Kineto-
plastida) of freshwater fishes from Brazil I. Description of three new species; Rev. Bras.
Biol. 38 461-468
Fores O M, Fortes E, Lima D F and Leite V R V 1979 Trypanasomes (Protozoa, Kinetc-
plastida) of freshwater fishes from Brazil. II. New trypanosomes from " Casudos" (Pisces
Loricaridae) ; Rev. Bras. Biol 39-425-429
Grogl M, Marinkelle C J M F, Sanchez N and Guhl F 1980 Trypanosome magdalenae sp. n
(Pratozua : Kinetoplastida) from freshwater teleosts, Patenia kraussi ; Colombia /.
Parasitol. 66 1022-1026
Hasan R and Qasim S Z 1962 Trypanosoma punctati n.sp. from the fish C. punctatus
common freshwater murrcl of India ; Z. Parasitenkd. 22 118-122
Hoare C A 1932 On protozoan blood parasites collected in Uganda ; Parasitol. 24 210-217
Joshi B D 1976 On two new species of trypanosomes from two fresh water teleosts ; Indian
J. Zootomy 17 5-10
Joshi BD 1978 Two new species of trypanosomes from freshwater teleosts ;/. Anim. Morphol
Physiol 25 1-7
Joshi B D 1979 On the occurrence of trypanosomes in the blood of some freshwater teleosts
of Lucknow (UP), India ; Proc. Indian Acad. Sci. B88 59-63
Katz M 1951 Two new haemoflagellates (Gen. Cryptobia) from some Western Washington :
/. Parasitol. 36 245-250
Laired M 1948 Trypanosoma heptatreti sp. nov. a blood parasite of hagfish ; Nature (London)
61 440-441
Laired M 1951 Some trypaoosomes of New Zealand fish ; Proc. ZooL Soc. London 12 285-309
Lavran A and Mesnil F 1901 On the flagellates with undulating membrane of the fishes (Gen.
Trypanosoma gruby and Trypanoplasma n.geo.) ; C.R. Acad. Sci. 133 670-675
Lingard A 1904 A short account of the various Trypanosomata found to date in the blood of
some of the lower animals and fish ; Indian Med, Gaz, 49-46-449
408 R C Rajalakshmi Bhanu et al
Heidenhain's Azan and Delafield's haematoxylin/eosin techniques were used
for routine histological studies. For histochemical studies the following tech-
niques mainly from Pearse (1968) have been employed. (1) Periodic acid/Schiff
(PAS) method of Hotchkiss and McManus, (2) PAS after diastase digestion, (3)
PAS after acetylation followed by deacetylation, (4) alcian blue 8 GX (2-5 and
1 -0 pH) for acid mucosubstances, (5) mercury bromophenol blue method of
Mazia et al, (6) Milion's reaction after Baker, (7) p-dimethyl aminobenzaldehyde
nitrite method of Adams, (8) potassium permanganate/AB method of Arvy and
Gabe, (9) ferric ferricyanide method of Chevremont and Frederic, (10) ninhydrin/
Schiff method of Yasuma and Itchikawa, (11) Congo red technique for glycopro-
teins, (12) Sudan black B technique for lipids after Chiffelle and Putt, (13) copper
phthalocyanin method of Kliiver and Barrera for phospholipids, (14) methyl
green/pyronin Y method. of Kurnick for nucleic acids, (15) Feulgen reaction of
Feulgen and Rossenbeck for DNA.
3. Observations
3.1. Histology
The albumen gland is a white opaque* mass lying dorsal and just posterior to the
pericardial cavity. In live condition, the albumen gland is creamish white in
colour and consists of large number of tubules (figure 1) separated from one
another by a thin layer of connective tissue. These tubules are spherical to oval
in shape. The wall of each tubule consists of large cuboidal to columnar cells
measuring about 58-5 /nn in height. Each cell contains a large basal nucleus and
is glandular in nature and secretory droplets are seen towards the apex of the
cell. The tubules lead into a number of small ducts which unite to form a
common duct which in turn opens into the capsular gland. The secretions of the
albumen gland are acidophilic as well as basophilic in nature. These secretions
are at their peak in the breeding season which is from December to June and
the albumen gland is found to be considerably small in the non-breeding season*
The oviduct opens into the albumen gland at a point below the kidney. The
albumen gland turns abruptly downwards making an acute angle with the ovi-
duct and it then recoils on itself passing ventrally to open into the capsule gland.
The albumen gland opens into the ventral wall of the capsule gland by a short
duct which is lined by a columnar ciliated epithelium, interspersed with few muco-
cytes. It is surrounded by a thick layer of circular muscles which on contraction
close the passage between the two folds.
The capsule gland is a creamish yellow glandular mass. In reproductively active
individuals this gland attains a thickness of about 2 mm and a length of about
5mm. The lateral walls are thickened and composed of groups of gland
cells lying at various heights (figure 2). They are packed together tightly with
a thin layer of connective tissue in between. The ducts of the walls run
parallel to one another and open between the columnar ciliated cells lining the
lumen. This ciliated epithelium covers the narrow dorsal wall, under which a
layer of gland cells is developed (figure 3). The lobes are composed of gland cells
filled with small colourless spherules. These spherules stain lightly with iron
Histological and histo chemical studies on Thais bufo
409
Figures 1-2. 1. Schematic representation of the albumen gland. 2. Schematic
representation of the capsular gland.
410 R C Rajatakshml Bhanu et al
Figures 3-4. 3. Transverse section of the capsular gland showing mucous cells
4. Transverse section of the capsular gland (Azan).
Histological and histo chemical studies on Thais bufo
411
Figures 5-8. 5. Albumen gland tubules showing the PAS reactive secretary drop,
lets. 6. Capsule gland showing PAS reactive substances. 7. Capsular glani
showing mucous secretions (AB 2' 5 pH). 8. Capsular gland showing proteinaceous
secretions (BPB).
Histological and histo chemical studies on Thais bufo 413
.aematoxylin and faint blue with alcian blue. After Heidenhain's Azan technique
pherules are reddish or orange and others blue. It appears that two types of
ecretions are produced by these cells. Near the posterior end of the capsule
land two narrow transverse strips of tissue one on either side arise near the
opening of the albumen gland. These strips separate the right and left posterior
ip from the main mass. These and the posterior tips of the gland are made up
>f mucous cells. Similar cells are found at the anterior extremity of each lateral
obe (figure 4). Beneath the ciliated epithelium of the strips a circular muscle
ayer is present. The cells constituting the main part of the gland are filled with
irge colourless granules. They stain with iron haematoxylin but are negative to
Ician blue. With Heidenhain's Azan the granules take a deep red stain, whereas
he cytoplasm stains deep blue. The distal tip of each duct is filled with the
nucoid substances and no granules are visible. Two types of secretions are
>roduced, a mucoid substance and a protein.
5.2. Histo chemistry
Joth the albumen gland and the capsular gland are intensely positive to PAS tech-
lique (figures 5 and 6). This PAS reactivity was resistant to saliva treatment
luggesting the absence of glycogen. This reactivity was abolished after acety-
ation and was restored after deacetylation indicating the presence of 1 : 2 giyco-
;roups. They showed a moderate positivity to Schiff's reagent without prior
>xidation. Those cells lining the posterior tips and the anterior extremity of each
ateral lobe of the capsular gland showed positivity to alcian blue at 1-0 and
1-5 pH (figure 6), whereas the albumen gland showed a negative response.
Both the gland cells were positive to mercury bromophenol blue, a technique
or basic proteins. Thus the proteinaceous nature of the gland cells is indicated
figure 7). This was further confirmed by subjecting the slides to deamination
vith Vanslyke's reagent. When subjected to ^-DMAB nitrite method and Miilon's
:eaction a negative response was observed. With ninhydria/Schiff and chlora-
nine T/Schiff the albumen gland showed an intense positivity whereas the capsule
jiand showed a moderate positivity, thus suggesting the presence of large and
noderate quantities of protein bound NH2 groups respectively. The presence
)f disulphides was indicated by their response to KMnO</AB technique. The
ilbumen gland showed an intense reaction to ferric ferricyanide for S-H groups,
;vhile the capsule gland stained faintly.
There is no considerable quantity of lipid as suggested by Sudan black B tech-
lique, but copious volumes of phospholipids are present as evidenced by a very
itrong positivity to copper phthalocyanin.
The presence of nucleic acids such as RNA and DNA were traced by methyl
$reen/pyronin Y reaction and Feulgen reaction respectively.
5 From the ensemble of these reactions it could be stated that both the albumen
>land and capsule gland are highly proteinaceous. They seem to contain large
luantities of basic protein, cystine, sulfhydrils, amino bound proteins, carbo-
wdrates lipids and phospholipids. The secretions of the albumen gland are rich
n carbohydrates and protein whereas that of the capsule gland is a mucoprotem.
Elesuits of the above histochemical reactions are presented in table 1.
414 R C Rajalakshmi Bhanu et al
Table 1. Histochemical reactions of the albumen and capsular glands of Thais
bitfo.
Results
Albumen Capsular
gland gland
Periodic acid/Schiff (PAS)
Carbohydrate
+++ +++
PAS/saliva
Glycogen
+++ +++
Acetylation/PAs
1 :2 glycols
— —
Deacetylatioa/PAS
1 :2 glycols
+ + + +
SchifFs without prior oxidation
Aldehydes
+ 4. + +
AB 2-5 pH
Mucins
— + + +
AB 1*0 pH
Mucins
— + + +
Mercury bromophsnol blue
Basic protein
+ + + + +
Millon's reaction
Cystine
— —
DMAB nitrite method
Tryptophan
— —
Ninhydrin/Schiff
NH2 groups
+ + + + +
Permanganate/ AB
Bisulphides
+ + + +
Ferric ferricyanide
Sulfhydryls
+ + + +
Sudan black B
Lipids
+ + + +
Copper phthalocyanin
PhoGpholipids
+++ +++
Methyl green/pyronin Y
Nucleic acid
+ + + +
Feulgen reaction
DNA
+ + + +
Intensely positive ; ++ = Moderately positive; + =* Faintly positive; — — Negative.
4. Discussion
The albumen gland is composed of a large number of tubules which are lined by
secretory cells. A change in its secretory activity was noticed with season. The
secretions of the albumen gland seem to contain PAS positive granules without
glycogen, protein and phospholipids. The eggs as they pass through the albumen
gland are bathed by the albuminous secretions of the gland cells. These secre-
tions are helpful in nourishing the embryos. The fact that the secretions of the
albumen gland contain the polysaccharide galactogen rather than glycogen was
established by the studies of May (1934) and Baldwin and Bell (1938) in the snail
Helix pomatia. Fantin and Vigo (1968) reported the presence of galactogen
and protein in the secretions of the albumen gland of L. stagnalis. Plesch et al
(1971) observed PAS positive secretory droplets in the albumen ggland of
L. stagnalis.
The capsule gland in T. bufo is large and attains a thickness of about 2 fan,
when the animals are in the active reproductive phase. As the eggs pass down
the albumen gland into the capsule gland along with the albuminous secretion
Histological and histo chemical studies on Thais bufo 415
a capsule is formed around a group of eggs along with the albumen. All the
gland cells of the capsule gland, except those of the posterior tip and anterior
border of each lobe, produce a double secretion. By the intervention of these
two secretions the fibrous wall of the egg capsule is produced. The capsule is
thus mucoprotein in nature.
Studies on the capsule gland in particular are meagre. This gland secreting
the egg capsule is present in some prosobranchs and the capsule is finally hardened
in the pedal gland in Cypraeacea, Lamellariaceae and in most Stenoglossans.
The capsule gland is absent in Onchidella and Pulmonates (Fretter 1943). The
mucoprotein secretions of the capsular gland in Tf bufo aid in the formation
of the fibrous wall of the capsule.
Acknowledgements
RCRB is thankful to the Council of Scientific and Industrial Research, New Delhi,
for financial assistance.
References
Baldwin E and Bell D J 1938 Preliminary investigation of galactogen from the albumen gland
of Helix pomatia ; J. Chem. Soc. 1938 1461-1465.
Fantin BAM and Vigo E 1968 Histoohemistry of ths glands associated with the reproductive
tract of Lymnaea stagnalis ; Histochemie 15 30D-311
Fretter V 1941 The genital ducts of some British stenoglossan prosobranchs ; /. Mar. Biol.
Assoc. U.K. 25 173-211
Fretter V 1943 Studies on the functional morphology and embryology of Onchidella celtica
(Forbes and Hanley) and their bearing on its relationships; /. Mar. Biol. Assoc. U.K. 25
635-730
Fretter V 1946 The genital ducts of Theodoxus, Lamzllaria and Trivia and a discussion on their
evolution in the prosobranchs ; /. Mar. Biol. Assoc. U.K. 26 312-351
Fretter V and Graham A 1962 British yrosobranch molluscs ; Roy. Soc. Land. 144 series pp. 755
Kugler O E 1965 A morphological and histochernical study of the reproductive system of the
slug, Phillomycus carolinianus (Bosc) ; /. Morphol. 116 117-132
May F 1934 Chem'sche und biologische untersuchungen uber Galactogen ; Ztschr. f. Biol. 95
287-297
Pearse AGE 1968 Histochemistry : Theoretical and applied (London : Churchill) Vol. 1
Plesch B, Marijke de J B and Boer H H 1971 Histological and hlstochemical observations
on the reproductive tract of tte hsrmophrodite pond snail Lymnaea stagnalis (L.) ; Neth.
J. Zool. 21 180-201
Rangarao K 1963 The poly sacchar ides of the reproductive system of the land snail Arhphanta
tigulata in the formation of egg capsules ; J. Anim. Morphol. Physiol. 10 158-163
Proc. Indian Acad. 831". (Aniin. Sci.), Vol. 91, Number 5, September 1982, pp. 417-422.
© Printed in India.
Effect of temperature on food intake, growth and conversion
efficiency of Eupterote mollifera (Insecta: Lepidoptera)
S PALANICHAMY*, R PONNUCHAMY f and T THANGARAJ tt
* Department of Zoology, Arulmigau Palaniandayar Arts College, Palni 624 602 ,
India
t Department of Zoology, Bangalore University, Bangalore 560056, India
ff Department of Zoology, University of Madras, Madras 600005, India
MS received 12 August 1981
Abstract. The effect of temperature on food intake, growth and conversion
efficiency has been studied in the final instar male and female larvae cf Euptercte
mollifera. Food consumed, assimilated and metabolised decreased with increase
in temperature. The larval duration decreased from 12 days for the group reared
at 22° C to 5 days for the group reared at 37° C. While the rates of feeding, assimi-
lation and conversion increased with increase in temperature, high conversion
efficiencies (K± and K2) w^re observed for the larvae reared at 27 and 32° C.
Keywords. Temperature ; food intake ; Eupterote mollifera.
I. Introduction
Many species of lepidopterous larvae are known to cause serious damage to
economically important plants (Ayyar 1963). While the energetics of food utili-
zation in relation to temperature have been reported for a few lepidopterans
(Waldbauer 1968 ; Mathavan and Pandian 1975 ; Pitchairaj et al 1977), there
are no such studies on Eupterote mollifera which is a common pest on drum-stick
plant. This paper, based on the earlier studies of energy intake and expenditure
pattern (see Palanichamy et al 1979), reports the effect of temperature on food
utilization in the tropical moth E. mollifera.
Lepidopterous larvae consume more than 70% of the total food intake during
final instar (Waldbauer 1968 ; Mathavan and Pandian 1975) and accumulate
sufficient energy (Delvi and Pandian 1972 ; Pandian 1973) to tide over the non-
feeding pupal stage. Palanichamy et al (1979) reported that the final instar larvae
of Eupterote mollifera consumed 71-4% of the total food intake at 30 ± 2° C.
Hence, the effect of temperature on food utilization has been studied only in the
final fifth instar larvae of E. mollifera.
417
418
S Palanichamy, R Ponnuchamy and T Thangaraj
2. Materials and methods
Newly hatched first instar larvae of Eupterote mollifera were collected from the
field and reared as a group in 8 litre glass trough. As soon as the larvae entered
the final instar, the males and females were separated out, weighed and reared
individually in 1 litre glass container at four different temperatures (22, 27, 32
and 37° C) with an accuracy of 1° C. Sex identification was confirmed after adult
emergence and any larvae identified wrongly was discarded from the experiment.
The larvae were fed ad libitum with fresh leaves of Moringa pterygosperma
(drum-stick plant) daily throughout the experimental period. Daily food intake
was measured by a standard gravimetric method (Waldbauer 1968) with all
weighings accurate to O'Olmg. Food, faeces and larvae were dried overnight
at 90 ± 2° C to constant weight for purposes of calculations (see Palanichamy
et al 1979).
3. Results
3.1. Larval duration and growth
The changes in the instar duration, live body weight and growth in relation to
four different temperatures are indicated in table 1. While there were distinct
differences in the live body weight of male and female larvae at all temperatures,
least differences were observed between the two sexes reared at different tempera-
tures. However, the instar duration decreased from 12 days for the larvae reared
at 22° C to 5 days for the larvae reared at 37° C. While the maximum weight
of male (710 mg) and female (916 mg) larvae was observed when reared at 22° Cr
highest growth was observed (male : 124 ; female : 164mg) for the larvae
reared at 32° C.
Table 1. Initial and final live weight of fifth instar larvae of male and female
Eupteroie mollifera fed on the leaves of dram-stick plant Moringa pterygosperma
at different temperatures.
Tempe-
Fifth
i n ^"f fl T
Initial weight (mg)
Final weight (mg)
Growth (mg)
(°C)
duration
Male Female
Male Female
Male Female
(days)
22 12-Oil-OO 229±36-2 253±32'6 710±90'4 916±84'5 10l±10*9 134±14*2
27 7'5±0-50 227±39*6 240±38*9 649±82'2 855±94*7 103±12-1 142 ± 9*7
32 77-0±0-00 216±32'S 231±27'4 691±88'1 748±92'9 124±ll-3 164± 8'6
37 5-0±l-00 151 ±29- 7 167 ±24- 1 562±79'3 601±7T'9 82± 9'0 87± 8*1
Effect of temperature on Eupterote mollifera
419
3.2. Food utilization M
The amount of food consumed and assimilated were high for either sexes of larvae
reared at 22° C and least for those reared at 37° C (figure 1). However, the
values remained similar for males and females reared at 27 or 32° C. In spite of
higher food consumption and assimilation for the larvae reared at 22° C, maximum
growth (male : 124 ; female : 164 mg) was observed for the larvae reared at
32° C. These values are higher than those reported for the same species (male :
107-7 ; female : 148 '5 mg) reared at 30 ± 2° C (see Palanichamy et al 1979).
3.3. Rates of bioenergetics of feeding
At all the temperatures tested, the rates of bioenergetics of feeding did not vary
much between the male and female larvae (figure 2). While the rates of bio-
1000
"22 27 32
TEMPgRATUBE CO
1 Effect of temperature on food intake, assimilation and conversion in
i t?ac0of"ale and female E^erote ^/-^ontKeJ^vesct
plant Morlnga pierygosperma. Each
10 larvae (— ± S.D.).
^resents an average of
420
S Palanichamy, R Pomuchamy and T Thangcttaj
Rate « mg dry/g Uv« larva /day
Q MALE
n FEMALE
200k
120
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27 32
TEMPERATURE CO
Figure 2. Histogram represents the effect of temperature on the rates of feeding,
assimilation and conversion in male and female of fifth instar larvae Eupierote
mollifera fed on the leaves of drum-stick plant. Each value represents an average
of 1C larvae (= ± S.D.).
energetics of feeding indicated gradual increase with increases in temperature,
the conversion rate did not show much variation for the larvae reared at 32 and
37° C. However, distinct differences were observed in conversion rate for the
larvae reared at 22 and 27° C.
3.4. Assimilation and conversion efficiencies
The changes in the assimilation and gross and net conversion efficiencies are
represented in figure 3. Assimilation efficiency decreased for either sexes with
increase in temperature (male : 50 ; female : 49% at 22° C to male : 36 ;
female : 31% at 37° C). However, high gross (^ :%) and net (AT2 : %)
Effect .of temperature on Eupterote moUifera
421
H
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22 27 32 37
TEMPERATURE (*C)
Figure 3. Histogram represents the effect of temperate e on the assimilation and
conversion (£"1 and £2) efficiencies in male rnd female of fifth instdr larvae Eupterote
mollifera fed on the leaves of drum-stick ^lant. Each value represents an average
of 10 larvae (= ± S.D.).
conversion efficiencies were found for either sexes reared at 32° C. As observed
in the rates of bioenergetics of feeding, assimilation and conversion efficiencies
remained similar for both the sexes at all the temperatures tested.
4. Discussion
The final body weight of fifth instar female Eupterote mollifera showed an increase
of 51% at 22° C as against the larvae reared at 37° C ; whereas the increase in
weight was only 32% for the fifth instar female Danaus chrysippus reared at
19° C as against the larvae reared at 37° C (see Mathavan and Pandian 1975).
Probably the variation in temperature (22 and 19° C) provided for these two
422 S Pahmichamy, R Ponnuchamy and T Thangarqr
species may account for the difference. This information supports the fact thai
many species attain larger final body size in the cooler parts of their distribution
(Kinnc 1970) and explains the variation in the maximum weights of different
ecotypes of insects with seasonal and geographical distribution (Odum 1971).
Food consumed, assimilated, converted and metabolized by Eupterote mollifera
showed distinct differences between the larvae reared at 22 and 37° C. However,
the values did not exhibit distinct differences for the larvae reared at 27 and 32° C.
This is in confirmity with the findings reported for the lepidopteran Danaus
chrysippus (Mathavan and Pandian 1975), The high conversion observed for the
larvae reared at 27 and 32° C indicates that these temperature ranges are optimum
to elaborate best growth.
On an average, the larvae of Eupterote mollifera showed nearly 1 J times higher
rates of feeding (353 mg/g live larvae/day) and assimilation (138 mg/g live larvae/
day) at 37° C than those reared at 22° C. However, this temperature proves to
be lethal for the larvae as evidenced by high mortality. The mortality was
observed due to reduction in the thickness of the skin which in turn changes
the colour from dark brown to reddish brown. The larvae reared at 37° C
consumed less food (680 mg) and assimilated with 33-5% efficiency. Thus the
assimilated food which are available for metabolic and growth processes is not
proportionally increased. This explains the finite body size of the larvae reared
at 37° C though the net and gross conversion efficiencies were more than those
reared at 22° C.
References
AyyarT V R 1963 Handbook of economic entomology (Go.vt. of Madras Publications) pp. 249-
250
Delvi M R and Pandian T J 1972 Rates of feeding and assimilation in the grasshopper
Poecilocems pictus ; J. Insect PhyshL 18 1829-1S43
Kinne O 1970 Temperature in animals. In Marine ecology (ed) O Kinne (London : Wiley
Interscience) pp. 407-514
Mathavan S and Pandian T J 1975 E^ct of temperature on food utilization in the monarch
butterfly Danaus chrysippus ; Oikos 26 60-64
Odum E P 1971 Fundamentals of ecology (Saunders Co.) pp. 574
Palaruchamy S, Thangaraj T and Ponnuchamy R 1979 Studies on food utilization by Eupterote
mollifera ; The Indian Zoologist 3 89-92
Pandian T J 1973 Food intake and energy expenditure patterns in two insect primary con-
sumers ; Citn. Sd. 42 423-425
Pitchairaj R, Mathavan S and Muthukrishnan J 1977 Observations on the effect of tempe-
rature on food utilization in a picrid butterfly ; Comp. Physio! . Ecul. 2 48-50
Waldbauer G P 1968 The consumption and utilization of food by insects ; Adv. Insect PhysioL
5 229-288
Proc. Indian Acad. Sci. (Aiiiin, ScL), VoU 91, Number 5, Septsoibsr 1982, pp.. 4-23-426,
© Printed ia India,
Seasonal variations in the phosphorus contents of the muscle of
catfish Clams batrachus L.
YAGANA BANG
Department of Zoology, Aligarh Muslim University, Aligarh 201001, India
MS received 13 October 1980 ; revised 30 May 19&1
Abstract. Seasonal variations were observed in total acid soluble phosphorus,
inorganic phosphorus and phosphoiipid in the muscle of C. batrachus L. The
maximum concentration of these constituents were recorded during April, May
and June. Thereafter values decreased and the levels remained low during winter
months. The observed changes have been correlated with feeding intensity, gonad
maturation and spawning. The rise and fall of different phosphorus contents were
found to coincide with high and low rate of feeding. There was a gradual rise in the
values when gonads advanced towards maturity. The maximum concentration
corresponded to the period of peak ripeness (April, May and June). The values
declined during the spawning period which possibly indicate the utilization of these
reserves for energy. The low phosphorus co.nten.ts observed in post-spawning and
winter appear to be the result of exhaustion of spawning.
Keywords. Seasonal variations ; phosphorus contents ; C. bairachus.
J . Introduction
Although phosphorus has been, studied in tissues of many fish species (Nakano
1960 ; Nakano and Tsuchiya 1960 ; Chang and Idler 1960 ; Jafri 1965 ;
Bhushana Rao 1965), there seems to be no earlier account on the changes in
muscle phosphorus contents with season except that in some fish such changes
were reported on blood (Shell 1961 ; Siddiqui and Siddiqui 1965 ; Siddiqui and
Naseem 1971 ; Siddiqui 1972). In this paper similar observations are being repor-
ted in the muscle of catfish Clatias batmchus L. a commercially important fresh-
water fish. In earlier papers seasonal variations in other chemical constituents
of this fish have been reported (Bano 1977 ; Bano and Hameed 1979).
2. Materials and methods
Specimens of C. batmchus ranging from 18-26 cm in length were procured at
monthly intervals from a freshwater pond at Aligarh and maintained alive in
a large laboratory aquaria. The fish were left for twenty-four hr for acclamati-
zation before starting the sampling. After that they were removed, killed by
decapitatipn and tissue taken out constantly from anterior trunk region taking
423
424
Jagana Scmo
care that only white muscle is removed The maturity stage of gonad was deter-
mined arbitrarily from the scheme suggested by Qayyum and Qasim (1964). The
two sexes were analysed separately and it was ensured that the muscle were free
of bones. The total acid soluble phosphorus, inorganic phosphorus and phos-
pholipid were determined by the methods described earlier by Bano (1975).
Extractions of these fractions were made at cold temperatures,
3. Results and discussion
Monthly values of different phosphorus contents showed a wide range of fluctua-
tion. The mean values are given in the form of annual cycle in figure 1 . As is
500
400
300
200
160
r
120
330
270
210
150 -
TOTAL ACID SOLUBLE PHOSPHORUS
\
o '•
-o
INORGANIC PHOSPHORUS
PHOSPHOLIPID
NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT
Figure 1. Seasonal variations in the phosphorus contents of the muscle of
Qarias batrachus L.
Variation of phosphorus in Ctarias batrachm L. 425
evideqt from the figute, ia both the sexes, the total acid soluble phosphorus- values
in the muscle were higher from April to June, the maximum being recorded in
May. From June onwards there was a gradual decrease and the minimum values
were recorded in October. Thereafter a regular rise was noted. Almost a
similar trend of change was followed by inorganic phosphorus, being highest in
April and lowest in October. The phospholipid content was highest in the month
of June, the values in females were relatively higher than in males. A subsequent
fall in phospholipid content occurred during July and August and the level
remained low during winter period (September to January).
Mineral contents are influenced by a number of factors such as age, sex and
sexual maturity (Vinogradov and Odum 1953). The present observed variations
appear to be correlated mainly to feeding, gonad maturation and spawning.
Though the synthesis of phosphorus contents takes place inside the body, their
chief source outside the body is food. Jn C. batrachus higher values of total acid
soluble phosphorus, inorganic phosphorus and phospholipid were recorded in the
period (April, May and June) when feeding intensity of fish was high. This high
rate of feeding indicates increased metabolic activity of fish during these months.
Similar observations have been reported by other investigators (Siddiqui and
Siddiqui 1965 ; Siddiqui 1972). Similarly low values observed during winter
period appear to be the result of less active feeding.
There was a marked relationship between the muscle phosphorus contents and
the cycle of gonad maturation. A gradual increase in total acid soluble phos-
phorus, inorganic phosphorus and phospholipid was recorded when gonads
advanced towards maturity. The highest concentration in the muscle from April
to June coincided with the period of peak ripeness. Thereafter the constant
decline corresponded to the period of spawning and in spent fish, values were
quite low (September, October). The fall in phospholipid content was from June
to August. These findings are in accordance with the observation of Siddiqui
and Siddiqui (1965) and Siddiqui and Naseem (1971). They reported maximum
value of phosphorus content in the fish with ripe gonad and declining values in
spawning fish.
It has been observed that during spawning period, feeding activity of fish is res-
tricted and fish needs a great amount of energy. This energy is derived from
various sources. Phosphorus content may be one of the sources as the inorganic
and organic phosphorus play a very important role in energy transfer and enzyme
system (Harper 1963). Hence a gradual depletion in different phosphorus contents
during spawning is quite justifiable. Besides, through intermediary formation of
lecithin, phosphorus is associated with fat metabolism and through the formation
of hexosephosphates of adenylic acid and of creatine phosphate it plays a primary
role in carbohydrate metabolism. During maturation cycle, variations have been
reported in carbohydrate and fat contents (Valtonen 1975; Petersen and Ernrnersen
1977 ; Fernandez and Planas 1980).
Acknowledgement
The author is grateful to Prof. S M Alam for providing necessary laboratory
facilities.
426 Yagana Sano
Kefereaces ,. _.... _ -
Bano Y 1975 Variations in the chemical composition of different sections of the flesh of
Clarias batrachiis L. ; Indian /. Zool 3 39-42
Bano Y 1977 Seasonal variations in the biochemical composition of Clarias batrachiis L. ; Proc.
Indian Acad. Sci. 85 147-155
Bano Y and Hameed T 1979 Seasonal changes in cholesterol content of the muscle of cat-
fish Clarias batmchits L. ; Indian J- Exp. Biol 17 2,14-215
Bhushana Rao K S P 1965 Biochemical studies on red and white muscles of Caranx sexfasciatus
Quoy and Gaimard ; Proc. Indian Acad. Sci. 62 &7-91
Chang V M and Idler D R 1960 Biochemical studies on sockeye salmon during spawning
migration. XIH. The distribution of phosphorus compounds, creatine and inositol in
the major tissues ; J. Fish. Res. Bd. Can. 17 565-582
Fernandez J and Planar J 19&Q Annual variation of some carbohydrate and lipid parameters
in the fish Spicara chrysdis during captivity ; Comp. Biochem. Physiol. 67 383-389
Harper H A 1963 Review of physiological chemistry (Bombay : Lange Medical Publication)
Jafri A K 1965 Studies on tJie biochemical composition of some freshwater fishes ; Ph.D. Thesis,
Aligarh Muslim University, Aligarh
Nakano T 1960 Studies on the physiological chemistry of phosphorus compounds in fish
muscle. IL On the individual and regional variations of phosphorus compound contents
in fish muscle ; Bull Jpn. Soc. Sclent. Fish. 26 1192-1197
Nakano T and Tsuchiya Y 1960 Physiological chemistry of phosphorus compounds in fish
muscle. I. Distribution of various phosphorus compounds in fish muscle ; Nippon Sutsaii
GtJtkaishi 26 1095-1098
Petersen I M and Ernmersen. B K. 1977 Changes in serum glucose, lipids, liver glycogen and
phosphorylase during vitello genesis in nature in the flounder Platichthys flesus L. ; Comp.
Biochem. Physiol. B58 167-171
Qayyum A and Qasim S Z 1964 Studies on the biology of some freshwater fishes. Part I.
Ophiceplialus punctatus Bloch ; /. Bombay NatL Hist. Soc. 61 74-9$
Shell E W 1961 Chemical composition of blood of small mouth bass ; U.S. Fish Wildlife Serv.
Res. Rep. No. 57 1-36
Siddiqui A Q and Naseem S M 1971 Seasonal variations in the biochemical composition of
blood serum of Heteropneustes fossilis (Bloch) (Teleostomi, Heteropneustidae) *, Kashmir
Science 8 41-50
Siddiqui M A and Siddiqui M A 1965 Seasonal variations in calcium, inorganic phosphate
and alkaline phosphatase content of O. punctatus (Bloch) ; Indian J. Exp. Biol. 4 122-123
Siddiqui N 1972 Studies on the chemical constituents of the blood plasma of some freshwater
fishes ; Ph,D. Thesis, Aligarh Muslim University, Aligarh
Vinogradov A P and Odum V 1953 The elementary chemical composition of marine organisms ;
Sears Foundation for Marine Research, Yale Univ.-, New Haven. Connecticut
Valtonen T 1974 Seasonal and sex bound variation in the carbohydrate metabolism of the
liver of white fish ; Comp. Biochem. Physiol A47 713-727
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 5, September 1982, pp. 427-43J..
© Printed in India.
The tannery industrial effluent effect on succinate dehydrogenase
activity pattern in a freshwater snail, Pila globosa
M GURUPRASADA RAO and N V NANDA KUMAR
Department of Zoology, S V University, Tirupati 517502, India
MS received 19 January 1982 ; revised 24 July 1982
Abstract. A high, degree of pollution by tannery effluent contamination has been
recorded in an irrigation reservoir in North Arcot district, Tamil Nadu. Seepage
of contaminants into drinking water wells has also been observed. The tannery
effluent is found to inflict changes in succinate dehydrogenase activity levels in
the hepatopancreas of Pila globosa, a common inhabitant of the polluted environ-
ment.
Keywords. Pila globosa ; tannery effluent ; chromium ; tannin ; succinate dehydro-
genase.
1. Introduction
A major irrigation reservoir namely Chennasamudram reservoir of Chenna-
samudrani village along with its hamlets and drinking water wells contaminated
by tannery effluents were identified in Walajapet taluk, North Arcot District,
Tamil Nadu. Physico-chemical analysis was carried out for a calendar year at
the above work spot (Guruprasada Rao and Nanda Kumar 1981) which revealed
that tannery effluents contain many toxic substances such as chromium compounds,
tannins, sodium chloride, calcium chloride and other compounds in considerable
quantities which adversely affect the biological systems thereby posing a threat
to the ecosystem (Eye and Lawrence 1971). Hence an attempt is made in the
present investigation to study the effect of untreated tannery effluent (TE) at different
concentrations and also its toxic ingredients like chromium (VI), sodium chloride
arid other compounds on succinate dehydrogenase (SDH) activity pattern of a
freshwater snail, Pila globosa, a common inhabitant of the polluted area which
shows low resistance to polluted freshwater environment and is also found to be
sensitive to chromium (Guruprasada Rao and Nanda Kumar 1982).
2. Materials a»d methads
Pila globosa were collected from tmcontaminated water resources. They were
acclimated to the laboratory conditions for eight days and maintained as reported
427
428 M Gumprasada Rao and N V Nanda Kumar
earlier (Muralimohan and Sasirababti 1976). The animals were exposed to the
media reported earlier (Guruprasada Rao and Nanda Kumar 1982). The ratio
of one animal in 500 rnl of medium was maintained throughout the exposure
period in a glass jar. The media employed consisted of different percentages of
TE, and also media containing different concentrations of potassium chromate,
sodium chloride, calcium chloride, and tannin (Wattle extract spray dried)
prepared in freshwater as they form the main ingredients of TE. The animals
were exposed to the above media at selected concentrations and for different
periods separately as mentioned in table 1. After the exposure period, they were
removed and the hepatopancreas were isolated on ice blocks and immediately
transferred to the refrigerator maintaining an ambient temperature of 0° C. The
tissue was used for assaying SDH activity. The concentration of the media chosen
to expose the animals were either found in the effluent or in the reservoir water
under natural conditions. These identifying concentrations chosen are indi-
cated in table 1. Higher concentrations were also chosen to magnify the extent
of implication on the enzyme chosen.
Hepatopancreas were homogenised in ice cold 0-25M sucrose solution, centri-
fuged at 2,500 rpm and the supernatant used as enzyme source. Enzyme assay :
SDH was assayed by the method of Nachlas et al (i960) while employing INT as
electron acceptor and extracting formazan in toluene layers (Nanda Kumar
et al 1973).
3. Results and discussion
The succinate dehydrogenase activity (SDH) showed an enhancement when the
snails were exposed to TE for 3 and 24 hr. Whereas SDH activity showed a signi-
ficant decrease when exposed for 10 days. Hence alteration in the enzyme acti-
vity level in animals exposed for short periods in the effluent contaminated
environment cannot be taken as an index. The change in the enzyme activity
level is an overall expression of combined action of all ingredients of TE. However
a detailed study was done on the effect of various ingredients of tannery effluent
separately on the SDH activity in snails. The effect of potassium cliromate,
tannin, sodium chloride and calcium chloride separately were studied on SDH
activity. Potassium chromate at 3 hr enhanced the SDH activity (table 1) at
various concentrations (1-100 ppm). The SDH activity level showed no increase
at all concentrations chosen (table 1) at 3- hr period and also at 2' 5 ppm level
after 10 day period. However the decrease at 24 hr was not significant. Stimula-
tion of dehydrogenase systems in rats (Horecker et al 1939), oxidation of NADH
to generate NAD by potassium chromate (Gruber and Jennette 1978) and enhanced
oxygen consumption (Ergeshev 1974 ; Sheer and Armitage 1973) with subsequent
oxidation of citric acid metabolites may be cited for the observed increase in SDH
activity.
Tannin was also found to enhance SDH activity (at 3 hr) with a subsequent
depressing effect (24 hr and 10 days). Earlier reports of Luciani (1973) on SDH
inhibition by tannic acid support the "present investigation. Corroborative
evidences of the inhibitory action of tannic acid on succinate transportation into
rat liver mitochondria (Johnson 1972) in isolated erythrocytes (Mitzavilla et al
1977) also strengthen this observation.
tannery effluent effect on SDH m snail 429
Table 1. Succinatc dehydrogenase activity levels in the hepatopancreas of Pila
globosa exposed to different media (% change in enzyme activity is calculated from
7* moles formazan forme d/mg protein/hr).
Medium Concentration*
(1) (2)
Exposure time
(3)
Per cent change in
SDH activity level
(4)
Tannery effluent
10%
3 hr
-•!- 9'86±3'45
N.S.
do.
10%
24 hr
+ 17-69±l-52
P< 0-05
do
10%
10 days
— 16-43±l-72
P< 0-005
Potassium chromate
1 ppm
3 hr
+ 20-71 ±2-4
P< 0-05
do.
10 ppm
3 hr
+ 25- 28 ±3-1
P< 0-05
do.
50 ppm
3 hr
+ 27'89±2-2
P< 0-001
do.
100 ppm
3 hr
+28'67±2-4
P< 0-05
do.
25 ppm
24 hr
—15- 98 ±4- 3
N.S.
do.
2'5 ppni
10 days
+20'72±M5
P< 0-05
Tannin (Wattle extract spray dried)
100 ppm
3 hr
+ 30-76±3'0
P< 0*05
Tannin
100 ppm
24 hr
— 5'30±0-48
P< 0*05
do.
20 ppm
10 days
— 19'76±2-34
P< 0-05
Sodium chloride
5000 ppm
3 hr
+ 9-42+4-2
N.S.
do.
5000 ppm
24 hr
+ 8-76±M7
P< 0-05
do.
1 000 ppm
10 days
— 31-16±3-67
P< 0-05
Calcium chloride
1000 ppm
3 hr
+ 2-55±l-5
N.S.
do.
1000 ppm
24 hr
+ 1-28±1-1
N.S.
do.
500 ppm
10 days
+ 13'99±1-61
P< 0-05
4- or — indicate % increase cr decrease in enzyme activity ovei control respectively.
Control activity is normalised to 100% = 0' 0527 ± 0' 001 p. moles of Formazan formed/mg
protein/hi.
± S.D. from mean of six observations.
N.S. Not significant at the level of 5%.
* Concentrations comparable to effluent/irrigation reservoir water (Guruprasada Rao and
Nan<Ja Kumar 1981),
430 M Guruprasada Rao and N V Nanda Kumar
Sodium chloride enhanced the SDH activity at 3 and 24 hr whereas at 10 day
the SDH activity showed a decrease. Variations in the salinity of the environ-
mental medium are known to exert considerable influence on the activity behaviour
and metabolism of invertebrates (Gilles et al 1971 ; Negus 1968). Sodium chlo-
ride was found inhibitory to TCA cycle (Korff et al 1954) and to SDH in vitro
(Gilles et al 1971). The decrease in the SDH activity in Pila globosa at 10 day
exposure observed in the present investigation might be due to the accumulation
of chloride ion in the medium. Corroborative evidence comes from the works
of Venkata Reddy (1976) who demonstrated a decrease in hepatopancreas SDH
activity in crab under sodium chloride stress.
Enhancement in the SDH activity was observed in Pila globosa exposed to
calcium chloride (table 1). At present experimental evidence is lacking on the
calcium chloride stimulation of SDH system. However it is suggested that the
triggering of glycolytic pathway, activation of ATP hydrolysis and increase in
glucose amounts (Hochachka and Somero 1973 ; Meenakshi 1956) might be
responsible for generation of raw material required for oxidative metabolism
(Adams and -Quastei 1956: Fruton and Simmonds 1965) and the possible
increase in SDH system.
Acknowledgements
We thank Prof. R Ramamurthi for providing facilities and CSIR, New Delhi, for
financial support to one of the authors (MGPR),
References
Adams D H and Quastel J H 1956 Proc. Roy. Soc. B145 472-479
Ergeshev I 1974 Chromium effect on the intensity of O3 consumption by guinea pigs ; Biol
Abstr. 58 28980
Eye J D and Lawrence L 1971 Treat me at of wastes from a sole leather tannery ; /. Wat.
Pollut. Cont. Fed. 43 2291-2303
Frutan J S and Simmonds S 1965 General biochemistry (Madras : Asia Publishing House)
Gilles R, Hogue P and Keauny B 1971 Effects of various ions in the succinate dehydrogenase
activity of Mytilus caUfomeanus ; Life Sciences 10 1421-1427
Gruber J E and Jennette K W 1978 Metabolism of carcinogen chromate by rat liver
. mjcrosomes ; Biochem. Biophys. Res. Commw. 82 700-706
Guruprasada Rao M and Nanda Kumar N V 1981 Analysis of irrigation reservoir contaminated
by tannery effluents ; Indian J. Environ. Health 23 239-241
Guruprasada Rao M and Nanda Kumar N V 1982 The tannery industrial effluent effect on
choHnesterase activity pattern in a freshwater snail, Pila globosa ; /. Environ. Biol. (in
press)
Hochachka PWand Somero GN 1973 Strategies of biochemical adaptation (Philadelphia: W B
Saunders Company) pp. 132-143
Horecker B L> Stotz B and Hogness T R 1939 The promoting effect of chromium and
the rare earths in the succinic dehydrogenase cytochrome system ; /. BioL Chem. 128
251-2*6
Johnson D J 1972 Effects of tannic acid on ion transport in rat liver mitochondria ; Arch.
Biochem. Biophys. 151 316-321
Korff R W V, Macpherso.n E H and Glaman G V 1954 Potassium ion stabilization of respi-
ration by heart muscle mitochondria ; /• BioL Chem. 209 1 51-1 53
Tannery effluent effect on SDH in snail 431
Luciani S 1973 .Inhibition by tannic acid of succinate and malate trans location across mito-
chondrial membrane ; Biochem. Pharmacol. 22 1821-1S2&
Meenakshi V R 1956 Studies on physiology of Pila virens (Lamark) with special reference to
aestivation. Ph.D. Dissertation, Annamalai University
Mitzavilla S, Blanquat G D and Derache R 1970 Effect of tannic acid 0.33 intestinal absorp-
tion in the mouse ; Food Cosmet. Toxkol. 8 27-33
Muralimohan P and Sasirababu K 1976 Modification of enzyme activities by body fluids,
arnino acids and divalent cations in the nervous system of aestivating snail ,PIJa globosa*
Indian J. Exp. Biol. 14 232-238
Nachlas M M I960 A colorimetric method for the determination of succinic dehydrogenase
activity ; /. Biol. Chem. 235 499-503
Nanda Kumar N V, Radhakrishnamurthy Ch, Vijayakumari D and Swami K S 1973 Axonal
protein changes and succinate dehydrogenase activity in sheep medulla oblongata ; Indian
J. Exp. Biol. 11 525-52$
Negus MRS 1968 Oxygen consumption and amino acid levels in Hydrobis ulvae (Pennat)
in relation to salinity and behaviour ; Comp, Biochem. PhysioL 24 317-325
Sheer C A and Armitage K B 1973 Preliminary studies of the effects of dichromate ion on
survival and Oa consumption of Daphnia pit] ex ; Crustaceans 25 51-69
Vcnkata Reddy V 1976 Studies on mechanisms underlying acclimation to. salinity in the fresh-
water field crab, Paratelphnsa hydi-odromous ; Ph.D. Thesis (Tirtipati : SV University)
Proc. Indian Acad. Sci. (Aaiiii. Set.), Vol. 91, Number 5, September 19S2, pp. 433-438.
© Printed in India.
Durational effects of hemispaying on ovarian hypertrophy and
estrous cycle in albino rats
SARASWATI B PATIL and M APPASWAMY RAO*
Department of Zoology, Gulbarga University, Gulbarga 585 106, Karnalaka, India
* Retired Professor of Zoology, 5th Main, Yadavgiri, Mysore, Karnataka, India
MS received 18 May 1982
Abstract. Ovarian hypertrophy is studied by hemispaying the rats for 7, 15, 20,
25 and 30 days- The compensatory hypertrophy of the ovary is calculated
in relation to their respective sham operated controls. The maximum hypertrophy
is observed 20 days after hemispaying, as indicated by ovarian weight and its
histological observations. Thereafter the hypertrophic response though significant,
decreases gradually, indicating that once the circulating estrogen seer -ted by the
hypertrophied ovary comes to preoperativc level, the pituitary gonadotrophin level
also falls down. The hemispaying has no significant effect either on the duration
or number of estrous cycle.
Keywords. Ovarian compensatory hypertrophy ; hemispaying ; estrous cycle.
1. Introduction
Unilateral ovariectomy or hemispaying causes compensatory fojlicular proliferation*
ovulation and hypertrophy in rats, mice and hamsters (Arai 1920 ; Mandl and
Zuckerman 1951; Greenwald 1961 ; Pepler 1975). This may be due to the
unchanged availability of pituitary gonadotrophins (FSH and LH) to the remain-
ing single ovary and/or increase in the pituitary release of gonadotrophins due
to decrease in the circulating estrogen after semiovariectomy ( Edgren et al 1965 ;
Welchen 1970, 1972; Howland et al 1,974). The compensatory response of the
ovary may continue till the ovary gets the increased amount of pituitary gonado-
trophins and once the gonadotrophins level falls down to normal, due to increase
in the steroid output by the hypertrophied ovary, this compensatory hypertrophy
may also decrease. Therefore, the present investigation is to study the duration
required to obtain the optimum hypertrophy in albino rats.
2. Material and methods
Nulliparous, female albino rats of Holtzman strain, with regular established
estrous cycle, weighing 130-150 g, 70-80 days old were hemispayed. The right
433
434 • '••- Saraswdtf'B-P.atii and-M Appaswamy Rao
ovary was exposed by dorsolateral route, major blood vessels were ligated and
after split opening the bursa, the ovary was carefully removed. Sham opera-
tion was performed by just exposing the right ovary. All operations were carried
at estrous, under mild ether anaesthesia.
The experimental rats were maintained in individual cages, with Hindustan
Lever rat feed, at water ad libitum, at a room temperature of 27 ± 1° C and
12 hr light/darkness.
The estrous cycle of all the experimental rats were studied everyday morning
by vaginal smear observations. The rats were autopsied after 7, 15, 20, 25
and 30 days. Ovaries were dissected out free from adherent tissue, weighed,
fixed in Bouin's fluid, sectioned and stained with heamatoxylin eosin.
3. Results
3.1- Ovarian hypertrophy
The present investigation is to study the durational effects of hemispaying on
ovarian hypertrophy. In sham operated controls, there is no appreciable change
in the ovarian weight from day 7 to 30. In hemispayed rats compensatory hyper-
trophy is observable as early as 7 days after the operation, wherein per cent hyper-
trophy is 25 -56 (F<0'1). This hypertrophic response gradually increases by
15 and 20 days wherein the respective per cent hypertrophy is 50*41 (P<0'0\)
and 96 '"40 (P< 0-001) in relation to respective sham operated controls. There-
after though the ovarian hypertrophy is significant as evidenced by the hemispay-
ing for 25 and 30 days wherein 65.54% (P< 0.001) and 64.74% (P<0«001)
hypertrophy, is seen respectively, it is slightly less compared to that of 20 days
(table 1).
Table 1. Durational effect of hemispaying on ovarian hypertrophy in a) bine rats.
Ovary wt.
Duration mg/100 g Body wt. M ± S.E.
% hypertrophy
(dayb)
Sham operated
Hemispayed
7 17-18±0"95
21-57±1'91*
25-56
15 18-46±1'85
27-77±0-98**
50-43
20 15-40±0'30
30'24±l-94*'**
96*40
25 16- 92 ±0- 94
28-06±l-99**»
65-54
30 17-78±0-26
29-31±l'14***
64--74 , ,
%, hypertrophy is calculated in relation to respective sham operated controls.
M ± S.E. =* Mean ± standard error.
*P=0-1; **P=0-01; ***P=- 0-001.
Effects of hemispaying in albino rats 435
3.2. Ovarian weight
The ovarian weight in the hemispayed rats also goes on increasing from day 7
to 20, and by day 20 the ovarian weight is almost doubled in hemispayed rats
(30-24 ± l'94mg) in relation to their sham operated controls (15 '40 ± 0'30mg)
with 96-40% compensatory hypertrophy. Then onwards gradually the ova-
rian weight falls down along with a decrease in the ovarian compensatory hyper-
trophy.
3.3. Ovarian histology
Histological observations indicate that the initiation of the ovarian hypertrophy
after hemispaying begins as early as 7 days, wherein the ovary shows large
corpora lutea and graafian follicles. Significant ovarian hypertrophy is seen by
15 days, but it is maximum by 20 days wherein the ovaries are large with well
developed corpora lutea and graafian follicles, indicating the increased follicular
proliferation and ovulation. Similar observations in the ovarian histology is made
after 25 days and 30 days of hemispaying, though the ovarian hypertrophic res*
ponse is slightly reduced.
3.4. Estrous cycle
The cyclical changes observed in the study of estrous cycle gives a fair index of
the ovarian activities (table 2). In the present experiment hemispaying has
no significant effect on estrous cycle either in the duration of diestrus or on the
number of estrous cycles. In sham operated rats the duration of diestrus ranges
from 2' 8 to 3*0 days whereas it is 2' 5 to 3' 1 days in hemispayed rats. The
number of estrous cycles goes on increasing gradually with the increase in the
Table 2. Durational effect of hemispaying on estrous cycle in rats.
duration of diestrus Number of cycles
M ± S.E. M ± S.E.
Duration
(days) Sham operated Hemispayed Sham operated Hemispayed
7(5)
3'0±0'0
3-l±0-2
I'OiO'O
l-2±0-2
15(5)
2'9±0-5
2'0±0-3
2-8±0-2
2'8±0-2
20(5)
2'8±Q'3
2-5±0-l
3'0±0'2
4-0 ±0-0
25(5)
3'0±0'2
3'l±0-3
4'4±0-2
4'6±G'l
30(5)
2'7±0'2
2*9±0'2
6'0±0-3
5-8±0-2
M ± S.E. - Mean ± standard error.
Number in parenthesis denotes the number of rats,
436 Saraswati B Patil and M Appaswamy Rao
duration of the experiment in both sham operated and hemispayed rats. The
number of estrous cycles ranges from 1*0 to 6*0 from day 7 to 30. It is evident
from the above results that these rats are regular 5 days cyclers with 3 days
of diestrus. The hemispayed rats though having single ovary can maintain the
hormonal balance which is essential for the vaginal cornification.
4. Discussion
Ovarian compensatory hypertrophy and ovulation after hemispaying is observed
by several investigators in rats, mice, hamsters and guinea pigs (Arai 1920 ;
Greenwald 1961 ; Hermerck and Greenwald 1964 ; Peppier 1975). The compen-
satory hypertrophy is evident even in the neonatal rats, pregnant and pseudo-
pregnant rats, but not so apparent in aged rats, since there is a decline in the
pituitary output of FSH and LH during that period (Labhsetwar 1967, 1969 ;
Chatterjee and Greenwald 1971; Peppier 1971). In spite of several investigations
the mechanism of ovarian compensatory hypertrophy is still debatable. It is
alluded to relative increase in the availability of serum gonadotrophins to the
remaining single ovary after hemispaying, since no increase in the pituitary gonado-
trophins is observable after hemispaying (McLaren 1963, 1966 ; Edgren et al
1965). However, this contention is questioned as there is an increase in the
gonadotrophin output, due to decrease in the circulating estrogen after hemis*-
paying which is responsible for the ovarian compensatory hypertrophy (Grady
and Greenwald 1968 ; Benson et al 1969 ; Walshen ,1970, 1972 ; Rowland and
Skinner 1973). But according to Greenwald (1968) and Peppier (1972) the
mechanism of ovarian hypertrophy involves not only an increase in the output
of pituitary gonadotrophins, but also the time of exposure to the available
gonadotrophins.
In this paper the ovarian compensatory hypertrophy in relation to sham opera-
ted controls is enhanced with the duration of hemispaying. Therefore the ovarian
compensatory hypertrophy obtained after 7 days is not significant (P<0'1),
significant after 15 days (P<0'01) and highly significant thereafter (P<0'001).
These results agree with those of Greenwald (1968) and Peppier (1972), wherein
the significant compensatory hypertrophy is obtained with an increase in the
time of exposure of the ovary to the constant gonadotrophfc levels. The maxi-
mum ovarian hypertrophy is observed by 20 days after hemispaying. These results
appear to be in agreement with those of Benson et al (1969), wherein an increase
in the initial surge of serum FSH is seen on day 4, comes to preoperative levels
by day 20 to 24. Therefore ovarian hypertrophy increases up to day 20 and
once the circulating estrogens come to preoperative level there will be no increase
in the gonadotrophin output, hence the hypertrophic response of the ovary also
decreases after 20 days.
The study of estrous cycle indirectly indicates the gonadotrophins output from
the pituitary, preceded by the ovarian estrogen secretion. In the present investi-
gation hemispaying has no effect on the duration of estrous cycle wherein the
diestrus extends from 3-4 days both in sham operated and hemispayed rats, which
is in full agreement with the opinion held by Greenwald (1960) and Peppier and
Greenwald (1970). The number of estrous cycles increases from 1 to 6 as tbe
Effect of hemispaying in albino rats 437
duration of the experiment increases from day 7 to 30, in both sham operated
and hemispayed rats. This indicates that the steroid hormone production from
the remaining single ovary in hemispayed rats is sufficient for the vaginal corni-
fication even before the significant ovarian hypertrophy takes place. .
Acknowledgements
The authors are thankful to the Department of Zoology, Karnatak University,
for providing the necessary facilities and one of the authors (SBP) is grateful to
UGC for the award of Jr. Research Fellowship during the tenure of this investi-
gation.
R
Arai A 1920 On the cause of the hypertrophy of the surviving ovary after hemispaying (albino
rats) and on the number of ova in it ; Am. J. Anat. 28 59-79
Benson B, Sorrentono. B and Evans J S 1969 Increase in serum FSH following unilateral
cvariectomy in rat; Endocrinology 84 369-374
Chatterjee A and Greenwal'd G S 1971 Compensatory ovarian hypertrophy of the pseudo-
pregnant and pregnant lats ; Endocrinology 88 191-196
Edgren R A, Parlow A F, Peterson D L and Jones R C 1965 On the mechanism of ovarian
hypertrophy following hemicastration in rats ; Endocrinology 76 97-102
Grady K L and Greenwald G S 1968 Studies on the interaction between the ovary and
pituitary follicle stimulating hormone in golden hamsters ; J. Endocrinol. 40 85-90
Greenwald G S I960 The effect of unilateral ovariectomy on follicular maturation in the hamster;
Endocrinology 66 89-95
Greenwald G S 1961 Quantitative study of follicular development in the ovary of intact and
unilaterally ovartectomised hamsters ; /. Reprod. FertiL 2 351-361
Greenwald G S 1968 Influence of one or two ovaries on ovulation and ovarian weight in the
hypophysectomised rat ; Endocrinology 82 591-596
Hermerck A S and Greenwald G S 1964 The effect of unilateral ovariectomy on follicular
maturation in guinea pigs ; Anat. Rec. 148 171-176
Rowland B E and Skinner K R 1973 Effect of hemiovariectomy on serum FSH and LH levels
during the estrous cycle in the rat ; /. Reprod. FertiL 32 501-503
Howland B E, Jack M I and Beaton D B 1974 Effect of hemiovariectomy and strain of rat
on serum gonadotrophin levels ; Experientia 30 653-654
Labhsetwar A P 1967 Age dependent changes in the pituitary -gonadal relationship : A study
of ovarian compensatory hypertrophy ; /. Endocrinol. 39 387-393
Labhsetwar A P 1969 Age dependent changes in the pituitary-gonadal relationship n : A study
of pituitary FSH and LH content in the female rat ; /. Reprod. FertiL 20 21-28
Mandl A M and Zuckerman S 1951 Number of normal and atretic oocytes in unilaterally
spayed rats; /. Endocrinol. 6 112-119
McLaren A 1963 Mechanism of ovarian compensation following unilateral ovariectomy in mice ;
/. Reprod. FertiL 6 321-322
McLaren A 1966 Regulation of ovulation rate after removal of one ovary in mice ; Proc.
R. Soc. B166 316-340
Peppier R D 1971 Effect of unilateral ovariectomy on follicular development and ovulation in
cycling, aged rats ; Am, /. Anat. 132 423-429
438 Saraswati B Patil and M Appaswamy Rao
Peppier R D 1972 FSH and LH levels in the intact and unilaterally ovariectomized cycling
rat ; Acta EndocrinoL (kbh) 69 267-280
Peppier R D 1975 Effect of removing one ovary and a half on emulation number in cycling
rats ; Expsrientia 31 243-245
Peppier R D and Greenwald G S 1970 Effects of unilateral ovariectomy on ovulation and
c>cle length in 4- and 5-day cycling rats ; Am. /. Anat. 127 1-8
Welchen R 1970 Compensatory ovarian growth and compensatory ovulation after unilateral
ovariectomy in rats with an ovarian autograph in the region of the portal vein ; Acta
EndocrinoL (kbh) 65 509-516
Welchen R 1972 Effect of unilateral ovariectomy on follicular growth in hypophysectomized
rats treated with pregnant mare serum gonadotrophins (37518) ; /. EndocrinoL 55 227-228
Proc. Indian Acad Sci. (Anim. ScL), Vol. 91, Number 5, September 1982, pp. 439-450
© Printed in India.
Structure and seasonal changes in the testes of a freshwater crab,
Potamon koolooense (Rathbun)
P C JOSHI and S S KHANNA*
Department of Zoology, Government PG College, Pithoragarh 262 501, India
* Joint Secretary, Ministry of Education, Lucknow, India
MS received 2& July 19&1 ; revised 22 July 1982
Abstract. The paired 4H '-shaped testes of Potamon koolooense show histo-
morphologtcal changes during various stages of maturity. Seminiferous tubules
show different stages of spermatogenesis. A few undifferentiated or resting spermato-
gonia supply a new crop of germ cells for the next breeding season. In a tubule
the meiotic divisions occur more or less synchronously in all the primary or secon-
dary spermatocytes. Sperm consists of a head and nutochondrial vesicle which
encloses axial filament and distal centrosome. Spermatogenetic activity is seasonal.
Spermatogenesis begins in January-February, progresses slowly through March,
reaching its peak in April-May. However, all the tubules do not mature simul-
taneously. Spermiation occurs during May and June, the spermatogenesis ceases
gradually, and by December the testes enter a brief period of rest.
Keywords. Potamon koolooense ; testes ; histology ; seasonal changes.
1. Introduction
The structure of male reproductive organs in Crustacea has been described by
Spaiding (1942), Cronin (1947), King (1948), Ryan (1967), Wolfe (1971), Chiba
and Honma (1971) and Gupta and Chatterji (1976). There has been little infor-
mation about the seasonal histomorphological changes in the testes of crustaceans
as investigators concerned themselves with specific study, such as spermatogenesis
(Binford 1913 ; Fasten 1926 ; Baker and Rosof 1927 ; Nath 1932) or measure-
ment of male gonad index for assessment of reproductive cycle (Subrahmanyam
1963 ; Rahman 1967 ; Chandran 1968). Recently seasonal histological changes
have been reported in the crab, Pachygrapsus crassipes (Chiba and Honma 1972),
Barytelphusa cunicularis (Diwan and Nagabhushanam 1974) and crayfish, Orco-
nects limosus (Wielgus 1976). The present paper describes the seasonality in
the testicular activity in a freshwater crab, Potamon koolooense.
2. Material and methods
15 to 20 live specimens of adult male P. koolooense (carapace width 37mm
to 45mm) were collected every month during 1976-78, from a stream near
439
P.(B)-5
440 P C Joshi and S S Khanna
Pithoragarh. The weight of each specimen was recorded immediately before dis-
section. The testes were removed and placed immediately in fixative. Their
length and weight were recorded after fixation. The gonad index (GI) was cal-
culated using the formula (Giese 1959) :
_ weight of the gonad
GI — • t~T FTi • 1 * JLUU.
weight of the animal
For histology, different regions of the testes were fixed in Bouin's fluid, Allen's
Bouin solution or Helly's fluid. Paraffin sections of 5-6 /nn thickness were cut
and stained with Delafield's haematoxylin or Mayer's haemalum, using eosin as
counterstain in all the cases. Heidenhain's Azan, Mallory's triple stain and
periodic acid Schiff (PAS) were also used.
3. Observations
3.1. Morphology
The testes are paired elongated bodies, lying attached with the hypodermis of the
overlying carapace but the ventral side freely rests upon the hepatopancreas.
Testes -of both the sides are connected together at their middle region by a cross
band of testicular tissue so that the pair appears 'H '-shaped. Occasionally
the testes of "two sides are unequal in length and thickness. 'Each testis leads into
a Ipng highly coiled vas deferens which opens outside through the penis situated
on the ventral subterminal region of the coxal segment of 5th leg.
3.2. Histology -
Each testis is* made up of numerous convoluted siminiferous tubules of varying
sizes, held together by a thin layer of connective tissue. The intertubular space
contains a few blood vessels. Each tubule is covered by a thin layer of connec-
tive tissue, and in a transverse section shows two distinct areas, the germinative
region and the lumen (figure 1). Different tubules in the same section consist
of germ cells in different stages of development (figures 6, 7). Tubules at
posterior region of testes become narrow and have small or no germinal area,
while their lumen is full of sperms (figure 2). The tubules appear to be conti-
nuous, opening directly into the vas deferens.
In spermatogenesis, the sperm mother cells or primary spermatogonia are the
germ cells of first stage and are the largest of all Each spermatogonium contains
a thin rim of cytoplasm around a vesicular nucleus containing peripheral chroma-
tin granules (figure 3). The number of primary spermatogonia gradually increa-
ses soon after spawning (June), becoming abundant during November and
December (figure 11). Most of them later on divide mitotically and give rise to
the secondary spermatogonia that will differentiate into spermatocytes, but a few
remain undifferentiated till sperm formation and spermiation (figure 5). These
are the resting spermatogonia which divide soon after spermiation and supply a
new crop of germ cells for the next breeding season. The secondary spermato-
gonia are smaller than the primary ones and chromatin granules distributed homo-
geneously in nucleoplasm (figure 3). These cells undergo mitotic division (figure 4),
so that a large number of primary spermatocytes are formed.
Testicular cycle of crab
441
area
ced x
testes •
°f
442
P C Joshi and S S Khanna
Testicular cycle of crab
443
;ures ld-12. Portions of sections of testes. 1<*. Showing stage I in July x 90.
Showing tubules filled with spermatogonia in December (stagt I) x 140-
Showing tubules packed with spermatids (ST) and sperms (s) in May (stage
) x 90. (L, Lumen; PSP, Primary spermatocytes ; RS, Residual sperms : SPG,
3rmato.gonia ; T, Tubules.)
Testicular cycle of crab
445
The primary spermatocytes are smaller than the spermatogonia but have
eosmophihc cytoplasm and basophilic chromatin threadf T the
(figure 6). A secondary .spermatocyte is nearly half the size of tte
matocyte and its cytoplasm is poorly stainable but cell boundary i c
in newly formed cells. The nuclei are small and have dense chromati i
t T found
the same tubule (figure 6). It is interesting to note that meiotic division occurs
synchronously in all the primary spermatocytes of a tubule and the s^e is Sie
for secondary spermatocytes. This is indicated by the presence of all the primal
or secondary spermatocytes of a tubule at approximately the same stage of deve-
opment (figures 5, 6). Furthermore, a tubule at any time contains either one
type of spermatocytes or spermatids (figures 5, 6, 7).
The spermatids are small rounded bodies having deeply stained nuclei (figure 7)
the cytoplasm stains grey with haematoxylin eosin. During spermiogenesis
they undergo morphological changes and at the end dome-shaped sperms are
formed. Each sperm consists of a head and the so-called mitochondrial vesicle
(figure 8). The head is deeply stainable with haematoxylin and represents the
nucleus. The vesicle is eosinophilic and contains a feebly staining axial filament
At distal tip of axial filament is a thick blue-black staining transverse piece the
so-called distal centrosome (figure 8). The proximal centrosome is not visible
as it is reported to be fused with nucleus. Pseudopodial rays which spread out
from head were not visible with the methods employed. When stained with PAS
all the cells except spermatids and sperms are negative to the stain. In sperms
both head and vesicle are PAS positive, while the axial filament is PAS negative
(figure 9).
3.3. Seasonal cycle
The annual testicular cycle of P. koolooense can be divided into the following
four stages on the basis of histomorphological characters :
3 . 3a. Stage I (July to December) : The size and weight of testes gradually
decreases reaching a minimum value in November and December (figure 13). The
testes are thin and translucent during September to December. On puncturing a milky
seminal fluid comes out through the vas deferens. Some of the tubules have large
number of spermatogonia and few residual sperms (figures 3, 10). Spermatogenesis
still continues to exist in some of the tubules of the same section (figure 10), but
the number of spermatocytes and spermatids decreases gradually and finally absent
during December. This indicates gradual cessation of spermatogenesis in testes.
The spermatogonia greatly increased in most of the tubules in October and were
in preponderance during November and December (figure 11). During this period
the dimension of tubules decrease and the wall of tubules becomes thick and
undulated (figure 11).
3 . 3b. Stage II (January to March) : The spermatogenesis begins during January
and February with an accompanying increase in size, weight and opaqueness
of testes. Tubules slightly increase in diameter and their walls become compa-
ratively thin. They contain a few primary spermatogonia and- a large number of
secondary spermatogonia. Mitotic figures are generally seen in such tubules
446
P C Joshi and S S Khanna
O'9
0'8
0'7
Q'&
0'5
0'4
0'3
0'2
tn
M A
M J J
MONTHS
O
figure 13. Showing seasonal changes in the gonad index (GSI) of male P. koolooense.
(figure 4). The primary and secondary spermatocytes are produced in some of
the tubules. A few residual sperms are still retained in the lumen of the tubules
and almost disappear in March. During March the spermatogonial population
decreases and actively dividing primary and secondary spermatocytes become
dominating cells in the tubules (figure 6). In a few tubules spermatids are formed
but sperms are not yet developed.
3.3c. Stage III (April to May) : The testes have greatly increased in their size
and weight, being maximum during April (figure 13). They appear turgid and
opaque and their wall becomes so thin that the seminiferous tubules are visible.
The vas deferens also appears swollen, opaque and highly coiled and when rup-
tured the seminal fluid does not ooze out from it. This stage is characterized
by spermatogenetic and spermiogenetic activity. Spermatids and sperms are in
preponderance (figure 7). Sperms are developed for the first time in April and
tubules fully packed with sperms are seen in April and May (figure 12). Owing
to this the tubules are greatly enlarged and turgid and as a result their walls
become thin and intertubular spaces are decreased. However, the maturational
Testicular cycle of crab 447
changes do not occur simultaneously in all the tubules of testes, as most of the
tubules are filled with spermatids and sperms while some others are still at primary
or secondary spermatocyte level. This results in the production of sperms in
successive waves.
3.3d. Stage IV (May to June) : During this stage, the testes appear opaque and
vas deferens is packed with seminal fluid. Both maturing and mature tubules
are seen in the same section. Maturing tubules consist of dividing primary or
secondary spermatocytes or spermatids and produce sperms a little later. Mature
tubules undergo spermiation in May or June. In some of the specimens collected
during May, testes show decrease in size and weight (figure 13). Some of the
tubules contain primary spermatogonia and residual sperms (figure 1). This
indicates that spermiation has taken place. All the specimens collected during
June show spawned conditions in a number of tubules of their testes.
Gonad index is minimum in November-December and reaches a peak in April
and is in conformity with the histomorphological changes in the testes
(figure 13).
4. Discussion
The above study on the testes of P. koolooense reveals many interesting features.
In the testes of crustaceans studied so far, the spermatogenetic cells are confined
in discrete bodies which are variously described as cysts, clusters, seminiferous
tubules or lobules, each containing germ cells at different stages of spermato-
genesis (Binford 1913 ; Fasten 1926 ; Baker and Rosof 1927; Ryan 1967; Gupta
and Chatterji 1976) or at the same stage of maturation (Iyer 1933 ; Wolfe 1971;
Wielgus 1976). In Callinectes sapidus (Cronin 1947), Portunus sanguinolentus
(Ryan 1967), Paratelphusa masoniana (Vasisht and Relan 1971) and Scylla
serata (Gupta and Chatterji 1976) testes have several lobes and their semini-
ferous lobules or tubules open into a branched (Cronin 1947) or unbranched
seminiferous duct (Ryan 1967). This duct continues posteriorly as vas deferens.
In P. koolooennse the testes are not lobulated and contain numerous semini-
ferous tubules having germ cells at various stages of spermatogenesis. The tubules
become narrower towards posterior side of the testis, where their germ cell area
is reduced or absent. These tubules appear to open directly into the vas deferens.
Binford (1913) and Gupta and Chatterji (1976) observed the presence of both
spermatocytes and spermatids in the same tubule. Cronin (1947) and Gupta and
Chatterji (1976) found that all the spermatocytes of a tubule occur at the same
stage of differentiation. In P. koolooense the meiotic division occurs more or less
synchronously, as the individual maturing tubule contains either spermatids or
only one type of spermatocytes which occur at approximately the same stage of
differentiation.
The decapod sperm is bicentrosomal or tricentrosomal and aflagellated consist
ing of a head or nucleus and a vesicle variously described as primary vesicle
(Fasten 1926), mitochondrial vesicle (Nath 1932 ; Dhillon 1966) or acrosomal
vesicle (Brown 1966 ; Langreth 1969). In brachyura having bicentrosomal sperm,
the vesicle contains an axial filament (Nath 1932) or acrosomal tubule (Brown
448 PC Joshi and S S Khanna
1966 ; Langreth 1969) which extends up to the distal centrosome (Nath 1932 ;
Dhillon 1966). In P. koolooense also the sperm is aflagellate consisting of a
head and a mitochondrial vesicle which encloses an axial filament and distal
centrosome. When stained with PAS all the cells except spermatids and sperms
were found negative to this stain. In sperms, the head and mitochondrial vesicle
are PAS positive and the axial filament is PAS negative, as also observed by Brown
(1966); Dhillon (1966) and Langreth (1969) on other species of Decapoda.
There is paucity of information regarding the origin of new crop of germ
cells in crustacean testes. A few primary spermatogonial cells (Binford 1913)
or residual spermatogonia (Aoto 1952) which remained undifferentiated till the
spermatogenesis is over, undergo divisions shortly after spermiation so as to
produce a new batch of secondary spermatogonia. In P. koolooense also some
undifferentiated or resting spermatogonia are found throughout the year. It
appears that after spermiation the new crop of germ cells is supplied by the
division of such existing germ cells.
The testes of P. koolooense undergo seasonal histomorphological changes asso-
ciated with change in testicular weight. The measurement of male gonad index
revealed two types of spawning patterns among decapods. In continuous bree-
ders like Penaeus indicus (Subrahmanyam 1963) and Portunus pelagicus (Rahman
1967), the male gonad index was found constant throughout the year whereas in
Charybdis variegata (Chandran 1968) and Barytelphusa cunicularis (Diwan and
Nagabhushanam 1974) which breed discontinuously, definite peaks in gonad
index were observed. Histological studies revealed the presence of both conti-
nuous (Baker and Rosof 1927 ; Spalding 1942 ; Black 1966 ; Ryan 1967 ; Haley
1973) and discontinuous spermatogenetic cycle (Black 1966 ; Chiba and Honma
1972 ; Wielgus 1976) in crustaceans. Such variation in the testicular activity
may be due to the genetic differences and the local ecological conditions.
P. koolooense shows discontinuous spermatogenetic cycle. Spermatogenesis
begins during January-February, progresses slowly through March, reaching a
peak in April or May. However, all the tubules do not mature at the same time,
as both maturing and mature tubules were seen in the same section. The mature
tubules become filled with sperms whereas maturing tubules consist of dividing
spermatocytes or spermatids which produce sperms a little later. This results
in the production of sperms in successive waves and spermiation starts before all
the tubules are fully packed with sperms. The mature tubules undergo spermia-
tion in May or June. Tubules soon after evacuation of sperms undergo spermato-
gonial proliferation. In other tubules of the same section of testis, meiosis is
still continued. The production of sperms in successive waves during breeding
season indicates that one male crab attempts to copulate more than once in a
single breeding season. This seems to be advantageous since the fertilization is
internal and that the number of males is comparatively fewer than females in a
population of P. koolooense.
Diwan and Nagabhushanam (1974) reported long resting phase in the repro-
ductive cycle of Barytelphusa cunicularis. In P. koolooense the spermatogenesis
slows down gradually from July onwards and almost ceases by November. The
testes enter a brief period of rest during December. In Pachygrapsus crassipus
also spermatogenesis continues for a longer period during post-spawning period
$s a result the recovery phase tykes place gradually (Chiba and Hojima 1972),
Testicular cycle of crab 449
Acknowledgements
One of the authors (PCJ) is thankful to the UGC, New Delhi, for financial assis-
stance and to Dr T S Gill, Kumaun University, Nainital, for encouragements.
References
Aoto T 1952 Sexual phases in prawn, Pandulus kessleri (Zern), with special reference .to the
reversal of sex ; /. Fac. Set. Hokkaido Univ. 11 1-20
Baker R C and Ro-sof J A. 1927 Spermitogenesis in Branchipus vernalis. Part I. The testes and
spermatogonial division ; Ohio J. Sci. 27 175-186
Binford R 1913 The germ cells and process of fertilization in the crab, Menippe mercenaria '
/. Morphol. 24 147-202
Black J B 1966 Cyclic male reproductive activities in dwarf crayfishes, Cambarellus shufeldti
(Faxon) and Cambarellus puer (Hbbs.) ; Trans. Am. Micro. Sci. 85 214-232
Brown G G 1966 Ultrastructural studies of sperm morphology and sperm-egg interaction in
decapod, Callinectes sapidus ; /. Vltrastr. Res. 14 425-440
Chandcan M R 19 68 Studies on marine crab, Charybdis variegata. 1. Reproductive and nutri-
tional cycle in relation to breeding periodicities ; Proc. Indian Acad. Sci. B67 215-223
Chiba A and Honma Y 1971 Studies on go-nad maturity in some invertebrates. II. Structure
of reproductive organs of the lined shore crab ; Bull. Jpn. Soc. Sci. Fish. 37 699-706
Chiba A and Honma Y 1972 Studies on the gonad maturity in some marine invertebrates.
VI. Seasonal changes in testes of the lined shore crab ; Bull. Jpn. Soc. Sci. Fish. 38 317-322
Cronin L E 1947 Anatomy and histology of the male reproductive system of Callinectes sapidus
(Rathbun) ; /. Morphol. 81 209-233
Dhillon B 1966 Cytochemical changes in cell organelles in spermatogenesis of decapod
Crustacea ; Res. Butt. Punjab Univ. 17 55-56
Diwan A D and Nagabhushanam R 1974 Reproductive cycle and biochemical changes in the
gonad of freshwater crab, Barytelpkusa cunicuJaris (Westwood) ; Indian J. Fish. 21 164-176.
Fasten TSf 1926 Spermato genesis of the black clawed crab, Lophopanopeus bellus ; Blol. Bull.
(Woodshole) 1 277-293
Giese A C 1959 Annual reproductive cycle of marine invertebrates. Comparative physiology ;
Ann. Rev. Physiol. 21 547-576
S R and Chatterji N B 1976 Anatomical observations of the internal male reproductive
organs of Scylla serrata ; Indian J. Physiol. Allied Sci. 30 34-42
Haley S R 1973 On the use of morphometric data as a guide to reproductive maturity in the
ghost crab, Ocypode ceratophthalamus (Pallas) (Brachyura, Ocypodidae) ; Pac. Sci. 27
350-362
Iyer Muthuswamy M S 1933 Spermato genesis of Paratelphusa hydrodromous with a note on
oogenesis ; /. Mysore Univ. 7 206-232
King J E 1948 A study of the reproductive organs of common marine shrimp, Penaeus seti-
ferous (Linn.) ; Biol Bull 94 244-250
Langreth S G 1969 Spermiogenesis in cancer crabs ; /. Cell Biol. 43 575-603
Nath V 1932 The spermatid and sperm of the crab, Paratelphusa spinigera ; Quart. J. Micro-
Sci. 75 543-556
Nath V 1965 Animal gametes (Male) (Bombay : Asia Publishing House) pp. 78-93
Rahman A A 1967 Reproductive and nutritional cycles of the crab, Portunus pelagicus (Linn.)
of Madras coast ; Proc, Indian Acad. Sci. B65 76-8?
450 P C Joshi and S S Khanna
Ryan E P 1967 Structure and function of the reproductive system of the crab, Portunus sangumo-
lentus (Horb.)- I. The male system; Proc.Symp. Crustacea Ernakulum, Mar. Biol. Assoc.
India 2 506-521
Spalding J S 1942 The nature and formation of spermatophore and sperm plug in Carcinus
maenas ; Quart. J. Micro. Sci. 183 399-422
Subrahmanyam C B 1963 A note on the reproductive cycle of the prawn, Penaeus indicus
(N. Edw.) of Madras coast ; Curr. Sci. 4 165-166
Vasisht H S and Relan U 1971 Anatomy of Paratelphusa masoniana (Hend.). V. Reproductive
system; Res. Bull Punjab Univ. 22 13-18
*Wielgus S E 1976 Morphological and histological changes in the male gonad of the American
crayfish, Orconectes limosus (Refinesque) in annual cycle ; Acta Biol. 19 87-105
Wolfe A F 1971 A histological and histochemical study of the male reproductive system of
Anemia (Crustacea, Branchiopoda) ; /. Morphol 135 51-70
* Not consulted in original.
Proc. Indian Acad. Sci. (Anira. Sci.), Vol. 91, Number 5, September 1982, pp. 451-462.
© Printed in India.
Seasonal changes in the ovary of a freshwater crab, Potamon
koolooense (Rathbun)
P C JOSHI and S S KHANNA*
Department of Zoology, Government PG College, Pithoragarh 262 501, India
* Joint Secretary, Ministry of Education, Lucknow, India
MS received 25 November 1981 ; revised 22 July 1982
Abstract. The ovaries of P. koolooense which are paired H-shaped structures
undergo seasonal morphometric and histological changes. A minute oviduct leads
into the seminal receptacle which receives sperms during breeding season. Oogonia
and young oocytes develop in the germinal zone, present in the centre of the
ovary. The resting or residual oogonia which occur throughout the year divide
shortly after ovulation and supply new crop of germ cells for the next breeding
season. Five maturational stages of ova have been described on the basis of changes
that occur in their nuclei and cytoplasm. They are oogonium, premeiotic oocyte,
previtellogenic oocyte, vitellogeriic oocyte and the lipe ovum. Spawning occurs
during May or June. The weight of the ovaries, gonad index and ova diameter
were minimum in June and reached a maximum value in April.
Keywords. Potamon ', ovarian histology ; ovarian cycle ; vitellogenesis.
1. Introduction
The structure of female reproductive organs has been described in some species
of crabs (Weitzman 1966 ; Rouquette 1970 ; Chiba and Honma 1971 ; Laulier
and Demeusy 1974). The process of yolk formation differs in various species of
crustaceans (Harvey 1929 ; Bhatia and Nath 1931 ; Hinsch and Cone 1969 ;
Hinch 1970). Most of the studies on the female reproductive cycle are based
on the morphometric characters of the ovaries only. Nevertheless, relatively
little emphasis has been laid on the seasonal histological changes in the ovaries
of the crabs and other decapods (Weitzman 1966 ; Chiba and Honma 1972 ;
Laulier and Demeusy 1974 ; Badawi 1975 ; Goldstein and Lauria 1975 ; Rao
et al 1981). In the present investigation the structure and seasonal histomorpho-
logical changes in the ovaries of a hill stream crab, Potamon koolooense were
studied.
2. Material and methods
15 to 20 live specimens of adult female P. koolooense (carapace width 3 '8 to
4-5 cm) were collected locally from a stream each month during 1976-78. The
451
452 P C Joshi and S S Khanna
weight of each specimen was recorded immediately before dissection. Ovaries
along with oviducts and seminal receptacles were removed and placed in fixative.
Ovarian weight was recorded after fixation. The gonosoniatic index (GSI) was
calculated using the formula (Giese 1959) :
_ weight of the gonad Q
GSI ~ weight of the animal X 1UU>
For histology, different regions of ovaries were fixed in Bouin's or Helly's fluid.
Paraffin sections of 5-6 micra thickness were cut and stained with Delafield's
haematoxylin or Mayer's haeinalum, using eosin as counterstain. Heidenhain's
Azan or Mallory's triple stain was also used. The average oocyte diameter was
calculated by measuring rounded oocytes having complete nuclei. The moulting
stages of the crabs are not determined in this paper.
3. Observations
3.1. Morphology of reproductive organs
The female reproductive organs include paired ovaries, oviducts and seminal
receptacles. Ovaries are elongated ' H '-shaped structures, situated between
hypodermis of carapace and the hepatopancreas. Just behind the pyloric stomach,
the two ovaries are connected together by a cross connection. Ovary leads into
a very minute oviduct. In sections the ovarian wall appears to continue as the
oviduct (figure 2), which opens into a large thick walled pouch, the seminal
receptacle. Each receptacle leads into a narrow vaginal tube which further opens
outside through a small circular gonopore situated on the 6th sternal segment of
the cephalothorax. Ovaries show morphological changes associated with their
degree of maturity, as reflected in their size, shape, colour and weight.
3.2. Histology of ovary
The ovarian wall is continuous with ovarian stroma (figure 1) and is thicker
during post-spawning period but becomes thin at maturing and mature stages
of the ovary. Ovarian stroma consists of connective tissue, muscle fibres and blood
vessels, and is abundant during post-spawning period but greatly reduced in
mature ovaries (figure 1). A germinal zone is present all along the centre of the
ovary (figures 1, 3). During early phases of maturation, the germinal zone
consists of oogonia and young oocytes whereas the developing oocytes are dis-
placed towards outer region of the ovary (figure 1). As maturity advances the
germinal zones become greatly reduced consisting of a few residual oogonia, the
rest of the ovary is filled with maturing oocytes.
3.3. Stages of developing ova
The oogonium passes through different maturational stages before it becomes
the ripe ovum. This process involves changes in the nucleus and cytoplasm.
According to the classification of Raven (1961) and Laulier (1974)? the following
developmental stages have been observed :
Seasonal changes in the ovary of crab
453
ovw
Figures 1-5. 1. Structure of ovary in transverse section, June specimen x 40.
2. Section passing through oviduct (OVD) x 40. 3. L.S. of ovary showing
germinal zone (GZ) x 140. 4. Oogonium (OG) showing mitotic figure (MF), yolk
droplets (YD) appear in peripheral ooplasm of primary vitellogenic oocyte (PVO)
x 380. 5. Showing premeiotic oocyte in germinal zone x 380. (DF, discharge
follicle ; GZ, germinal zone ; OG, oogonia ; os, ovaiian stroma ; ov, ovary ;
ovw, ovarian wall ; PMO, premeiotic oocytes ; PO, previtellogenic oocytes;
PVO, prim?ry vitellogenic oocytes; SK, synezesis knot; SR, seminal receptacle).
454
P C Joshi and S S Khanna
Figures 6-10. 6. Primaiy vitellogenic oocyte showing yolk droplets^ (YD) extended
towards perimiclear region (PR). Displacement of nucleolus is due to mechanical
disturbance during section cutting X 140. 7. An enlarged portion of above showing
vacuoles (v) inside yolk droplets (YD) x 380. 8. Yolk granules (YG) and yolk
vesicles (YV) in periphery of secondary vitellogenic oocyte x 70. 9. Vacuolation
of yolk dropkts (YD) to form yolk vesicles (YV) x 380. 10. Portion of secondary
vitellogenic oocyte showing light yolk granules (YG), dark yolk droplets (YD) and
yolk vesicles (YV) x 380, (DF, discharged follicle ; FL, follicular layer ; N, nucleus,
VM, vitelline membrane.)
Seasonal changes in the ovary of crab
455
Figures 11-15. 11. Secondary vitellogenic oocyte showing vacuolation of yolk
droplets (arrow). Perinuclear region is yolkless x 40. 12. Showing vacuolation
(v) and eccentiic position of nucleolus (NL) x 380. 13. Secondary vitello.genic
oocyte showing yolk globules (YGL) and yolk vesicles (YV). Yolk droplets (YD) are
in the background x 60. 14. Tertiary vitellogenic oocyte filled with yolk globules.
(YGL) and yolk vesicles (YV). Perinuclear region (PR) is occupied by yolk x 60.
15. Ripe ovum filled with yolk globules (YGL) and yolk vesicles (YV) x 40.
(FL, follicular layer ; NM, nuclear membrane ; PR, perinuclear region ; YG, yolk
granules ; YV, yolk vesicles).
Seasonal changes in the ovary of crab 457
3.3a. Oogoniwn (figures 3, 4, 5) : A few primary or residual oogonia which
occur throughout the year in the germinal zone, divide mitotically (figure 4)
shortly after ovulation and give rise to additional oogonia to provide for
further growth of ovary. Oogonium is a small spherical cell with a pale
nucleus and thin rim of poorly basophilic cytoplasm. Oogonia develop into
premeiotic oocytes but a few remain undifferentiated (residual oogonia) till the
ovulation.
3.3b. Premeiotic oocyte (figures 3, 5) : The oogonia which enter into prophasic
activities are termed as premeiotic oocytes or primary oocytes. The chromosomes
condense at one side of the nucleoplasm and form synezesis knot (figure 5)
which corresponds with zygotene or synapsis stage of meiotic prophase. At the
final developmental stage (diakinesis) the chromatin appears in the form of discrete
clumps lying close to nuclear wall (figure 5) and the nucleolus is not visible . The
oocyte measures 20 micra in diameter.
3.3c. Previtellogenic oocyte (figure 3): As the premeiotic activities come to an
end, the nucleus increases in volume and oocyte acquires a large amount of
basophilic cytoplasm. The nucleus appears vesicular containing peripherally
arranged chromatin clumps and a centrally placed nucleolus which appear solids
and stain blue-black with haematoxylin or red with Mallory's triple or Azan
stains. The yolk formation has not yet begun and oocyte attains a diameter of
95 micra.
3 . 3d. Vitellogenic oocyte : The oocyte now enters a synthetic or vegetative
phase resulting in the formation of yolk. The nucleus, nucleolus and ooplasm
undergo marked changes in their cytology as described below :
(a) Primary vitellogenic oocyte (figures 4, 6, 7) : Further increase in amount of
basophilic ooplasm and the volume of nucleus and nucleolus accompanies the
appearance of few small yolk droplets in peripheral ooplasm (figure 4). The
yolk droplets stain purple to black with haematoxylin or blue with Mallory's
triple or Azan stains. Chromatin clumps become finely granular arranged in
a network in nucleoplasm. Nucleus is solid and central in position. A thin
layer of follicular cells forms around the oocyte (figures 6, 7). Further increase
in oocyte diameter is accompanied with increase in amount of yolk droplets which
progressively extends towards the yolkless and homogeneous perinuclear region
(figures 6,7). The yolk droplets swell and minute unstainable vacuoles begin
to appear in them (figure 7). Average diameter of oocyte is 250 micra.
(b) Secondary vitellogenic oocyte (figures 8, 9, 11, 13) : The oocyte shows a rapid
growth and the unstainable vacuoles in yolk droplets increase in number and size
(figures 8, 11). This results in marked decrease in stainable contents of yolk
droplets which thus appear reticulated or spongy. Vacuoles fuse with each other
and ultimately form large unstainable yolk vesicles, initially in peripheral ooplasm
but later on in other regions (figures 9, 11, 13). This is followed by the appearance
of small eosinophilic yolk granules in the extravesicular ooplasm (figures 9, 11).
In Mallory's triple or Azan stains the yolk granules stain red. They increase in
size probably by their fusion and thus gives rise to large oval yolk globules
458 P C Joshi and S S Khanna
(figure 13). Oolemma and follicular layers are well differentiated (figure 10).
While the yolk granules are appearing in the ooplasm, the nucleolus enlarges and
becomes eccentric and vacuolated without any change in its tinctorial properties
(figures 12, 13). Yolk droplets gradually disappear. Oocytes measure 600 micra
in diameter.
'(c) Tertiary vitellogenic oocyte (figure 14) : With further growth of oocyte, the
amount of yolk globules increases. Yolk granules still continue to appear in
peripheral ooplasm. Yolk droplets are absent because of their conversion into
yolk vesicles. Ooplasm thus becomes entirely acidophilic. Yolk globules become
polygonal and occupy most of the ooplasm including the perinuclear region.
Nucleolus is eccentric in position and poorly stainable due to its extensive vacuali-
zation. Follicular layer is stretched to a thin membrane and its nuclei become
spindle shaped, probably due to the turgidity of oocyte.. Vitelline membrane
is distinct. Oocyte is 950 micra in diameter.
3.3e. Ripe ovum (figure 15): It is largest in size measuring upto 1400 micra
in diameter. Ooplasm is heavily impregnated with large yolk globules and
yolk vesicles.
3.4. Seasonal changes in ovaries
The annual ovarian cycle can be conveniently divided into following four stages
on the basis of histomorphological features of ovaries :
3*4a. Spawning and spent stage (May- June)- During this period females
carry their spawn attached onto their pleopods. Ovaries are small, smooth
and cream coloured. Seminal receptacles contain sperms. Discharged follicles
are found in stroma, indicating that the spawning has taken place (figure 1).
Ovarian wall becomes thick and the germinal zone consists of numerous oogonia,
premeiotic and previtellogenic oocytes (figures 1, 3). A few primary vitellogenic
oocytes also develop at peripheral region of ovary.
3.4b. Early maturing stage (July-October) : Ovaries increase in size and are
coloured deep yellow. Externally they appear granular owing to the preponde-
rance of primary and secondary vitellogenic oocytes in them. Germinal zone
contains a few residual oogonia and previtellogenic oocytes.
3.4c. Advanced maturing stage (November-February)'- Ovaries become enlarged,
convoluted and orange in colour. Large orange ova bulge out on the surface
of ovaries. Tertiary vitellogenic oocytes are commonly present but a few
secondary ones also occur. Premeiotic and primary vitellogenic oocytes are rare
in the ovaries.
3.4d. Mature stage (March-April): Ovaries attain a maximum size and deep
orange mature ova are visible through the thin ovarian wall. The gonosomatic
index is minimum in June and reaches a maximum value in April and is in
conformity with the average ova diameter (figure 16).
Seasonal changes in the ovary of crab
459
7
6
5
o
t/>4
3
2
I
©-© GSI
©•••© OVA DIAMETER
0'
'4
•2.o
'°2
z
0'6
04
0'2
0
m
79
J F M A M J JASOND
MONTHS
Figure 16. Seasonal changes in the ganosomatic index (GSI) and ova diameter.
4. Discussion
The female reproductive organs of P. koolooense are built on the same general
plan as observed in other crabs by Hartnoll (1968), Vasisht and Relan (1971)
and Chiba and Honma (1971). Several decapods possess a germinal zone in the
centre of ovary (Kessel 1968 ; Hinsch and Cone 1969 ; Rouquette 1970 ; Laulier
and Demeusy 1974 ; Rao et al 1981) whereas in others the germinal zone is
peripheral (Cronin 1942 ; King 1948) or is in the form of nests of germ cells
distributed throughout the ovary (Weitzman 1966). In P. koolooense the
germinal zone occurs at the centre of ovary.
Little is known about the origin of new crop of germ cells in adult decapods-
It is generally agreed that the new crop of germ cells arise by division of existing
oogonia. In Gecardnus later alls (Weitzman 1966) and Pachygrapsus mormoratus
(Rouquette 1970) oogonial mitosis occurs throughout the year. In P. koolooense
the resting oogonia or residual oogonia occur throughout the year but they divide
shortly after ovulation and produce a new drop of oogonial cells for further growth
of ovaries, as observed by Aoto (1952) and Laulier and Demeusy (1974) in other
decapods.
The process of yolk formation varies considerably in different decapods. The
yolk vesicles and yolk globules in the oocyte of P. koolooense correspond with
the fatty yolk vacuoles and proteinous yolk bodies of Carcinus maenas (Harvey
1929), Palaemon lamarrei and Paratelphusa spinigera (Bhatia and Nath 1931) and
Paratelphusa hydrodromous (Vasisht and Relan 1971). Bhatia and Nath (1931)
observed that the fatty yolk vacuoles appear initially at the peripheral ooplasm
while the protein yolk bodies near perinuclear region. In shrimp, Chirocephalus
bundyi (Linder 1959) the fatty yolk droplets and proteinous yolk granules first
appear in the central region of ooplasm. In P. koolooense both yolk vesicles
460 P C Joshi and S S Khanna
and yolk granules first appear at peripheral ooplasm and then extend progressively
towards perinuclear region as has also been observed by Harvey (1929) in
Cardnus maenas.
Besides the structural changes, the yolk bodies and nucleolus in some crabs
undergo parallel changes in their cytochemical nature and staining affinity
(Otsu 1963 ; Carmignani et al 1973). In P. koolooense, neither the yolk globules
nor the nucleolus undergo changes in their staining reactions. The source of
yolk material differs in various species of crustaceans according to several
authors. Generally the yolk is formed from both extra oocyte sources (perinuclear
endoplasmic reticulum and' golgi bodies in collaboration with nucleolar extrusions
(Kessel 1968 ; Hinsch and Cone 1969 ; Hinsch 1970 ; Dhainaut and Leersynder
1976) and extra oocyte sources (yolk precursors are incorporated from haemolymph
into ooplasm) by diffusion through follicular cell layer (Linder 1959 ; Beams and
Kessel 1963) or by micropinocytosis at oocyte surface (Hinsch and Cone 1969;
Dhainaut and Leersynder 1976).
The nucleolus during vitellogenesis undergo progressive increase in size and
shows vacuolation (Harvey 1929 ; Hinsch 1970 ; Dhainaut and Leersynder 1976).
The nucleolar extrusions or granules which pass into ooplasm are believed to
take part in yolk formation (endogenous yolk) in crabs (Harvey 1929 ; Bhatia
and Nath 1931 ; Hinsch 1970). In P. koolooense, neither the nucleolar granules
were seen nor the nucleolus was found moved into the ooplasm. Although the
increase in the size of nucleolus, its eccentric position and progressive vacuoli-
zation during vitellogenesis in this species indicates that it has some role in yolk
ormation, nothing can be said about its functions in the absence of direct
evidence.
Seasonal morphometric studies revealed that the breeding cycle in crabs and
other decapods varies widely, even in species having close taxonomic relation-
ships or similar ecological niches, i.e., (i) continuous breeder around the year
(Boolootian et al 1959 ; Knudsen 1964 ; Rahman 1967 ; Badawi 1975),
(ii) Seasonal breeders having one spawning season (Boolootian et al 1959 ;
Hartnoll 1963 ; Otsu 1963 ; Knudsen 1964 ; Diwan and Nagabhushanam 1974 ;
Bomirski and Klek 1974 ; Badawi 1975) or two distinct spawning seasons
(Knudsen 1964 ; Chandran 1968 ; Adiyodi 1968, Goldstein and Lauria 1975).
P. koolooense is a seasonal breeder, as all the oocytes in the ovaries become fully
mature at the onset of breeding season and are laid simultaneously during May
or June, followed by the next ovarian cycle. The gonad index begins to decline
in May, reaching a minimum level in June and increases slowly during the
following months with a peak in April (figure 16). Such variations in the breeding
season among decapods may be due to the genetic differences and the local
ecological conditions.
Studies on the seasonal histological changes in the ovaries revealed that in post
spawning period the vitellogenesis takes place late and is preceded either by a
long resting stage (Weitzman 1966 ; Diwan and Nagabhushanam 1974) or by
recovery or previtellogenic stages (Chiba and Honma 1972 ; Badawi 1975). In
biannual breeders, the vitellogenesis of second ovarian cycle begins soon after
the completion of the preceding cycle while in first ovarian cycle the vitellogenesis
is preceded either by long resting phase (Adiyodi 1968) or a brief resting stage
Seasonal changes in the ovary of crab 461
(Bomirski and Klek 1974). In P. koolooense vitellogenesis occurs shortly after
spawning and it appears that the resting stage in this species is of very short
duration probably of a few days only.
Acknowledgements
One of the authors (PCJ) is thankful to the UGC, New Delhi, for the financial assis-
tance. Thanks are due to Miss Maya Deb and Dr M Koshy, zsi, Calcutta,
for the identification of the species.
References
Adiyodi R G 1968 On the reproduction and molting in the crab, Pamtelphusa hydrodromous',
Physiol. ZooL 41 204-209
Aoto T 1952 Sexual phases in the prawn, Pafldulus kessleri (Z«rn.) with special rtference to
the reversal of sex ; /. Fac. Sci. Hokkaido Univ. 11 1-20
Badawi H K 19 75 On maturation and spawning in some penaeid prawns of Arabian Gulf ;
Mar. Biol (Berlin) 32 1-6
Beams H W and Kessel R G 1963 Electron microscopic studies on developing crayfish oocytes
with special reference to the origin of yolk ; /. Cell Biol. 18 621-649
Bhatia D R and Nath V 1931 Studies on the origin of yolk. VI. The crustacean oogenesis ;
Quart. J. Micro. Sci. 74 669-701
Bomirski A and Klek E 1974 Actio-n of eyestalks on the ovary in Rhiihropanopeus harrisii and
Cragon cragon (Crustacea, Decapoda) ; Mar. Biol. 24 329-337
Boolootian R A, Giese A C, Farmanfarmaian A and Tucker J 1959 Reproductive cycle of
five west coast crabs ; Physiol. ZooL 32 213-220
Carmignani M, Albanese P, Batognari A and Zaccove G 1973 Morphological stud} on the
yolk globules in the growing oocyte of Maja verrucosa (Edw.) (Crustacea, BrachYura);
Acta Histochem. 46 195-201
Chandian M R 1968 Studies on marine crab, Charybdis variegata. I. Reproductive and nutri-
tional cycle in relation to breeding periodicities ; Proc. Indian Acad. Sci. B67 215-223
Chiba A and Ho-nrna Y 1971 Studies on gonad maturity in some marine invertebrates.
II. Structure of reproductive organs of the lined shore crab ; Bull. Jpn. Soc. Sci. Fish. 37
699-706
Chiba A and Honma Y 1972 Studies on gonad maturity in some marine invertebrates.
VII. Seasonal changes in the ovary of the lined shore crab ; Bull. Jpn. Soc. Sci. Fish. 38
323-329
*Cronin L E 1942 A histological study of the development of ovary and accessory reproductive
organs in the blue crab, Callinectes sapidus (Rathbun) ; M.S- Thesis Univ. Maryland
Dhainaut A and Leersynder De M 1976 Cytochemical study of the oocyte development in
crab, Eriocheir sinensis. I. Natural oogenesis ; Arch. Biol. 87 261-282
Diwan A D and Nagabhushanam R 1974 Reproductive cycle and biochemical changes in
gonads of freshwater crab, Barytelphusa cunicularis (West wood) ; Indian J. Fish. 21
164-176
Giese A C 1959 Annual reproductive cycle of marine invertebrates ; Ann. Rev. Cytol. 21
547-576
Goldstein B and Lauria L 1975 Cycle of sexual maturation and preliminary studies on spawning
of the freshwater shrimp, Palaemonetes argentinus. I. Female ; Physis. Agnas Cont. Org.
Sect. B33 165-176
HartnoU R G 1963 The biology of manx spider crabs ; Proc. ZooL Soc. London 141 423-496
Hartnoll R G 1968 Morphology of the genital ducts in female crabs ; /. Linn. Soc. (ZooL) 47
279-300
Harvey L A 1929 The oogenesis of Carcinus maenas (Penn.) with special reference to volk
formation ; Trans. Roy. Soc. Edinburgh 56 157-176
46.2 f C Joshi and S S Khanna
Hinsch G W 1970 Possible role of intranuclear membranes in nuclear cytoplasmic exchange
in spider Crab oocytes ; /. Cell Biol. 47 531-535
Hinsch G W and Cone M V 1969 Ultrastructural observations of the vitellogenesis in spider
crab, Libinia emarginata ; J. Cell Biol. 40 336-342
Kessel R G 1968 Mechanism of protein yolk synthesis and deposition in crustacean oocyte ;
Z. Zellforsch. 89 17-38
King J E 1948 A study of the reproductive organs of the common marine shrimp, Penaeus
setiferous (Linn.) ; Biol. Bull. 94 244-250
Knudsen J W 1964 Observations of the reproductive cycle and ecology of the common
brachyura and crab like anomura of Puget Sound coast, Washington ; Pac. Sci. 18 3-33
Laulier M 1974 Caracteres cytologique de la cellule sexuele female du crabe, Carcinus maenas
L. au cours de la gametogenese ; Cah. Biol. Mar. 15 159-167
Laulier M and Demeusy N 1974 Etude histologique du fonctionment ova; i an an cours d'une
maturation de ponte chez le crabe, Carcinus maenas L. (Crustacea, Decapode) ; Cah. Biol.
Mar. 15 343-350
Linder H J 1959 Studies on freshwater fairy shrimp, Chirccephalus bundyi (Forbes). I. Structure
and histochemistry of ovary and accessory reproductive tissue ; /. Morphrl. 104 1-60
Otsu T 1963 Bihormonal control of sexual <ycle in the freshwatei ciab, Potamon dehaani ;
Embryologica 8 1-20
Rahman, A A 1967 Reproductive and nutritional cycle of the crab, Portunus pelagicus (Linnaeus)
(Decapods , Brachyura) of Madras coast ; Proc. Indian Acad. Sci. B65 76-82
Rao Ch Narasimha, Shakuntala K and Reddy S R 1981 Moult-reproduction relationship in
the freshwater prawn, Machrobrachium lanchesteri (de Man) ; Proc. Indian Acad. Sci.
90 39-52
Raven C P 1961 Oogenesis : The storage of developmental information (London and New
York : Pergamon Press)
Rouquette M 1970 Etude du tissu cvarien chez le crabe Pachygrapsus marmoratus (Fabricius)
Premiers resultats cor.oernant les role de la temprature et des pedoncules oculaires ;
Bull Soc. ZooL 'France 95 233-240
Vasisht H S and Relan U 1971 Anatomy of Paratelphusa masoniana (Henderson). V. Reproductive
system ; Res. Bull. Punjab Univ. 22 13-18
Weitzman M C 1966 Oogenesis in tropical land crab, Gecarcinus lateralis (Freminville) ; Z.
Zdlforsch. 75 109-119
* Not consulted in original.
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 5, September 1982, pp. 463-46$.
© Printed in India.
Evaluation of warfarin against Tatera indica and Meriones hurrianae
R P MATHUR and ISHWAR PRAKASH
Coordinating and Monitoring Centre for Rodent Research and Training,
Central Arid Zone Research Institute, Jodhpur 342 003, India
MS received 15 September 1981
Abstract. Warfarin was evaluated in laboratory against Indian gerbil, Tatera
indica and desert gerbil, Meriones hurrianae. Chronic LDSO for the two species
was found to. be 4 x 19 '1 and 4 x l5'9mg/kg respectively. Feeding for 14 days
on 0-025% warfarin treated bait provided complete kill in the gerbils but the
poisoned bait was less palatable than the plain bait. A period of 18 and 19
days feeding on 0*025% waifarin bait was found suitable to detect resistance to
warfarin among T. indica and M. hurrianae respectively.
Keywords. T. indica ; M. hurrianae ; oral toxicity ; no-choice tests; base-line
susceptibility ; palatability ; warfarin.
1. Introduction
The Indian gerbil, Tatera indica Hardwicke and the desert gerbil, Meriones
hurrianae (Jerdon) are dominant rodent species in the Indian desert and inflict
severe damage to crops and grasslands (Barnett^ and Prakash 1975). Since
they induce bait shyness after a single exposure of zinc phosphide (Prakash and
Jain 1971) the need to evaluate other rodenticides as alternative poisons for their
control has arisen. The present study was, therefore, undertaken to evaluate
warfarin [3-(l-phenylethyl-2 acetyl) 4-hydroxy-coumarin] against T. indica and
M. hurrianae.
2. Material and methods
The gerbils were captured from fields around Jodhpur (Lat. 26° 18'N ; Long.
73° 1'E). They were sexed, weighed and caged individually for 3 weeks for
acclimatization and were fed on bajra (Pennisetum typhoides) and jowar (Sorghum
valgare). Average body weights of T. indica and M. hurrianae (g ; mean ± SE)
were 124:16 ± 5*73 and 62-44 ± 3 -62 respectively. Each of the four doses (5-0,
15-0, 25-0 and 50-0 rug/kg) of technical warfarin of 98% purity was administered
by oral tube for four consecutive days to calculate the chronic LDSO. No-choice
and choice feeding trials were conducted using 0*0125% and 0*025% warfarin-
463
P. (B)-6
464 R P Mathur and Iskwar frakash
treated bajra grains. The former trials were conducted for different lengths of
feeding periods. In choice tests an alternative unpoisoned bait was also provided
to the gerbils. The trials were conducted as recommended by WHO (1976) and
the LD50/8> lethal feeding periods (LFPSO and LFP9S) and their 95% confidence
limits were calculated by probit analysis (Finney 1971).
3. Results
Sex difference in the mortality was not observed in any of the trials and hence
combined sex mortality data were analysed.
3.1. Oral toxicity
Chronic LD50 and 95% confidence limits for T. indica and M. kurrianae are
4 x 19 -1 (13-8-27-61) and 4 x 15*9 (11'0-24-Q) -nag/kg respectively. Slopes
of the probit regression line with respect to two species are 1 *48 ± S.E. 0 • 12 and
1-61 ±0-12 respectively.
3.2. No-choice tests
In no-choice feeding tests complete kill was observed with 14 days, feeding on
0-0125 and 0*025% warfarin treated bait in both the species (table 1) except that
with the former concentration one T. indica survived.
In both the gerbils, T. indica and M. hurrianae, mortality started from day 4
and 5 and lasted upto days 18 and 16 respectively and maximum kill occurred
between 5 to 10 days (table 1). Bait intake in no-choice test was fairly high
upto 6-7 days after which it declined possibly due to the development of ffae
symptoms of anticoagulant poisoning.
3.3. Base-line susceptibility
Table 2 gives the lethal feeding periods (LFPSO and LFP98), their 95% confidence
limits and slopes of the probit regression lines. The slope of the probit regression
line and LFPSO does not differ significantly between the sexes and concentration
but significant difference was found between species (P < 0'02) with respiect to
0-025% concentration (table 2) which indicates that M. hurrianae is more suscep-
tible to warfarin than T. indica.
3.4. Acceptability of poisoned bait
Poisoned bait was less palatable than the plain bait (table 3). The difference
was not significant between the two concentrations in both the species. How-
ever, with both the concentrations the intake of poisoned bait by M. hurrianae
was significantly more (P < 0-01) than T. indica (table 3) and hence the morta-
lity was higher in the former species.
Evaluation of warfarin
465
Table 1. Mortality in T. indica and M. hurrianae feeding on warfarin-treated
pearl millet in no-choice tests.
Feeding
period
(days)
Cone, of Anticoagulant consumed (mg/kg),
poison Mean±S.E.
(percent)
- Mortality
Days to
death
Died
Survived
Mean
Range
Tatera indica
2
0-0125
14-3C±0 -84
0/10
4
24-48± 3-78
26-06± 2-56
2/10
4-5
4-5
7
42-38± 5-43
46-98± 5-20
6/10
10-0
7-13
10
41-70± *57
65-38
9/10
9-1
5-12
14
39-16± 4-07
81-25
11/12
8-2
5-11
2
0-025 38-65
21-30± 2-17
1/10
18-0
4
41 -61 ±11 -23
56-80± 3-17
4/10
5-2
5l*6
7
104- 16± 5-51
108- 83± 6-67
6/12
6-6
4-8
10
72'20± 3-99
60-75
9/10
8-5
7-13
14
86'27± 9-23
12/12
86
5-14
Meriones hurrianae
2
0-0125 22-32
!9-98± 2-24
1/10
11-0
4
28-17± 2-36
22-47± 5-90
4/10
6-7
5-10
7
79-40± 9-43
80'67± 7-19
6/10
7-3
4-11
10
85-45±14'60
99-95± 2-65
7/10
9'7
6-15
14
96*46± 9'96
...
10/10
10-5
7-15
2
0-025 21-51
30-58± 4-06
1/10
6-0
4
47-22± 8-40
57-56± 5-37
5/10
56
4-7
7
114-10±l4-95
103'57±23«45
S/12
8-4
5-12
10
114-41 ±21-59
148-83±16'18
S/10
8-1
5-14
14
173-16±16'98
...
10/10
11-4
5-16
Table 2. Lethal feeding periods (LFP) for T. indica and M. hur.ianae and their
95% fiducial limits using warfarin.
Species
Cone, of
poison
(percent)
Slope of the
probit regression
line (b) ± S.E.
LFP60
(days)
L*P98
(days)
T. indica
0-0125
0-025
1-92±0-10
M3±0*08
6-0(4-4-^-1)
5-7(4-2-7-9)
16-6 (8-9-30-9)
.13-2(10-0-17-4)
M. hwrianaz
0-0125
0-025
1-81 ±0-07
1-89±0-08
4-6(3-8-5-7)
3-7(2-8-4-7)
13 -5 (10- 0-18 -2)
12-9 (9-1-18-2)
466
R P Mathur and Ishwar Prakash
Table 3. Bait acceptability and mortality in T. indica and M. hurrianae given
'choice' between plain and warfarin-treated bait.
Concen- Duration Mean daily bait intake
Significance
Days to
tration of test (g/100 g body wt)
of
Morta- death
of poison (days) Mean ± S.E.
student's
lity Mean
/ ^ A 4-\
't*
(range)
vpcr ccnij --- """"• •"
Poison (1) Plain (2)
between
1 and 2
Tatera indica
0-025 14(2) l-95±0-35 4- 21 ±0-41 O'OOl 6/12
0*0125 14(2) 2-48±0-45 4'20±0-59 0'05-0'02 5/12
Meriones hurrianae
0-025 14(2) 5-46±0-98 7'16±1*07 030-0 '20 11/12
0-0125 14(2) 4-07±0-76 6'94±0'81 0- 02-0' 01 8/12
11-3
(5-19)
9*4
(4-14)
9'7 (
(5-15)
6-1
(4-16)
(Figures in parenthesis indicate the number of days for which bait consumption data were
analysed.)
4. Discussion ;
Our data on toxicity of warfarin Against T. indica are fairly comparable with that
of Greaves and Rehman (1977) in as much as that complete kill was achieved
in 14 days feeding on 0*025% warfarin. Comparing the suceptibility of warfarin
to gerbils with that of other species it is revealed that they are less susceptible
than R. norvegicus (Bentley and Larthe 1959 ; Brooks and Bowerman 1974) and
Bandicota bengalensis (Deoras 1967 ; Greaves and Rehman 1977 ; Sridhara
1979 ; Brooks et al 1980).
The two gerbils under study are also less susceptible than Arvicanthis niloticus
where 6 days feeding on 0-025% warfarin produced 100% kill (Gill and Redfern
1977). Mukthabai and Krishnakumari (1976) reported 100% kill in R. rattus
in 7 days, in the same period Mathur and Prakash (1981 a) achieved 92% kill,
whereas Krishnamurthy et al (1968) and Chaturvedi et al (1975) observed °1 00%
kill in 13 days. Similar results are also obtained with Rattus argentiventer where
10-12 days feeding is required to kill all experimental animals (Buckle et al 1980)
and Mastomys natalensis giving complete 'kill in 13 days (Gill and Redfern 1979).
However, the northern palm squirrel, Furiambulus pennanti, tois found fairly less
susceptible to warfarin as compared to the two gerbils where, even 14 days feeding
could not kill more than 58% squirrels (Mathur and Prakash 1980). Mus
musculus also required 28 days of feeding on 0-025% warfarin-treated bait for
Evaluation of warfarin 467
complete kill (Rowe and Redfern 1964). Significant difference was not observed
in the mortality among the two gerbils when the two concentrations, 0*0125%
and 0 • 025% of warfarin were used and hence the former is recommended for the
control of T. indica and M. hurrianae.
Taking the upper 95% confidence limits of LFP98 (precluding 0'0125% concen-
tration) the data suggest that feeding on 0-025% warfarin for 18 and 19 days
would be suitable to test resistance to warfarin among T. indica and M. hurrianae
respectively. This period is quite comparable with that for T. indica (21 days)
reported by Greaves and Rehman (1977), R argentiventer (18 days, Buckle et al
1980) and cotton rat, Sigmodon hispidus (20 days, Gill and Redfern 1980). How-
ever, B. bengalensis (8 days ; Brooks et al 1980) and R. norvegicus (7 days ; Brooks
and Bowerman 1974) are more prone to develop resistance to this poison than
the gerbils. Greaves and Rehman (1977) also reported that Tatera has the
potential to develop a significant degree of resistance to anticoagulants than
R. rattus which requires 28 days feeding on 0'025% warfarin as a suitable test
for resistance. It is evident that warfarin provides good results against a number
of species but it requires longer period of feeding than brodifacoum and chloro-
phacinone (Mathur and Prakash 1981b, 1982).
The widespread use of such poison as the sole mean to control gerbils and
other rodent pests for a considerable period can eventually lead to pockets of
warfarin-resistant animals which is a serious problem in most of Europe and
United States of America. It is, therefore, recommended that intermittently
other poisons which kill rodents in shorter time should also be used.
Acknowledgements
Authors are thankful to Dr H S Mann, Director, Central Arid Zone Research
Institute, Jodhpur, for providing necessary facilities and encouragement.
References
Barnett S A and Prakash 1975 Rodents of economic importance in India (New Delhi and
London : Arnold-Heinemann) Pp. 1-175
Bentley E W and Larthe Y 1959 The comparative rodenticidal efficiency of five anticoagulants ;
/. Hyf. Camb. 57 135-149
Brooks J E and Bowerman A M 1974 An analysis of susceptibilities of several populations of
Rattus norvegicus to warfarin ; /. Hyg. Camb. 73 401-407
Brooks J E, Htun P T and Naing H 1980 The susceptibility of Bandicota bengalensis from
Rangoon, Burma to several anticoagulant rodenticides- ; J. Hyg. Camb. 84 127-135
Buckle A P, Rowe F P and Yong Y C 1980 Laboratory evaluation of 0-025% warfarin against
Rattus argentiventer ; Tropical Pest Management 26 162-166
Chaturvedi G C, Madsen C R and Thakore K M 1975 Studies on efficacy "of different anti-
coagulant rodenticides on Rattus rattus (Hoi se rat) ; Proc. All India Rodent Seminar,
Ahmedabad pp. 139-141
Deoras P J 1967 Tolerance status of some rats to an anticoagulant rat poison ; Curr. Set. 36
207-208
Finney D J 1971 Probit analysis 3rd edn. (Cambridge University Press)
Gill J E and Redfern R 1977 Some laboratory tests cf five rodenticides for ths cor.trol of
Arvkanthis niloticus ; PANS 23 33-37
468 R P Mathur and Ishwar Prakash
Gill J E and Redfem R 1979 Laboratory tests of seven rodenticides for the control of
Mastomys natalensis ; /. Hyg. Camb. 83 345-352
Gill J E and Redfern R 1980 Laboratory trials of seven rodenticides for use against the
cotton rat (Sigmodon hispidus) ; /. Hyg. Camb. 85 443-450
Greaves J H and Rehman A B 1977 The susceptibility of Tatera indica, Nesokia indica and
Bandicota bengalensis to three anticoagulant rodenticides ; J. Hyg. Camb. 78 75-84
Krishnamurthy K, Uniyal V and Pingale S V 1968 Studies on rodents and their control. IV.
Susceptibility of Rattus ratius to warfarin ; Bull. Grain. Technol. 6 133-137
Mathur R P and Prakash I 1980 Laboratory evaluation of anticoagulant treated baits for the
control of the Northern palm squirrel, Fun&mbulus pennanti Wroughton ; /. Hyg. Camb.
85 421-426
Mathur R P and Prakash I 1981 a Comparative efficacy of three anticoagulant rodenticides on
desert rodents ; Prof. Ecol. 3 331-335
Mathur R P and Prakash I 198lb Evaluation of brodifacoum against T. indica, M. hurrianae
and R. rams ; /. Hyg. Camb. 87 179-184
Mathur R P and Prakash I 19S2 Laboratory evaluation of chlorophacinone against Tatera
indica and Meriones hurrianae ; Tropical Pest Management 28 291-295
Mukthabai K and Krishnakumari M K 1976 Responses of Rattus species to anticoagulant
poisoning ; Comp. Physiol. Ecol. 1 129-135
Prakash I and Jain A P 1971 Bait shyness of the two gerbils Tatera indica indica Hardwicke
and Meriones hurrianae Jerdon ; Ann. Appl. Biol. 69 169-172
Rowe F P and Redfern R 1964 The toxicity of 0-025% warfarin to wild house mice (Mus
musculus L.) ; /. Hyg. Camb. 62 389-393
Sridhara S 1979 Rodenticidal action of poisons on two rodent pests in India ; Pest Control
47 3*-31
World Health Organisation 1976 Instructions for determining the susceptibility or resistance
of rodents to anticoagulant rodenticides (WHO Technical Report Series No. 443)
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 5, September 1982, pp. 469-472.
© Printed in India.
Effects of handling on oxygen consumption and random activity in
the freshwater mullet Rhinomugil corsula (Hamilton)
M PEER MOHAMED
Central Inland Fisheries Research Sub-station, 24 Pannalal Road,
Allahabad 21 1002, India
MS received 19 October 1981
Abstract. Handling caused excitement which resulted in lower random activity
associated with higher rate of oxygen consumption. The routine and standard
oxygen consumption rates were increased by 260 and 23£%, and 291 and 277%
at 30° and 35° C respectively. The temperature effect (30-35° C) did not cause a
marked difference (P< 0*05) in the rate of oxygen consumption and random activity
in R. corsula.
Keywords. Handling ; oxygen consumption ; random activity ; respirometer ;
Rhinomugil corsula.
1. Introduction
Although the rate of oxygen consumption in relation to several factors has been
extensively studied in fishes (Spoor 1946 ; Beamish 1964 ; Kutty 1968, 1972 ;
Kutty and Peer Mohamed 1975 ; Peer Mohamed and Kutty 1981 ; Peer Mohamed
1981), information on the impact of handling is inadequate (Kutty 1968, 1972 ;
feett 1964 ; Smit 1965). In majority of the studies, the experimental fish was
kept in the respirometer for some time in order to recover from the effects of
handling, if any, because handling causes excitement and/or random activity to
increase (Fry 1967). It has also been reported that the respiratory quotient (RQ)
of goldfish and rainbow trout (Kutty 1968) and Tilapia mossambica (Kutty
1972) is frequently over unity during periods of excitement. Wedemeyer (1972)
found that coho salmon and steelhead trout required 24 hr for normalization of
several blood chemistry imbalances after handling. Since it is not known how
handling would influence oxygen requirement in fishes, there is need for such
information especially on selected cultivable fishes. The present investigation
provides an insight on the effects of handling on oxygen consumption and random
(spontaneous) activity in the freshwater mullet, Rhinomugil corsula (Hamilton).
The observations were made at 30° and 35° C ; the high temperatures were
chosen because of its relevance to local conditions and because the mullet is
exposed to such high temperatures during a good portion of the year.
469
470 M Peer Mohamed
2. Material and methods
R. corsula, collected from Vaigai Reservoir in South India, ranged in total length
from 17*0 to 17'4cm (mean 17*3 cm ; N = 9) and in weight from 40-5 to
43 -5 g (mean 42 -lg) were used. Fish were acclimated and the observations
were made at 30° and 35° C. Two series of experiments were carried out by using
a modification of Fry's respirorneter (Kutty et a! 1971) at high ambient oxygen
(air saturation) ; (i) control fish (the fish was left in the respirometer overnight
after handling and before experiment and (ii) * handled ' fish (immediately
after netting and introducing into the respirometer). The experimental procedure
followed was as described in Kutty and Peer Mohamed (1975). Dissolved oxygen
in the water samples (50 ml), collected just before and after the closure of the
respirometer, was measured by using unmodified Winkler technique (APHA 1955).
The random activity was counted by the difference between the initial and final
figure of the electronic counter, noted after each sampling. Data obtained on
the rate of oxygen consumption and random activity were analysed for fitting
regression lines in semilogarithmic grid by least square technique.
3. Results
Regression equations of oxygen consumption (ml/kg/hr) against random activity
(counts/hr) in R. corsula at 30° and 35° C are given in table 1. Mean values of
routine and standard oxygen consumption, (extrapolated values to zero activity)
and random activity are also included in table 2. The high and low rates of
oxygen consumption were estimated to be 98 and 115 ml/kg/hr (30° C) and 105
and 128 ml/kg/hr (35° C) in control fish (series i) ; 459 and 350 ml/kg/hr at
30° C and 420 and 360 ml/kg/hr at 35° C in R. corsula immediately after handling
(series ii). The random activity of the fish in series (ii) was low (0-9 counts/hr)
and high (10-31 counts/hr) in series (i).
4. Discussion
In the results presented (table 1), a positive correlation between oxygen consump-
tion and random activity in jR. corsula is noted in both the series (i and ii), which
coincides with the observations made earlier in the same species by Kutty and
Table 1. Regression equations (log Y=a+bX) of oxygen consumption (ml/kg/hr)
(Y) against random activity (counts/hr) (X) in Ehinomugil corsula.
Series (i) — Control fish
log Y = 1 • 95704 + C- 00342 X (30° C)
log F = 1-98213 4- 0- 00414 X (35° C)
Series (ii) — ' Handled ' fish
log Y = 2' 54873 + 0' 01161 X (30° C)
log Y = 2' 55895 + 0- 00713 X (35° C)
Effects of handling in Rhinomugil corsuta 471
Table 2. Routine and standard oxygen consumption, and random activity in
Rhinomugil corsula.
30° C 35° C
Series • _
Routine Standarc Routine Standard
(mean ± S.E.) (mean ± S.E.)
Oxygen consumption
(ml/kg/hr)
i 105-7 (7) 90-6
=b 2-6
114-7 (7)
± 3-1
96
Oxygen consumption
(ml/kg/hr)
ii 382-1 (12) 354
± 8-3
387-5 (12)
± 6'0
362
Random activity
(counts/hi)
i 19-3 (7)
± 2*7
18'4 (7)
± 2*7
...
Random activity
(counts/hr)
ii 2-8 (12)
± 0-7
4-0 (12)
± 1-0
...
In the case of routine values mean ± S.E. is indicated. Values in parenthesis denote the
number of determinations.
Series i and ii denote control and ' handled ' fish respectively (see text).
Peer Mohamed (1975). But, the present data differ from those of Kutty et al
(1971) in that they observed much higher levels of random activity for JR. corsula
and also a break in activity- oxygen consumption relationship. It is possible that
the discontinuity in the relation of oxygen consumption and activity is not evident
in the present data because of the narrowness of the range of activity (Kutty 1968 ;
Kutty and Peer Mohamed 1975).
The routine and standard oxygen consumption of the control fish (series i) are
almost the same as observed earlier (Kutty and Peer Mohamed 1975). It is evident
from the results (table 2) that, on comparison of series (ii) with series (i), an
upward shift in the rate of oxygen consumption was observed. The routine
oxygen consumption rate was shifted by 260 and 238%, and standard rate by 291
and 277%, at 30° and 35° C respectively. The present results thereby suggest
that the fish were excited due to handling which resulted in lower random activity
associated with high rate of oxygen consumption, that is, the less active the fish,
the proportionately higher its energy cost. Fishes could, however, respire up
to a level as high as the active metabolic rate due to excitement (Fry 1967). It
is likely that the elevated oxygen consumption during lower random activity in
the e handled ' fish is accomplished in part by an increase in the transfer factor of
the gills (Randall et al 1967), that is, the effective exchange area of the gills is
increased which results in an increase in the osmotic movement of water. In
freshwater, the water moves down the osmotic gradient into the animal.
At both the test tempetatures, the rate of oxygen consumption and random
activity values are in close proximity and the test of significance showed that the
values at 30° and 35° C are not significantly different (P < 0'05), suggesting that
P. (B)-7
474 R K Par shad, G Grewal and S S Guraya
and other ovarian components. The present study was, therefore, "undertaken
to investigate the effects of wall of preovulatory follicle on the ovary of growing
chicks till the onset of lay.
2. Material and methods
Three-week old chicks were purchased from the local hatchery and were kept
in the laboratory under continuous light and provided with feed and water ad
libitum. The chicks were allowed to grow till they were 11 weeks old. Then
they were divided into three groups of 9 chicks each, keeping the average body
weight of a chick similar in all the groups. On alternate days intramuscular
injections of 0*5 ml doses of lipid and aqueous extract of wall of largest follicle
were given to each chick of the first and second group respectively. The chicks
of the third group were injected with saline which served as control. Three chicks
from each group were sacrificed at 17th, 23rd and 29th weeks to study the ovarian
changes. Killing of chicks before 17th week was avoided as our preliminary
studies had shown no visible effect on the ovary up to this time. After counting
the number of follicles from the ovarian surface they were fixed in Bouin's fluid
and calcium-formaldehyde and subjected to routine histological and histochemical
techniques for localization of lipids (Pearse 1968).
For preparation of f ollicular extracts the follicles measuring 3 • 8-4 -.0 cm diameter
were separated from the ovary of laying hens. The f ollicular walls which included
both the thecal and granulosa layers were obtained after removal of yolk as
described by Huang and Nalbandov (1979). The follicular walls from 10 follicles
were then homogenized in saline and after homogenization the material was
centrifuged for 15 min at 5000 rpm. The supernatant, thus obtained, was diluted
to 50 ml and was kept at 5° C during testing procedures. Similarly the lipid
extract of the follicular walls from largest follicles was obtained by extracting the
material with chloroform and methanol (2: 1 v/v). The lipid extract was dried
and was suspended in saline, with slight heating and stirring.
3. Results
Chicks administered with lipoidal extract and killed at 17th, 23rd and 29th week
continue to show higher body, ovarian and oviducal weights as compared to
control and aqueous extract injected birds (table 1).
At 17th week, no conspicuous differences were observed in the ovarian surface
morphology of the lipid and aqueous extract injected hens. But a study of folli- '
cular populations from the serial sections of the ovary revealed that the ovaries of
chicks, injected with lipid extract, contained some follicles having dimensions more
than 500 ^m. The other growing follicles like those of control and water extract
injected measure less than 400 /xm. The number of follicles at different stages of
growth (as given in table 1) is relatively more in lipid extract than those of control
and aqueous extract injected birds. Follicular atresia affecting mainly the follicles
ranging in size from 200-400 ju.m was common in the ovaries of all the three
groups of hens; no significant differences could be observed among them. The
interstitial gland cells existed in irregularly distributed patches (figure 1).
Effect of preovulatory follicle wall on chick ovary
475
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476 R K Parshad, G Grewal and S S Guraya
Ovaries of hens at 23rd week possessed follicles of larger size, some of which
could be counted easily from the ovarian surface. Marked differences were seen in
the ovarian surface morphology of hens which were continuously administered
with lipid extract because they started laying at 22nd week. Ovaries of these
birds were having normal hierarchical follicles whereas those of control and
aqueous extract injected chicks contained only large white follicles, their number
was also relatively less in the latter (table 1). Atresia of follicles is increased
considerably at 23rd week as compared to the ovaries of 17-week old chicks but
it was more in ovaries treated with the water extract and affected the previtellogenic
follicles (figures 2, 3). Vitellogenic atretic follicles were also observed in the lipid
extract administered hens. The ovarian stroma of the 23 -week old chick was
found to contain abundant interstitial gland tissue, but their amount was relatively
more in aqueous extract injected birds (figures 3,4). In the latter case',' the inter-
stitial gland tissue contained more sudanophilic lipids as compared to those of
control and lipoidal extract administered chicks.
At 29th week, the ovaries of control and lipid extract injected birds showed
the normal features of those of laying hens, since the control birds also started
laying at the 25th week. But the aqueous extract injected birds started laying
at the end of the 29th week. The ovaries of these birds contained more number
of medium-sized vitellogenic atretic follicles as judged from the shrinkage and
prominence of the stigmal site. The ovarian stroma at this stage appeared rela-
tively loose in all the three treatment groups. The interstitial gland cells were
abundant and continued to show more lipids in aqueous extract injected hens.
4. Discussion
The present observations have shown that the lipid and aqueous extracts of walls
of larger yellow follicles have antagonistic effects on the ovarian functions in the
growing chicks. The lipoidal extract initiates the follicular growth from the
pool and enhances the rate of growth of follicles at all stages leading to their early
maturity. However, the aqueous extract appears to have the reverse effect. The
enhanced rate of growth of follicles, thus, indicates the presence of some lipid-
like growth-promoting substance elaborated by the larger follicles. In vivo and
in vitro studies have shown that the largest follicle secretes progesterone (Furr
et al 1973 ; Shodono et al 1975 ; Shahabi et al 1975 ; Huang et al 1979) and
prostaglandins (Hammond et al 1980) shortly before ovulation. Thus the presence
of these two substances in the lipid homogenate of the follicular walls is expected
but the possibility of the existence of any other lipoidal substance cannot be
excluded. Prostaglandins do not appear to influence ovarian steroidogenesis in
hen (Hertelendy and Hammond 1980), but their role in initiating and promoting
follicular growth is not known. The involvement of progesterone in promoting
follicular growth cannot be overlooked since it is known to play a key role in
endocrine control of the hypothalamo-pituitary-ovarian axis.
The effect of growth-promoting substance expected to be present in the lipoidal
extract becomes more marked after 17th week. Thus it appears that pituitary-
ovarian axis after 17th week probably becomes more responsive to the growth-
promoting factor contained in lipid extract of the walls of the largest follicle,
Effect of preovulatory follicle wall on chick ovary
477
Figures 1-4. 1. Section of 17-week old chick administered with aqueous extract
showing small growing follicle(s) and patches of interstitial gland cells in stroma.
Sudan black Bx 50. 2. Section of ovary of 23-week old chick administered
with aqueous extract showing degenerating follicles (dg) and abundant lipid-rich
interstitial gland cells (igc). Sudan black B x 50. 3. Section of the ovary of
23-week old chick injected with aqueous extract showing abundant interstitial gland
cells (igc) in the stroma. Sudan black B x 50. 4. Section of ovary of 23-week
old chick injected with lipoidal extract showing normal growing follicles (ng) and
stroma with lesser interstitial gland cells (igc). Sudan black B x 50.
Effect of preovutatory follicle wall on chick ovary 479
In contrast to the lipid extract, the aqueous extract of the follicular wall inhibits
the follicular growth and simultaneously enhances follicular atresia. A water-
soluble factor was extracted from the largest preovulatory and postovulatory
follicles which could induce premature oviposition (Tanaka and Nakada 1975)
but no mention is made until now regarding its effect on the ovary itself. Preli-
minary studies on the estimation of soluble proteins have indicated that the amount
of proteins abruptly increases in the follicles of 3 -8-4* Ocm diameter (unpublished
observations). Possibly, there may be same protein in aqueous extract which
exerts inhibitory influence on the follicular growth and stimulation of follicular
atresia but this suggestion needs to be extended and confirmed.
Our observations on the ovary of growing chicks after treatment with lipoidal
and aqueous extracts have clearly shown that the larger yellow follicles in the
laying hen elaborate two different kinds of substances, one stimulates and the second
possibly inhibits the follicular growth. But the exact mechanisms of action of
these two factors in maintaining the normal and regular pattern of laying remains
to be determined.
References
Etches R J and Cunningham F J 1976 The interrelationship between progesterone and luteinizing
hormone during t,he ovulatory cycle of the hen (Gallus domesticus) ; /. Endocrinol. 71
51-58
Fun: B J A, Boimey R C, England R J and Cunningham F J 1973 Luteinizing hormone and
progesterone in peripheral blood during ovulatory cycle of the hen Gallus domestkus ;
/. Endocrinol. SI 159-169
Graber J W and Nalbandov A V 1976 Peripheral estrogen levels during the laying cycle of
the hen (Gallus domesticus) ; Biol. Reprod. 14 109-114
Hammond R W, Koelkebeck K W, Scanes C G, Biellier H V and Hertelendy F 1980 Prosta-
glandins and steroid hormones in the plasma and ovarian follicles during the ovulatory
cycle in domestic hen (Gallus domesticus) ; Gen. Comp. Endocrinol. 42 195-202
Hertelendy F, Yeh M and Biellier H V 1974 Induction of oviposition in the domestic hen by
prostaglandtns ; Gen. Comp. Endocrinol. 22 529-531
Hertelendy F and Hammond R W 1980 PGs do not affect steroidogenesis and are not produced
in response to oLH in chicken gianubsa cells ; Biol. Reprod. 23 918-923
Huang E S and Nalbandov A V 1979 Steroidogenesis of chicken granulosa and theca cells ;
In vitro incubation system ; Biol. Reprod. 20 442-453
Huang E S, Kao K J and Nalbandov A V 1979 Synthesis of sex steroids by cellular compo-
nents of chicken follicles ; Biol. Reprcd. 20 454-461
Haynes N B, Cooper K J and Kay M J 1973 Plasma progesterone in the hen in relation to
the ovulatory cycle ; Br. Poult. Sti. 14 349-357
Kumagai S and Homma K 1974 High estrogen production of the medium-sized follicles
during fcllicular growth and ovulation in laying quail; Endocrinol. Jpn. 21 349-354
Lance V and Callard I P 1979 Hormonal control of ovarian steroidogensis in non-mammalian
vertebrates. In The vertebrate ovary, (eds.) R E Jones (New York and London : Plenum
Press) Chap. 10, pp. 361-407
Pearse AGE 1968 Histochemistry— Theoretical and applied (London : J & A Churchill Ltd.)
Shahabi N A, Norton H W and Nalbandov A V 1975 Steroid levels in follicles and plasma
of hens during the ovulatory cycle ; Endocrinology 96 962-968
Shodono M, Nakamura T, Tanabe Y and Wakabayashi K 1975 Simultaneous determinations
of oestradial-17j#, progestrone and luteinizing hormone in the plasma duj ing the ovulatory
cycle of the hen, Acta Endocrinol 78 565-573
Tanaka K and Nakada T 1974 Participation of the ovarian follicle in control of time of
oviposition in the domestic fowl. ; Poult. Sci. 53 2120-2125
£roc. Indian Acad. Sci. (Anim, Sci.), Vol. 91, lumber 5, September 1982, pp.
© Printed in India.
Biochemical studies on the haemolymph and heart muscle of
normal and insecticide treated cockroach Periplaneta americana L.
G SURENDER REDDY and A PURUSHOTHAM RAO
Department of Zoology, Kakatiya University, Warangal 506 009, India
MS received 25 June 1981 ; reused 9 July 1982
Abstract. In this paptr quantitative estimations of free proteins, total carbo
hydrates/glycogen and fatty acids from cockroach haemolymph and heart muscle
are reported from normal and insecticide treated insects. High content of protein,
carbohydrate/grycogen and fatty acids are found in the haemolymph arid heart
muscle of nymphal insects. Higher amount of carbohydrates/glycogen are found
in adult males, while more protein and fatty acids were found in ttu- females.
After insecticide treatment, no sex variation was found in the percent depletion
of metabolites. The difference in the depletion rates between nymph and adult
was also insignificant. High percent depletion of the macro-molecules was found
with insecticides which are found more toxic in bioassay studies. A correlation
has been made between the rate of depeltion and insecticidal poisoning.
Keywords. Insecticide ; haemolymph ; heart muscle ; carbohydrates ; glycogen
proteins ; fatty acids.
1. Introduction
It is generally known that, insecticides interfere with the physiology of insect
nervous system, particularly with the nerve conduction mechanism. However,
with the lack of sufficient data the ultimate causes of death in insects are usually
difficult to prove.
Information on carbohydrate, protein and lipid levels from various tissues of
insect is scanty as compared to vertebrates. Only in recent years, insect blood
has been studied for both normal and after treatment with some insecticides,
especially the chlorinated hydrocarbons (Corrigan and Kearns 1963 ; Hawkins
and Sternburg 1964). These studies are limited since the observations were only
made on isolated fractions such as amino acids, free sugars or total lipids.
Despite years of research (Jones 1974 ; Florkin and Jeuniaux 1974), limited
information is available on the changes in heart-beat and much less on heart
muscle due to insecticide action.
481
P. (B)- 8
482 G Surender Reddy and A Purushotham Rao
The present studies were undertaken to find out the effect of certain insecticides
on basic metabolite content of cockroach haemolymph and heart muscle and its
significance in the poisoning of the insects. A description of the quantitative
variations in the total carbohydrates, proteins and fatty acids from the haemo-
lymph and heart muscle of the last instar nymph and adult cockroach, Periplaneta
americana L. are given. The effects of a plant extract 'Morindin' reported
to be toxic to insects (Surender Reddy et al 1978) is also mentioned.
2. Material aud methods
The test insects Periplaneta were collected and developed at room temperature
in glass cages with a wire mesh lid at the top. A layer of sawdust was laid on
the floor of the cage for the deposition of oothecae. Once in a week the oothecae
deposited were separated to another cage for hatching. The insects were fed on
glucose biscuits mixed with yeast powder and potato peels. The following insecti-
cides were used.
Fenitrothion : O,O-dimethyl-o-(3-methyl-4-nitrophenyl)-thionophosphate (Baeyer
India Limited, Bombay) ; Carbofuran : 2,3-dihydro-2,2-dimethyl-7-Benzofuranyl
methyl carbamate (Rallis India Limited, Bangalore) ; Ekalux : 25% (w/w)
Quinolphos (O,o-diethyl-o (quinoxalinyl-(2) Thionophosphate) and 75% (w/w)
stabilizers, emulsiflers and other adjuvants (Sandoz India Limited, Bombay) ;
Morindin : The glycoside morindin 6-primeveroside of morindone (C26H28O14,
1,5,6-trihydroxy 2-methyl anthraquinone) has been extracted and purified from
the root bark of Morinda tinctoria var. tomentosa Hook, as described by Rao
and Reddy (1977) ; Nicotine : Manufactured by E Merck, Dermstadt, Germany.
The insecticides were dissolved in ethyl alcohol and insects were treated
intraperitoneally (Menusan 1948) with the help of an Agla micrometer syringe.
Insects to be treated were weighed individually and the dosage was calculated per
gram (5 //1/g) of the body weight. Last instar nymphs of developing wing base
were selected to maintain uniformity of age. Adults belong to the age group
of 1-4 days after molt. After determining the lethal dosages, one producing
50% kill (LC50) was selected for the present experiments and the insects 'were
taken for biochemical estimations 4-6 hours after treatment. This particular
time lag was chosen because the initial symptoms of poisoning such as hyper-
activity and convulsions were complete in less than 4 hr, thus the insects were
with complete knock down effect. Besides the normal insects, control insects
were taken 4-6 hr, after treatment with ethyl alcohol (5/d/g).
Haemolymph was collected according to the method described by Sternburg
and Corrigan (1959) and the haemocytes were not allowed to sediment. For
cardiac muscle, insect heart was fully exposed with specially made needles and
carefully separated from the alary muscles throughout the length, then it was
gradually lifted on to a cover slip to weigh it gently before transferring into the
test tube. Care was taken to eliminate all foreign tissue associated with heart,
including the alary muscles and fat content.
To estimate the total carbohydrates from haemolymph and glycogen from heart
muscle, the modified anthrone method of Klicpera et al (1957) was adopted. For
proteins the procedure of Lowery et al (1951) and for total/esterified fatty acids,
the methods of Stern and Shapiro (1953) were followed.
Biochemical studies on Periplaneta 483
3. Results
The values of total carbohydrates/glycogen, total proteins and fatty acids
recorded from the normal nymphs and adult cockroaches of both sexes are
mentioned in tables 1 and 2. It may be seen that, the three constituents of
haemolymph of adult insects are relatively lower than those of nymphs. Haemo-
lymph from nymphal cockroach shows about 15-20% more of carbohydrates,
proteins and fatty acids. Among the adults, males show 15% more carbohydrates
than females, while females possess 18% more proteins and 27% more fatty acids
than males. In the heart muscle also it is observed that, nymphs of both the sexes
possess relatively higher values ofglycogen (25-30%), proteins (13-20%), and fatty
acids (17-23%) as compared with adults. In adult cockroaches, glycogen content
of heart is about 20% more in males while proteins and fatty acids are about 18%
and 20% higher in females. In the control insects treated with ethyl alcohol
slight decrease in the total content was seen as compared to normal.
In haemolymph, the percent depletion caused due to fenitrothion treatment
in relation to control values were 33-38% in proteins, 22-24% carbohydrates
and 20-39% fatty acids. With carbofuran, 52-60% proteins, 23-40% carbo-
hydrates and 33-42% fatty acids, which is considered to be highly significant.
After ekalux treatment, 20-30% of proteins, 14-42% of carbohydrates and 11-17%
of fatty acids were found to be depleted. After morindin application, 15-26%
proteins, 11-28% carbohydrates and 8-28% fatty acids were found to be depleted.
Similarly, nicotine caused 17-20%, 10-15% and 5-14% depletion in proteins,
carbohydrates and fatty acids respectively (table 1). The concentration of insec-
ticides employed are given in the table.
In heart muscle, the percent depletion observed after fenitrothion treatment
in the nymphal and adult cockroaches of both sexes were glycogen 24-33%,
proteins 18-21% and fatty acids 17-38%. After carbofuran treatment, glycogen
29-35%, proteins 24-47% and fatty acids 24-40%. After ekalux, glycogen 20-26%,
proteins 11-17% and fatty acies 10-18% were found to be depleted.- With nicotine
treatment, glycogen 10-16%, proteins 2-6% and fatty acids 4-8%, while with
morindin, glycogen 12-18%, proteins 4-10% and fatty acids 5-16% were depleted
(table 2).
4. Discussion
Quantitative studies on the total proteins, carbohydrates and fatty acids from
the haemolymph of normal cockroach give a general indication of higher content
in the nymphs than adults. It is suggested that the initial high values of protein
and aminoacids in the young ones and the rapid fall during adult stage was asso-
ciated with the completion of maturation processes involving protein synthesis
during the first few days after the final moult (Nowosielski and Patton 1965).
Relatively lower content of fatty acids and carbohydrates in adults, as found in
the present study, can be attributed to the higher rate of metabolism during
metamorphosis (Weis-Fogh 1952 ; Guthrie and Tindall 1968). High amounts
of total proteins and fatty acids found in female cockroaches are in agreement
with the findings of Nath et al (1958) and Anderson (1964).
As in case of haemolymph, the normal values of glycogen, total proteins and
fatty acid contents from the cardiac muscle of P. americana (alary muscles
484
G Surender Reddy and A Purushotham Rao
Table 1. Effect of different insecticides on carbohydrates, proteins and fatty
acids of cockroach haemolymph.
Content Normal Control Fenitro- Carbo- Ekalux Nicotine Morindin
std. error thion furan
*N 0-2
A 1-0
0-05
0 06
0-2
0-4
0-25
0-75
0-15
0-25
Total
carbohydrates N 1070±39 1020±50 780±38 700±38 840±32 910±21 9QO±23
A 910±47 35C±70 620±62 650±62 570±78 720±40 610±50
N 920±32 870±25 680±29 630±36 740±16 760±29 730±26
A 770±65 750±40 510±49 440±18 430±19 700±25 537±24
Proteins N 1000±39 900±50 600±26 360±19 720±26 740±?4 760±17
A S40±53 800±60 500±60 320±77 560±77 640±11? 600±77
N 1200±33 1100±30 700±29 520±27 820±30 880±16 900±29
A 1020±98 980±60 600±50 440±20 700±77 780±101 720±76
Fatty acids N 300±19 280±25 190±17 160d=26 230±27 240±26 200±26
A 240±34 210±30 160±20 140±22 185±18 195±19 180±21
N 400±23 380±15 280±24 230±28 320±21 360±18 340±20
A 330±32 300±25 240±19 200±21 260±20 285±28 275±44
Values represent the average of 20 individuals, expressed in mg per 100 ml haemolymph.
N : nymph ; A adult ; * concentration of insecticides expressed in ^g/insect.
Glycogen
Proteins
Table 2. Effect of different insecticides on glycogen, proteins and fatty acids of
cockroach heart muscle.
Content
Normal
std. error
Control Fenitro-
thion
Carbo -
furan
Ekalux
Nicotine Morindin
*N 0-2
A 1-0
0-05
0-06
0-2
0-4
0-25 0-15
0'75 0-25
N 2580±l04 2*00±95 1720±89 1630±92 1840±93 2lOO±78 2050±96
A 1918±80 1900±80 1470±92 1350±101 1520±98 1700±98 1658±101
N 2270±128 2220±100 1640±82 1560±86 1720±89 1917±89 18«8±89
A 1520±87 1470±100 1060±78 980±72 1140±51 1305±49 1250±82
N 2826d=97 2760±9C 2268±101 2089±98 2448±78 2695±79 2640±106
A 2250±110 2200±100 1726±89 1608±90 1833 ±104 2149 ±48 2043±89
N 3168±118 3090±115 2452±87 2286±ll9 2620±87 2896±112 2768±122
A 2742±120 2675±1102160±88 1920±92 2280±86 2509±88 2390±124
Fatty acids N 681 ±78 610±72 505±42 463±46 562±32 584±40 575±3S
A 520±50 480±65 388±36 320±39 409±29 440'±20 428±30
N 780±70 750±60 564±72 502±42 610±84 706±88 628±92
A 648±82 600±62 450±42 390±40 504±42 560±46 520±49
Values represent the average of 20 individuals, expressed in ^g per 100 mg w.w. of muscle.
N : nymph ; A : adult ; *' concentration of insecticides expressed in jig/insect
Biochemical studies on Periptanetct 485
excluded) also, show an increase in nymphs. A similar decrease in the glycogen
content of adult locust muscles was observed by Chari (1970). A decrease in the
content of metabolites of adult cockroach heart muscle (observed in the present
studies) appear to be due to their utilization during metamorphosis from young
to adult, as it was emphasized by Rockstein (1964).
It has been observed that sexual variation is higher than the influence of age
on the concentration of the basic constituents. Males have high amount of
carbohydrate/glycogen while females have higher amount of protein and fatty
acids. This is true for the heamolymph as well as for the heart muscle. Such
similarity in the metabolite ratios between the insect, haemolymph and heart
muscle reveals, perhaps, their physiological association. It is well known that
the heamolymph, having a number of reserve transport material, constantly
circulates between the dorsal tubular heart and body cavity. The heart is a
connective tissue, pulsating and pumping the blood which enters it through the
ostia and is emptied through the dorsal aorta.
In the insects treated with insecticides, haemolymph proteins were depleted
the most, followed by carbohydrates and fatty acids. While in heart muscle, the
difference in the percent depletion of three metabolites was however not signi-
ficant. This is applicable for nymphs and adults of both sexes. More percent
depletion was observed with carbofuran followed by fenitrothion > ekalux >
morindin > nicotine. In general, the degree of percent depletion found in the
three metabolites do not vary much between one another. However, the percent
depletion noted in blood proteins is found to be significantly higher.
As in case of vertebrates, binding of insecticides both to cellular components
and soluble proteins in insects is suggested by Olson (1973). The small and
insignificant difference found in macromolecule content of control insects may
be attributed to the dilution of heamolymph after solvent treatment.
Relatively low depletion of proteins, carbohydrates/glycogen and fatty acids
found with nicotine is in agreement with its low toxicity in the bio-assay studies
(Surender Reddy 1979). This may be attributed to quick metabolism and
excretion of nicotine from the insect body. Extensive metabolism of nicotine
when fed to grasshoppers or applied topically to house-flies was observed by Self
et al (1964). With tobacco hornworm it is reported that 90% of oral dose of
nicotine was excreted in about 4 hr while 83% of nicotine injected into the
body cavity was seen in feces in about 15 min (Self et al 1964).
From the present studies it appears that, besides the target tissue like central
nervous system, susceptibility of insects to an insecticide will also be necessarily
accompanied by biochemical variations in other vital tissues, proportionate to
the toxicity of the substance. A similar conclusion was drawn by Mansingh (1 965)
in his studies with Blatella germanica. It is also corroborated by the opinion of
other workers (Hollingworth 1976), that besides acetylcholinesterase there exist
other targets in the insecticide poisoning of insects.
Acknowledgements
Authors are thankful to Prof. VIA Novak and Dr K Slama of Czechoslovak
Academy of Sciences, Praha, for critical reading of the manuscript and valuable
486 G Surender Reddy and A Pwushotham
suggestions. Messrs. Baeyer India Ltd. (Bombay), Rallis India Ltd. (Bangalore)
and Sandoz India Ltd. (Bombay) are gratefully acknowledged for the gift of
insecticide samples.
References
Anderson E 1964 Oocyte differentiation and vitello genesis in the cockroach Periplaneta
americana ; Cell. BioL 20 131-155
Chari N 1970 Metabolic differentiation of functionally different muscles of some insects and
other invertebrates. C.Sc. Thesis CSAV Praha
Cornwell P B 1976 The cockroach, Vol. II (London : Assoc. Business Prv.gr. Ltd.) p. 318
Corrigan J J and Kearns C W 1963 Amino acid metabolism in DDT poisoned American
roaches ; /. Insect. PhysioL 9 1-12
Florkin M and Jeuniaux C 1974 Haemolymph composition. In The Physiology of Insccta Vol. 5
(ed.) M Rockstein (New York, London : Academic Press) pp. 256-302
Guthrie D M and Tindall A R 1968 The Biology of the cockroach [London : Edward Arnold
(Publishers) Ltd.] pp. 98, 287, 332
Hawkins W B and Sternburg J 1964 Some chemical characteristics of a DDT induced neuro-
active substance from cockroach and cray fish ; /. Econ. Entomol. 57 241-247
Hollingwoith R M 1976 The biochemical and physiological basis of selective toxicit}. In
Insecticide biochemistry and physiology (ed.) .C F Wilkinson (New York and London :
Plenum Press) pp. 431-497
Jones J C 1974 The circulatory system of insects. In The Physiology of Insecta, (ed.)
Vol. 3 M Rockstein (New York, London : Academic Press) p. 121
Klicpera H, Drahota Z and Zak R 1957 Notes on the determination of muscle glycogcn ;
Physiol Bohemoslov. 6 569
Lowcry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein estimation with
folinciocalteu reagent ; J. Biol. Chem. 193 256-272
Mansingh A 1965 The effect cf malathion on the metabolism of amino acids in the German
cockraoch £. gennanica ; /. Insect PhysioL 11 1389-1400
Menusan Jr H, 1948 Comparative toxicity of insecticides administered in various ways to several
species of insects; /. Econ. Entomol. 41 302-313
Nath V, Gupta B L and Lai B 1958 Histochemical and morphological studies of the lipids
in oo.genesis in Periplaneta americana ; Q> /. Micro sc. Sci. 99 315-322
Nowosielski J W and Patton R L 1965 Variation in the haemolymph protein, amino acid and
lipid levels in adult house cricket, Acheta domes ticus L. of diffeient ages ; /. Insect Physiol.
11 263-270
Olson W P 1973 Dieldrin transport in the insect— an examination of Gerolts hypothesis ;
Pestic. Biochem. Physiol. 3 384-392
Rao P S and Reddy G C V 1977 Isolation and characterization of the glycoside of morindone
from the root bark of Morinda tinctoria var. tomentosa ; Indian J. Chem. B15 497-498
Rockstein M 1964 Biology of the Insecta. In The Physiology of Insecta Vol. 1 (ed.) M Rockstein
(New York, London : Academic Press) pp. 3-8
Self L S, Guthrie F E and Hodgson E 1964 Metabolism of nicotine by certain insects ;
Nature (London) 204 300
Strenbarg J and Corrigan J J 1959 Rapid collection of insect blood ; /. Econ. Entomol. 52
538-539
Stern I S and Shapiro B 1953 Calorimttric estimation of esterified differences in insect blood
proteins ; Physiol. ZooL 30. 114-120
Surender Reddy G, Purushotham Rao A and Rao P S 1978 Preliminary observations of the
toxicity of morindin, a glycoside to cockroaches and houseflies ; J. Food Sci. Tech. 15 86
Surender Reddy G 1979 Biochemical and physiological effects of certain insecticides on cock-
roach, Periplaneta americana. Ph.D. Thesis, Kakatiya University, WarangaU A.P., India
Weis-Fogh T 1952 Lipid metabolism during insect metamorphosis; Phil. Trans. Roy. Soc.
London B237 1
Proc. Indian Acad. ScL (Anim. Sci,), Vol. 91, Number 5, September 19&2, pp. 487-491.
© Printed in India.
Fecundity of a hillstream minor carp Pontius chitinoides (McClelland)
from Garhwal Himalaya
H R SINGH, B P NAURIYAL and A K DOBRIYAL
Department of Zoology, Garhwal University, Srinagar Garhwal 246 l745 UP, India
MS received 19 May 1981; revised 29 June 1982
Abstract. One hundred mature specimens of P. chilinoides collected from the
Badiyar gaad, a tributary of the river Alaknanda were examined for fecundity. The
fish weight, ovary weight, and fecundity ranged from 25-11 5 g, 2* 1-1 4- 35 g,
and 2135-7974 respectively. The ovary weight was found from 8-4 to 16-34%
of the body weight. The relationships between fecundity and total length and
weight of fish, fecundity and length, weight and volume of ovary, fish length-
ovary weight, and fish weight-ovary weight were found to be of linear form.
Keywords. Fecundity ; Pimtius chilinoides ; fish weight ; ovary weight.
1. Introduction
Fecundity of a fish may be defined as the number of eggs that are likely to be laid
during a spawning period. Studies on the fecundity of fishes are useful for
increasing the yield of consumable fish species. However, so far no studies have
been made on the fecundity of coldwater fishes of Garhwal Himalaya. Hence
it was considered desirable to study the fecundity of P. chilinoides, an important
food-fish found in the tributaries of the Alaknanda.
2. Materials and methods
Hundred specimens of mature P. chilinoides were collected from Badiyar gaad,
a tributary of the river Alaknanda of Garhwal Himalaya during March- April 1980
and 1981. The total length and weight of each fish and ovary in fresh condition
were noted. The ovary of each fish was dissected out and preserved in 5%
formalin solution for 24 hrs. The fecundity of the fish was recorded by gravi-
metric method (Simpson 1959) and studied in relation to its weight and total
length,- and length, weight and volume of ovary. These relations have been
expressed as follows by applying the method of least square.
(i) The straight line 7 = a + bX (ii) Y = axh or in logarithmic form as
log Y = log a + b log -X
487
488 H R Smgh, B P Naurlyal and A K Dobriyal
3. Observations
3.1. Fecundity and fish length
The relationship between fecundity and total length of fish is shown in table 1.
According to mean values the number of ova varied from 2097 for a fish of 130 mm
to 7978 in the fish measuring 220mm, while the minimum fecundity was 2080
in a fish of 135 mm. The largest specimen of 217 mm had a fecundity of 8020.
The relationship between fecundity and total length in the logarithmic form can
be expressed as :
log F= 3-56 + 1-825 I,
where F= fecundity in thousands and L = total length in mm. The fecundity-
length relationship in P. chilinoides can be expressed as ;
F = ~ 0-15 + 100 L (r = 0 -91 12).
3.2. Fecundity and fish weight
The relationship between fecundity and fish weight is shown in table 2. Egg
production ranged from 21 15 in a fish of 2' 1 g to 8020 in a fish of 14 -6 g. The
fecundity-body weight relationship in P. chilinoides can be expressed as :
100 WF
where WF is the total weight of the fish in g. The relationship between fecundity
and body weight in logarithmic form can be expressed as :
log F = 3'16 + 2-227 log WF (r = 0-8767)
3.3. Fecundity and ovary weight
The relationship between ovary weight and fecundity was found to be close and
linear in nature. The correlation coefficient, r, is 0*9493, which indicates that
Table i. Relationship between fish length, ovary weight and fecundity in
P. chilinoides.
Total length
(mm) of fish.
Mean
(mm)
No. of
fish
Ovary weight (g)
Number of eggs
range
examined
Range
Average
Range
Average
125-135
130
2
2-00-2-100
2-050
2080-2115
2097
135-145
140
15
^2-100-4-200
2-733
2122-3035
2543
145-155
150
21
4:00- 6-500
5-128
3837-5747
4963
155-165
160
14
6-400-7-450
6-975
5680-6380
5956
165-175
170
20
6-700-10-600
8-327
6485-7090
6851
175-185
180
17
10-300-12-300
11-108
7081-7750
7398
185-195
190
6
11-900-13-200
12-400
7680-7788
7719
195-205
200
I
13-400
13-400
7820
7820
205-215
210
2
13-800-14-00
13-900
7845-7935
7890
215-225
220
2
14-100-14-600
14-350
7929-8020
7978
Fecundity of a hill stream minor carp
489
Table 2. Relationship between fish weight* fecundity and ovary waight in
P. cMUnoides.
Weight of
Mean
No. of
Fecundity
Ovaiy weight
(g)
% of ovary
fish(g)
(S)
fish
- weight in
Range
examined
Range Mean
Range
Mean
total
weight of
fish
20-30
25
5
2080-2186
2135
2-00-2-200
2*100
8-40
30-40
35
24
2285-4950
3618
2-400- 5*200
3-714
10-61
40- 50
45
18
5050-5992
5652
5-250- 7-200
6-363
14-14
50- 60
55
22
5921-6990
6661
6-900-10-600
7-468
13-57
60- 70
65
11
7020-7392
7201
9' 900-1 1-400
10-622
16'34
70- 80
75
15
7420-778$
7602
10- 300-1 3 -200
11-793
15-72
80-90
85
1
7820
7820
13-400
13-400
15-76
90-100
95
1
7845
7845
14'CO
14-00
14'73
100-110
105
1
7935
7935
13-800
13* £00
13-14
110-120
115
2
7929-8020
7974
14- 100-14- 600
14-350
.12-47
the fecundity is more directly related to the weight of the ovary. Egg production
ranged from 2115 in an ovary of 2-1 g to 8020 in an ovary of 14'6g. The
fecundity-ovary weight relationship may be expressed as :
F = 3350 + 354-1 WO ; where WO = weight of ovary
log F = 3'065 + 0-555 (r = 0«9493)
3.4. Fecundity and ovary length
The fecundity increased with length of ovaries. This relationship can be expressed
as :
jr = — 0-09 + 250 itf
log F = 2-09 + 2-794 log LO ; (r = 0*9629)
where LO is the length of ovary.
3.5. Fecundity and ovary volume
Fecundity increased with the volume of ovaries. The data on the volume of
ovary and fecundity can be expressed as :
F = 3100 + 423 VO ;
log F= 3-538 + 0-475 log VO ; r = 0*9384
where VO = the volume of ovary.
3.6. Ovary weight and fish weight
The relationship between the fish weight and ovary weight can be expressed as :
WO^— 2-8 + 2FW
The same relationship in logarithmic form may be expressed as :
log WO = 0-21 + 2-5 log WF ; r = 0*9597
where WF = weight of fish
490 H R Singh, B P Nauriyal and A K Dobriyal
3,7. Ovary weight and fish length
The relationship between total length of fish and ovary weight was found to be
fairly close and linear in nature, the '/-' being 0*9862 appears to be the highest
amongst all relationships. It indicates that fish length is more directly related to
ovary weight. The relationship between length and ovary weight may be expressed
as :
0*3+ 1-6 FL
log OW= —0-854 + 0-202 log FL; r = 0'9862.
where OW = weight of ovary and FL = length of fish.
4. Discussion
Various investigators like Clark (1934), Khan (1945), Smith (1947), Lehman
(1953), Alikunhi (1956), Mathur (1964), Saigal (1964), Bhatnagar (1964),
Alikunhi et Ml (1965), Rangarajan (1971), Devraj (1973), Varghese (1973,
1976), Chondar (1977), and Joshi (1980), have studied the fecundity of several
fish species. The relationships have been found to exist between the length and
fecundity of different species of fish. Clark (1934) suggested that the fecundity
of a fish increased in proportion to the square of its length. Simpson (1951)
concluded that the fecundity of plaice was related to the cube of its length.
Relationship between fish length and fecundity has been reported by Sarojini
(1957), Pantula (1963), Gupta (1968), Varghese (1973), and Joshi (1980).
However, in P. chilinoides, the fecundity increases with increase in fish length.
A straight line relationship between the fish weight and fecundity has been
reported by several workers including Begenal (1957), Sarojini (1957), and
Varghese (1961, 1973). A curvilinear relationship was found in Coilia ramcarati
(Varghese 1976), but in P. chilinoides a straight line relationship has been found
between the fish weight and fecundity. In Salvelinus fontinalis the fecundity is
related more to the weight than the length of fish (Smith 1947). A direct propor-
tional increase in the fecundity with the increase in the fish weight has been noted
by Simpson (1951) and Lehman (1953). In P. chilinoides also there is an increase
in the number of eggs with the increase in the body weight
This paper shows that the fecundity and fish length relationship (r = 0-9112)
is more closely related than the fish weight and fecundity (r = 0-8767). The
linear relationship between the volume of ovary and fecundity indicates an increase
in the number of ova produced with the volume of ovaries. Therefore, it appears
that the fecundity increases at a smaller rate in respect to the volume of ovary.
Acknowledgements
The authors are grateful to the Department of Science and Technology (DST),
New Delhi, for financial assistance.
Fecundity of a hill stream minor carp 491
References
AUkunhi K H 1956 Observations on fecundity, larval development and early growth of
Labeo bata (Ham.) ; Indian J. Fish. 3 216-229
Alikunhi K H, Sukumaran K K and Patfneswaran S 1965 Observations on growth, maturity
and breeding of induced bred, pond-reared silver carp, Hypophthalmichthys molitrix and
grass carp, Ctenopharyngodon idellus in India during July 1962 to August 1963 ; Cent.
lust. Fish. Education Bulletin No. 2
Begenal T B 1957 The breeding and fecundity of the long rough dab, Hippoglossoides plates-
soides (Fabr.) and the associated cycle in condition ; /. Mar. Biol. Assoc. U.K. 36 339-375
Bhatnagar G K 1964 Observations on the spawning frequency end fecundity of certain Bhakra
reservoir fishes ; Indian J. Fish. 11 485-502
Chondar S L 1977 Fecundity and its role in racial studies of Gadusia chapra ; Proc. Indian
Acad. Sci. B86 245-254
Clark F N 1934 Maturity of California Sardine (Sardinella caeruled) determined by ova diameter
measurements ; Fish. Bull. California pp. 42-49
Devraj M 1973 Biology of the large snake head Ophiocephalus marulius (Ham.) in the Bhawani
Sagar water ; Indian /. FisJi. 20 280-309
Gupta M V 1968 Observations on the fecundity of Polynemus paradiscus Linn, from the
Hooghly estuarine system ; Proc. Natl. Inst. Sci. India 34 330-345
Joshi S N and Klianna S S 1980 Relative fecundity of Labeo genius (Ham.) from Nanaksagar
reservoir ; Proc. Indian Acad. Sci. (Anim. Sci.) 89 493-503
Khan H 1945 Reproductive powers and breeding habits of some of the fishes of Punjab ;
Punjab Fish. Manu. (Lahore), Appendix 2 pp. 6-11
Lehman B A 1953 Fecundity of Hudson river shad ; Res. Rep. Fish. Bull. U.S. pp. 121
Mathur P K 1964 Maturity and fecundity of Hilsa ilisha ; Indian J. Fish. 11 423-448
Pantula V R 1963 Studies on the age growth and fecundity and spawning of Ostiogeiteiosus
militant (Linn.) ; /. Com. Int. Explor. Mer. 28 295-315
Rangarajan K 1971 Maturity and spawning of the Snapper, Lutlanus kasmira (Forskal) from
the Andaman sea ; Indian J. Fish. 18 114-125
Saigal B N 1964 Studies on the fishery and biology of the commercial cat fishes of the Ganga
river system. II. Maturity, spawning and food of Mystus aor (Ham.) ; Indian J. Fish.
11 1-44
Sarojini K K 1957 Biology and fisheries of the grey mullets of Bengal. I. Biology of Mugil
parsia (Ham.) with notes on its fishing in Bengal ; Indian J. Fisli. 4 160-207
Simpson A C 1951 The fecundity of plaice ; Fish. Inves. London 17 1-27
Simpson A C 1959 Method used for separating and counting the eggs in fecundity studies on
the plaice (Pleuronectes platessd) and herring (Clupea herengus) ; Occ. Pap. FAO, Indo-
Pacific Fish. Coun. No. 59/12
Smith O R .1947 Returns from natural spawning of cut throat trout and eastern brook trout ;
Trans. Am. Fish. Soc. 74 281-296
Varghese T J 1961 Observations on the biology of Raconda nisselliapa (Gray) ; Indian J. Fish.
8 96-106
Varghese T J 1973 The fecundity of the rohu, Labeo rohita (Ham.); Proc. Indian Acad. Sci.
77 214-224
Vargjiese T J 1976 Studies on the fecundity of Coilia ranicarati (Ham-Buch) ; Proc. Indian Acad
Sci. B83 47-54
Proc. Indian Acad. Sci. (Anim. Sci.)> Vol. 91, Number 6, November 1982, pp. 493-499.
© Printed in India.
Bionomics of Mil-stream cyprieids. III. Food, parasites and length-
weight relationship of Garhwal mahaseer. Tor tor (Ham.)
SANDEEP K MALHOTRA ,
Parasitological Laboratory, Department of Zoology, University of Garhwal,
Pauri Campus, Pauri (Garhwal) 246001, U P, India
MS received 27 October 1981 ; revised 27 July 1982
Abstract. 829 Tor tor (Ham.) were examined for food habits, parasites and length-
weight relationships. Parabolic equations describing the body length-body
weight relationships were J^ = 0-000929& Z,2'0553, W= 0-0013146 I,1-9769, and
1^=0-0010884 L1'9561 for females, males and pooled fishes respectively. The
regression coefficients of the < 15*0 cm, 1 5' 1-20- Ocm and >20.-lcm length classes
and sexes were found to be significantly different from one another and from 3.
The regression coefficients of the fishes of larger size classes were higher than
those of the fishes of < 15'Ocm size classes.
Keywords. Gut contents ; parasites ; length- weight relationship ; regression
coefficient ; parasitocoenosis ; variance ; Himalayan riverine ecosystem.
1. Introduction
Garhwal mahaseer, Tor tor (Ham.), is of economic value in the hilly area and it
is available almost throughout the year in the rivers of Garhwal Himalayas. The
present investigation was conducted to help fill the need for more information
on the general biology of this fish in the area. It deals with the bionomics
and helminthocoenoses of T. tor in Garhwal Himalayas ; this study is also part
of an investigation into the biology and fishery of hill-stream fishes, results of
certain aspects of which have already been published by the author and coworkers
(Malhotra 1981a, b ; Malhotra (in press) ; Malhotra et al 1980a, b).
2. Material and methods
Methods of collect^ a of samples and their analyses were published earlier
(Malhotra 1981a ; lauhan et al 1981). 829 T. tor of 4'5~79cm length range
(with one fish measuring 125cm) were used in the present investigation. The
length-weight relationship was estimated by the formula,
493
494 Sandeep K Malhotra
where W = weight, L = body length and a and n are constants. Logarithmic
transformation of this may be written as :
log W = log a + n log L
where, log W is the dependent variable (Y), log L the independent variable
(X), n the regression coefficient or slope (6) ; and log a the F-intercept.
Analysis of variance (Snedecor and Cochran 1967) was applied and the coeffi-
cient of determination (r2) (Croxton 1953) and the values of least squares regres-
sion slopes (Zeller and Carmines 1978) were computed.
3. Results
3.1. Food
Qualitative and quantitative (percentage by weight) analysis of gut contents inclu-
ding food and parasites showed 5 '49% worms, 8 -42% Cladophora sp., Spirogyra
sp., Sphaerocystis sp., Volvox colonies and plant debris and 86 '09% insects, their
larvae and nymphs, viz., coleopterans (Corixa sp., Psephenus sp.), dipterans
(Tendipes sp.), hemipterans (Gems sp.), trichopteran larvae, ephemeropteran
nymphs (Heptagenia sp.) and plecopteran nymphs.
3 . 2. Parasites
The frequency of parasites in alimentary canal of examined fishes was 0-20%
cestodes, 99-50% nematodes, and 0*30% trematodes. Bothriocephalus teleostei
(Malhotra 1981b) was the only cestode and Diplostomum minimum was the only
trematode recorded from the small intestine. However, 79*72% of the nema-
todes collected were females and 20-28% were males. Out of these 8 '09% female
and 11 "36% male specimens of Pseudanisakis sp. were gathered from stomach
while 61'85% female ; 50 '0% male specimens of Comephronema sp. and 30* 06%
female ; 38*64% male specimens of Cystidicoloides sp. were collected from small
intestine,
3.3. Length-weight relationship
The ratio of total and standard length of fish including body weight have been
computed in table 1. It illustrates a comparative account of various relation-
ships between different body measurements and body weight.
3.4. Estimated regressions
Altogether 829 fish of the length range 4* 5-79 cm (with one fish of 125 cm) were
analysed. An initial assessment suggested that the same equation would not
fit the data for the entire length range and that breaks occurred around 10*0-
15*0 cm ; 15* 1-20 -Ocm ; and > 20*1 cm groups. Separate parabolic equations,
their logarithmic transformations, and linear regression were, therefore, computed
for different groups as mentioned in table 2.
The significance of differences between the regression coefficients (b) was tested
by the method of analysis of variance. The relevant data have been presented in
table 3.
Bionomics of hill-stream cyprinids
495
Table 1. Mean values of body weight and ratios of total/standard lengths of
Tor tor (Ham.).
Mean ± S.E.
Sample size
Total length
(cm)
Standard length
(cm)
TL/SL ratio Body weight (g)
Female 474 19 '4 ±0'6691 16'0109±0'5921 l-2088dbO-001 272'S ± 52' 169
Male 35518-5 ±0-4739 15- 4746 ±0' 4345 1 • 2051 ±0" 0034 131-7 ±27-3614
<l5-0cm 549 12- 5593 ±0-0923 10-8927±0'0858 l-l530±0'1131 27'2993± 0*7517
15-1-20-0
cm 149 17-0639±0'1168 17- 6628 ±0' 1062 1'3458±0-1342 86'0302± 6-1856
>20-lcm 131 31-8632±M824 34- 5254± 1-5788 l'6186±0-2459 1 155- 6406±185- 1899
Pooled 829 19* 0041 ±0* 4349 15-7957 ±0*3864 1'2017±0'0029 212'1402± 32-1309
Table 2. Regression equations describing length-weight relationship in Tor tor
(Ham.).
Category
Logarithmic regression equations
Parabolic equations
Female
log W = 3- 0316 4 2- 0553 logL
W ==& 0009298 L2:°°5S
Male
log W = §• 8812 4 1 ' 9769 log L
HP =0- 0013146 L1'9769
<l5-0cm
log W= 1-4208 4 1 -4819 log L
PF = 0- 037949 L1'4819
15* 1-20- Ocm
log W = $- 1459 4 2- 00 logi,
Hr=0- 0071466 I/'00
>20'lcm
log HP = 3- 4281 4 2' 4156 log L
0P=0- 0003732 L2'4157
Pooled
log ^=2- 9632 4- 1- 9561 log £
PF = 0- 0010884 L1'9561
Table 3. Analysis of co-variance between the regression coefficients (6) for Ta-
tar (Ham.).
Female
N 474
Male
355
Pooled
829
<15cm
549
15' 1-20- Ocm
149
>20* 1 cm
131
£(;r— J)2 5-0027
4-4227
5-1108
3-3451
2-4386
4-6242
r(y—r)a 8-7873
7-9748
8-8412
5-0822
5-9265
8- 7579
Z1 (X— X) (y— T) 6- 8447
6- 1060
6-8854
4-0375
3-5456
6-6699
617 (JT— ^>(F— T) 14-0678
12-0710
13-4686
5-9832
7-0912
16-1113
o-2unexp. 1-6427
1-415
1-7228
0-3504
0-2417
1-5074
^ 0-7931
0- 6512
0-6607
0-4443
0-01532
0-8999
r2 0-7949
0-6101
0-6397
0-3158
0-0579
0-8684
« proportion of correlated variance ; <r2 = Unexplained variance.
496 Sandeep K Malhotra
The test of heterogeneity of regressions is given below : —
Source of variation
df Sum of squares Mean square
Between length classes :
Deviation from average
total regression
Deviation from individual
regression withio sample
Difference
Between sexes
Deviation from average
total regression
Deviation from individual
regression within sample
Difference
829
829
0-5725573
825 0*4380454
4 0-1345119
0-000530964
0-0336279
63-33
0.5 ft- 3* 72
0-0054845
825 0-0037392
4 0-0017453
0-0000045
0-0004363
96-96
Fo.50-3-72
The differences between the regression coefficients were significant at 0*5%
level.
A comparison of the regression lines of the length-weight relationship of T. tor
has been presented in table 4. According to the standardized least squares linear
regression, for each standard unit of length, the fish gained 0-890-0-891 ; 0*799-
0-836 ; 0-806-0-820 ; 0*562-0-791 ; 0-220-0-242 ; and 0-947-0*950 of a stan-
dard unit of weight for females (size group 6-78 cm and one fish of 125 cm) ; males
(size group 4 -5-79 cm) ; pooled ; < 15'Ocm ; 15 '1-20*0 cm ; and > 20 -1cm
groups of T. tor respectively. In both the sexes r is significant.
A logarithmic plot of weight (mean values) on length (mean values in 5*0 cm
length intervals in 829 fishes and the linear regression for separate groups and
pooled fishes are shown in figure 1.
4. Discussion
4.1. Food and parasites
The analysis of food reveals that T.tor is a carniomnivorous fish but predominantly
exhibits carnivorous habit. Nematode (N) parasites were more prevalent (99*50%)
than cestodes (C) (0-20%) and trematodes (T) (0*30%). Hence a relationship
C < T < N could be established for T. tor. A detailed analysis of trends in
parasitocoenoses in T. tor has been dealt with by Malhotra (in press) recently.
Bionomics of hill-stream cyprinids
497
Table 4. Comparison of the regression lines of the length-weight relationship of
Tor tor (Ham.)
Sample size Variance Covariance Standardized least squares Level of
Length Weight regression slope predicting significance
r (P)
X from Y Y from X
Female (474)
2-3269
6-1116
4-1689
0-8897
0-8914
0-8916
(P< 0-025)
Male (355)
1-8724
5-4245
3-5558
0-7794
0-8355
0-7811
(P< 0-100)
< 15- Ocm (549)
0-6055
2-3427
1-2979
0-7905
0-5619
0-5620
(P< 0-250)
15- 1-20- Ocm
(149)
0-2654
3-75342
1-3724
0-2422
0-2197
0-2408
(P< 0-50Q)
> 20- 1cm (131)
2-5069
6-6441
4-5526
0-9501
0-9471
0-9319
(P< 0-005)
Pooled (829)
2-1922
5-9226
3-9669
0-8201
0-8057
0-7998
CP< 0-100)
4.2. Length-weight relationship
In the present investigation no major difference was found in the ratio value of
total vis-a-vis standard length from that reported by earlier workers. There was
a highly significant correlation of body length to body weight for female (P <
0-025), male (P < O'lOO), pooled (P < 0-100) and > 20*1 cm length classes
(P< 0-005) of T. tor (table 4). Based on the coefficient of determination (r2)
(Croxton 1953), more than 79% of the variation in weight in females, 61% in
males, 63% in pooled, and 86% in > 20-1 cm length class was attributable to the
variation in length of the Garhwal mahaseer. However, only 31-58% and 0*06%
of the variation in weight in < 15 '0 cm and 15 -1-20-0 cm length classes respec-
tively was attributable to the variation in length of fish. Similarly the proportion
of correlated variance (/?2) suggests that 79*31% variance in length in females,
65-12% in males, 66*07% in pooled fishes and 89*99% in fishes of > 20-1 cm
length class was associated with weight while only 44-43% and 5*32% variance
in length in fishes of < 15 -0 cm and 15 • 1-20-0 cm length classes, respectively, was
associated with weight. The length-weight relationship for female, male, pooled
and fishes of < 15 -Ocm, 15 '1-20 -Ocm and > 20 -1cm length classes of T. tor
is defined and illustrated in figure 1.
The differences in regression coefficients between male and female fishes have
been reported by Sekharan (1968), Eggleston (1970) and Krishnamoorthi (1971).
The results of the present investigation show closeness to these studies in descri-
bing a significant difference between regression coefficients of different size classes
and the sexes. It, however, does not conform to the views of Sekharan (1968)
who regarded in Sardinella albella and S. gibbosa, higher values of regression
coefficients in smaller length classes than in larger ones. Contrary to this, in the
present study, the fishes of larger length classes, viz., 15 -1-20-0 and > 20-lcrrj
498
Sandeep K Malhotra
14000-
10000-
1000-
500-
£
o»
250-
100-
20-
10-
Y Y - <!5cm
YV-t& POOLED
y//Y/x. gi
Y°Y°- 15.1-20 cm
15 20
UENGTH (Cm)
Figure 1. Length-weight relationship in Tar tor (Ham.).
125
showed higher values of regression coefficient (b = 2-00-2-4156) than the smaller
ones (< 15 -Ocm). This perhaps indicates a relatively rapid change in body
outline of the fishes of 15-1 cm to 79cm size group including 125cm long fish,
when they increase more in length than those of the fishes of smaller size classes.
As 'a' depends upon the obesity of the fish (LeCren 1951), by comparing the
* log a ' values it is evident that the general fatness in the two sexes shows no
significant difference in the present study like those reported by Narsimham (1970),
Mojumdar (1971) and Vinci and Nair (1974). * Log a ' values also show appre-
ciable difference in general fatness of individuals of different length classes contrary
to the report of Sekharan (1968).
In this paper as per requirements of the exponential formula (W = a If) there
was a consistently significant correlation in length and weight of T. tor. The
values of regression coefficients indicate that the growth rate is lesser than the cube
pf length and represent an isometric trend (figure 1). Significant departures
Bionomics of hill-stream cyprinids 490
from the isometric growth value have been reported by Narsimham (1970), Vinci
and Nair (1974), Qadri and Mir (1980) and Malhotra (unpublished). This depar-
ture is statistically tested for the significance of the difference of the regression
coefficient from 3. The regression coefficient and its standard error for the
general relationship being 1-986 and 0-133 respectively, 'z'test (f = -7*61
obtained by subtracting 3 from the regression coefficient and dividing the result
by S.E.) indicated a high degree of significance, showing that the cubic law (W =
CL? ; W = weight, L = length, C = constant) does not hold good for T. tor
in the Himalayan riverine ecosystem.
Acknowledgements
The author is grateful to the Conservator of Forests, Garhwal Division, for permis-
sion of ichthyoparasitological survey and Dr R C S Rawat, Principal for facili-
ties. The assistance received from Shri R S Chauhan, SRF, UGC, in collections
is acknowledged.
References
Chauhan R S, Malhotra Sandeep K and Capoor V N 1981 The distribution and abundance
ofcestodes in eleven Species of teleosts from Garhwal Himalayas with a note On host
biology ; Himalayan J. Sci. 1 15-30
Croxton F E 1953 Elementary statistics with applications in medicine and the biological sciences
New York : Dover Publ., pp. 376
Eggleston D 1970 A symposium on the Japan current (ed.) / C Mar pp. 417-424
Krisfmamoorthi B 19?1 Length-weight relationships fox Nemipierus japonicus of Andhra-
Orissa coast; Indian J. Fish. 18 1-21
LeCren E D 1951 The length- weight relationship and seasonal cycle in gonad weight and
condition in the perch (Perca fluviatilis) ; /. Anim. Ecol 20 201-219
Malhotra Sandeep K 1981a Cestode infection in freshwater fishes of Garhwal Himalayas,
India ; Geobios. 8 90-92
Malhotra Sandeep K l981b Systems models for parasite pathways in ichthyoparasitobgy of
the Himalayan, riverine ecosystem ; Curr. Sci. 50 874-875
Malhotra Sandeep K (in press) Trends in ichthyoparasitobgy of Tor tor (Ham.) in the Himalayan
riverine ecosystem ; J. Himalayan Stud. Reg. Dev.
Malhotra Sandeep K, Chauhan R S and Capoor V N I9&0a Nematode infection in relation
to some ecological aspects of hill-stream fishes ; Geobios. 7 193-198
Malhotra Sandeep K, Chauhan R S and Capoor V N 1980b Statistical analysis of cestode,
infection in relation to some ecological aspects of hillstream fishes in Garhwal Himalayas,
India ; Indian J. Helmmthol. 32 43-52
Mojumdar P 1971 Length-weight relationship in the cat-fish Tachysurus thalassinus (Ruppell) ,
Indian /. Fish. 18 179-182
Narsimham K A 1970 On the length-weight relationship and relative condition in Trichiums
lepturus Linnaeus ; Indian /. Fish. 17 90-96
Qadri M Y and Mir S 1980 Length-weight relationship of Orienus plagiostomus (McClell.) ;
Geobios 7 158-159
Sekharan K V 1968 Length-weight relationship in Sardinella albella (Val) and S. gibbosa
(Bleek) ; Indian J. Fish. 15 166-174
Snedecor G W and Cochran W G 1967 Statistical Methods (6th edn) (Ames. Iowa : Iowa
State Univ. Press) pp.593
Vinci G K and Nair A K K 1974 Length weight relationship in the threadfinbream,
Nemipterus japonicus along the Kerala coast ; Indian J. Fish. 21 299-302
Zeller R A and Carmines E G 1978 Statistical analysis of social data (Chicago;; Rand
McNally College Publ. Co.,) pp. 398
Proc. Indian Acad. Sci. (Anim. Sci.)> Vol. 91, Number 6, November 1982, pp. 501-505,
© Printed in India.
Effects of sublethal levels of DDT, malathion and mercury on
tissue proteins of Sarotherodon mossambkus (Peters)
K RAMALINGAM* and K RAMALINGAM
Department of Zoology, University of Madras, Madras 600 005, India
* Research Associate, Entomology Research Institute, Loyola College,
Madras 600034, India
MS received 21 January 1982 ; revised 21 July 1982
Abstract. Liver and muscle total proteins declined in Sarotherodon mossambicus
subjected to sublethal concentrations of DDT, malathion and mercury. The results
indicate their role in maintenance of energy supply irrespective of the nature of the
toxicant. The qualitative variations in serum protein pattern also support the
quantitative changes in tissues.
Keywords. Toxic stress ; proteolysis ; iso-osmotic ; milieu interior.
X. Introduction
Tissue total proteins as energy sources for fishes during thermal stress, spawning
and muscular exercise have been demonstrated by several investigators (Fontaine
and Hatley 1953 ; Idler and Clemens 1959). However, studies on the impact
of toxicants on tissue energy sources are relatively few, though considerable
information is available dealing with the determination of acute toxic levels of
several pollutants and their influence on oxidative metabolism. In this paper,
an attempt has been made to determine the extent of changes in the level of
proteins in two principal tissues, liver and muscle and also the electrophoretic
pattern of serum proteins in the fish Sarotherodon mossambicus exposed to sub-
lethal concentrations of DDT? malathion and mercury.
2. M^teri^Is and methods
Sarotherodon mossambicus (Peters) (15-20g) were obtained from local ponds
maintained by Tamil Nadu state fisheries research station, and acclimated in the
laboratory for 15 days. They were fed with cooked rice mixed with dried prawn
powder. DDT (III-Trichloro 2-2-Bis (P-Chlorophenyl ethane) as 10% wettable
powder and malathion (S-1,2 Bis (ethoxy-carbonyl) ethyl 0, o-dimethyl phosphoro-
dithiate) as 5% wettable powder, supplied by M/s Parry and Company Limited,
L-:1-'*: •' V;:, •!*"} •.':...-.-: , ' ,-' -. - ," :• :. , V .;-/^; -•; .-. - ;. • ' ; ..
..','* ;-w^,i " " < 501
P.CB)-2
502
K Ramalingam and K Ramalingam
Madras, were employed for the sublethal tests. The chloride form of mercury
(HgCl2) was used as the heavy metallic compound. Acetone was used as the
solvent for DDT and water for both malathion and mercury. Two sets of fishes
each consisting of five were exposed to 0*01 ppm of DDT, 0'95 ppm of malathion
and 0*09 ppm of mercury, the respective sublethal levels representing the active
ingredients of the toxicants. The sublethal concentrations of them were calcu-
lated by multiplying an application factor of 0' 25 X with the respective LC 50
values determined from the acute toxicity tests, as recommended by the Ontario
Ministry of Environment (1974). The fishes were exposed for the 24 hr, 7 days
and 15 days simultaneously along with controls for each. At the end of respective
intervals, fishes were sacrificed and tissues were taken for total protein analysis.
The protein was estimated following the procedure of Lowry et al (1951).
For the qualitative study of serum proteins, disc electrophoresis using polyacryl-
amide gel was carried out. The pattern of fractions obtained after 15 days
exposure is indicated in figure 3.
3. Results
The levels of total protein in the liver -and muscle of control and toxicant exposed
groups are shown in figures 1 and 2. There appears to be no significant differ-
ence either in the liver or muscle of the control and the three toxicant exposed
groups at 24 hr interval. However, a significant decrease was noticed after
7 and 15 days in both tissues (P = 0-05). Electrophoretic studies revealed that
serum proteins in fishes kept under control showed eleven fractions. On the
contrary, in fishes exposed to DDT— a total of fourteen fractions, and in those
exposed to malathion and mercury, ten and nine fractions were discernible
respectively.
24
22
20
18
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I u
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o
^ 10
I *
6
4
2
12
Control
D.D.T,
Malathion
Mercury
15
U
~ 13
£ 12
o 11
O 9
2 8
,~, 5
5
4
3
2
1
24 HOURS
7 DAYS
Control
D.D.T
Malathion
Mercury
15 DAYS
Figure 1. Total protein (liver) (mg/100 mg
wet wt.).
Figure 2. Total protein (muscle) (mg/10
mg wet wt).
Effects of DDT, malathion and mercury on tissue proteins
503
Figure 3. Polyacrylamide-gel-ekctrophoretic patterns of serum pioteins of control
group vs. toxicant-exposed groups.
4. Discussion
The total proteins in the liver and muscle showed a steady decline after 7 and
15 days, in contrast to 24 hrs interval. The absence of considerable alterations
in the total protein content during the initial period of exposure (24 hrs) supports
the concept of Fry (1971) that fishes tend to resist a changed situation for a specific
period, but will eventually succumb as a result of their inability to continuously
adapt. The pattern of changes in the total carbohydrates in blood, the free
sugars in liver and muscle and the consequent depletion of glycogen in these
tissues at the initial period of exposure (24 hr) in this animal (Ramalingam 1980)
also lends support to the view extended by Umminger (1970) that carbohydrates
represent the principal and immediate energy precursors for fishes exposed to
stress conditions while proteins being the energy source to spare during chronic
periods of stress.
Depletion of tissue proteins in fishes exposed to various toxicants has been
reported by several investigators (McLeay and Brown 1974 ; Sakaguchi and
Hamaguchi 1975 ; Shakoori et al 1976). Besides the above changes, the protein
fractions in the serum of fishes exposed to toxicants, revealing an increase in the
case of DDT while a decrease in malathion and mercury-exposed ones also indicate
the conversion of tissue proteins into soluble fractions reaching the blood for
utilisation. Similar qualitative changes have been reported by Anees (1974)
in Channa punctatus exposed to diazinon, dimethoate and methyl parathion for
14 days.
The decline in the liver and muscle protein would suggest an intensive proteo-
lysis which in turn could contribute to the increase of free aminoacids to be fed
into the tricarboxylic acid cycle (TCA) as keto acids, thus supporting the
hypothesis of Kabeer Ahamad et al (1978). Such a possibility is further
strengthened by the investigations of Shaffer (1967)— Mehrle et al (1971),
Shakoori et al (1976) which revealed both qualitative and quantitative
variations in the tissue aminoacids of fishes exposed to toxicants. In addi-
tion, studies of Bell (1968), McKim et al (1970), Lane and Scura (1970), Sakaguchi
"504 K Ramalingam and K Ramalingam
and Hamaguchi (1975) have also revealed marked variations in the activity
of enzymes involved in transaminations in fishes at similar situations. However,
an understanding of the levels of aminoacids at different intervals during stress
imposed by toxicants, would be of interest in explaining the role of tissue proteins
either to meet the energy demand completely or to maintain an iso-osmotic
condition of the milieu interior also by increasing the aminoacids pool as suggested
by Kabeer (1979).
Acknowledgements
First author thanks UGC for awarding a fellowship.
References
Anees M A 1974 Changes in starch gel electrophoretic pattern of serum proteins of a freshwater
teleast Channa punctatus (Bloch) exposed to sublethai and chronic levels of three organo-
phosphorous insecticides ; Ceylon J. Sd. 11 53
Bell G R 1968 Distribution of transaminases (aminotransferases) in the tissues of Pacific salmon
(jOncorhynchus) with emphasis on the properties and diagnostic use of glutamic-oxaloacetic
transaminass ; /. Fish. Res. Ed. Can. 25 1247-1268
Fontaine M and Hatley J 1953 Contribution al etude du metabolisme glucidique du salmon
Salmo solar L. a liver ses etape$ de son development et de ses migrations ; Physiol. Comp.
Et Oecologia 3 37-52
Fry F E J 1971 In Fish Physiology (eds.) W S Hoar and D J Randall (New York :
Academic Press) Vol. 6 p. l.
Idler D R and Clemens W A 1959 The energy expenditures of Frasher river sockeye salmon
during spawning migration to Chilke and Stuart lakes ; Int. Pacific. Salmon Fish. Comm*
Prog. Rep. Jackson Printing New Westminster B C 80 pp
Kabeer Ahamad I, Begum Md, Sivaiah S and Ramana Rao K V 1978 Effect of malathion on
free aminoacids, total proteins, glycogen and come enzymes of pelecypod Lamellidens
marginalis (Lamarck) ; Proc. Indian Acad. Sci. 87 377-381
Kabeer Ahamad Sahib I 1979 Studies on some aspects of protein metabolism and associated'
enzyme systems in the freshwater teleost Tilapia mossambica to malathion exposure. Ph.D.
Thesis, Sri Venkateswara University, Tirupati
Lane G E and Scura E D 1970 Effects of dieldrin on glutamic oxaloacetic transarninase in
Poedlia latipinna ; /. Fish. Res. Ed. Can. 27 1869-1871
Lowry D H, Rosebrough N J, Fair A L and Randall R J 1951 Protein measurement with the
Folin phenol reagent ; /. Eiol Chem. 193 265-275
McLeay D J and Brown D A 1974 Growth stimulation and biochemical changes in juvenile
coho salmon (Oncorhynchus kisutcti) exposed to bleached kraft pulpmill effluent to 200
days ; /. Fish. Res. Ed. Can. 31 1043-1049
MaKim J M, Christensen G H and Hunt E P 1970 Changes in the blood of brook trout
(Salvelinus fontinalis) after short term and long term exposure to copper ; /. Fish. Res.
Ed. Can. 27 1883-1889
Mehrle P M, Stalling D L and Bloomfield R A 1971 Serum aminoacids in rainbow trout
(Salmo gairdneri) as affected by DDT and dieldrin ; Camp- Biochem. Physiol. 38 373-377
Ontario Ministry of the Environment 1974 Guidelines and criteria for water quality management
in Ontario. Supervised for publication by the Hon William G Newsman, Minister
and Everst Biggs, Deputy Minister, 135 St. Clair Avenue, West Toronto
Ramalingam K 1980 Studies on the effects of sublethai concentrations of a few toxicants on
Biochemistry, Physiology and Histology of Tilapia mossambica (Peters). Ph.D. Thesis,
University of Madras, Madras
Effects of DDT, malathion and mercury on tissue proteins 505
Sakaguchi H and Hamaguchi A 1975 Physiological changes in the serum and hepatopancreas of
yellow tail injected with carbon tefrachloride ; Bull. Jpn. Sac. Scient. Fish. 41 283-290
Schafer R 1967 The effects of pollutants on the free aminoacid content of the fish Leuciscus
cephdus (L.) albus ; Rev. Bi&l 59 3S5-407
'Shakoori A R, Saleem A Z and Muhammed S A 1976 Effect of malathion, dieldrin and
endrin on blood serum proteins and free aminoacids pool of Channa punctatus (Block) !
Pak. J. Zool. 8 124-134
Umminger BL 1970 Physiological studies on supercooled killifish fundulus heteraclitus. III.
Carbohydrate metabolism and survival at subzero temperature, /. Exp. Zool. 173 159-174
Proc. Indian Acad. Sci. (Anim. ScL), Vol. 91, Number 6, November 1982, pp. 507-513.
© Printed in India.
Effect of teleostean prey size and salinity on satiation amount,
satiation time and daily ration in the glassy perchlet Chanda
(j=Amba$sis) thomassi (Day) (Pisces : Centropomidae)
J RAJASEKHARAN NAIR and N BALAKRISHNAN NAIR
Department of Aquatic Biology and Fisheries, University of Kerala, Beach P.O.
Trivandrum 695 007, India
MS received 26 June 1982
Abstract. Results of the experiments conducted to. estimate the maximum single
food intake, satiation time and daily ration in the predator, Chanda thomassi using
different size groups of teleostean prey (guppies) and in six non-lethal salinities
are presented. The results suggest that satiation amount and satiation time vary
considerably with the size of the fish prey. It is seen that the appetite of the fish
is lost on consuming relatively fewer number of larger fish prey, while the predator
could accommodate a much larger number of smaller prey fish of greater gross
size. Also the satiation amount decreases when the prey is available in bulk
than when given at regular intervals. The computed daily ration of the predator
shows high values when compared with available data on other tropical predators.
The over all results project the destructive potential of this predatory species coupled
with its shoaling habits.
Keywords. Satiation ; teleostean prey ; predator ; Chanda thomassi.
\. Introduction
Within the genetic potential of any species to grow, many abiotic and biotic
factors limit maximum growth. The daily rate at which food can be consumed
is a prime factor. This in turn is related to the capacity of the stomach (satiation
feeding) and the rate of digestion. Thus, knowledge of food consumption in fish
populations is, therefore, essential for interpretation of the influence of a variety
of factors on fish production (Warren et al 1964 ; Windell 1966 ; Brocksen
et al 1968 ; Brett et al 1969 ; Swenson and Smith 1973).
Information on the satiation amount i.e., the amount of food necessary to
satisfy the fish (Brett 1971), the satiation time (time to attain satiation), and
details regarding daily ration, and the gastric evacuation rates of a piscivorous
predator are essential prerequisites for assessing the feeding capacity of these
predators on valuable fish fry and fingerlings in the natural waters and culture
systems. Chanda ( = Ambassis) thomassi (Day) is a medium sized piscivorous
predator found in shoals in the fresh and low saline waters of Kerala in South
507
508 J Rajasekharan Nair and N Balakrishnan Nair , • .
India. With a view to estimating the satiation amount, satiation time and daily
ration in the case of C. thomassi adults under laboratory conditions, a series of
tests were conducted using four different size groups of the fish prey (Poecilia
( == Lebistes) reticulata Peters) and six different salinities.
2. Materials and methods
Healthy individuals of C. thomassi immature adults (4-250 ± 0*250 g and
standard length (SL) 7*1 ± 0-5 cm) were acclimated and reared in large plastic
buckets (20 litre capacity). The temperature of the water was 27 ± 1° C and
the oxygen content maintained at air saturation level. The fish were fed with
an excess amount of fry, juveniles and adults of Poecilia reticulata for nearly
fifteen days prior to experiments. The prey fish (P. reticulata) were then grouped
into 4 size groups :—
Group I fry (average SL 8mm, average Wt. 16*3mg)
Group II juveniles (average SL 14 -2 mm, average Wt. 57-Omg)
Group III mature males (average SL 18 -6mm, average Wt. 98*4mg)
Group IV mature females (average SL 24 -8 mm, average Wt 175-4mg)
All the prey fish of each size group were almost of the same size and weight
and the averages were calculated after weighing and measuring more than 50 fish
collected at random from each group. Preliminary tests were conducted to find
out the feeding intervals for each size group and the rate of feeding at each
interval. They were estimated as 2 min and 5 fish (Group I), 5 min and 3 fish
(Group II), 7 min and 3 fish (Group III) and 10 min and 2 fish (Group IV).
(1) At intervals (Expt I)— The individuals of C. thomassi were starved for two
days prior to the experiment in order to effect complete stomach evacuation. The
precalculated numbers of prey fish were presented during each time interval
removing the excess until the fish completely stopped feeding. To accommodate
an initial high rate of feeding (Brett 1971), food was presented twice as fast
during the first time interval. Fish were considered satiated when they would
no longer accept any food, in the presence of excess, after a period of active
feeding. The time from start to voluntary cessation is defined as the satiation
time. Each experiment was done in triplicate.
(2) As a bulk (Expt. II)— Another experiment was done after a days starving
presenting each fish with a bulk of fish prey (more than twice the satiation
amount of the previous experiment) of each size group at a single instant. The
fish were considered satiated when they did not capture a prey for a fifteen
minutes time lapse. The satiation time was considered as the time from the
start of feeding to the time of the last feed. Rough estimates of daily ration
were made from the results of these experiments.
(3) In different salinities (Expt. Ill) — Also the fish were reared in 6 precal-
culated non-lethal salinities (0-96%0, 6-83%0, 9'75%0, 12*69%0, 15'62%0) and were
provided with a bulk of prey (Group II) and the total amount consumed during
the first 25 min, up to 12 hrs and up to 24 hrs were noted so as to roughly,
estimate the daily ration at different salinities.
Teleostean prey size and satiation in Chanda thomassi (Day) 509
3. Results
The satiation time, satiation amount, satiation amount as percentage of predator
body weight (wet) and the amount of food consumed per unit time for the different
prey fish size groups (feeding at intervals and in bulk) are illustrated in figures 1
and 2. It can be clearly discerned that with the increasing prey fish size there
•500
II in
PREY FJSH SIZE OROUPS
IV
Figure 1. Effect of teleostean prey size on satiation amount, satiation time and
consumption per unit time in Chanda thomassi when fed at intervals.
9.75-
9.5-
o
40-
35'
30H
2SH
2:
2 20-
'
15-
10-
-50
-40 J
^
-30 g
•20
-10
II III
PREY FISH SIZE GROUPS
IV
•410
•390r
•370 <
§
-350
Figure 2. Effect of teleostean prey size on satiation amount, satiation time and
consumption per unit time in C. thomassi when fed in bulk.
P.(B)-3
510
/ Rajasekharan Nair and N Balakrishnan Nair
is a decline in satiation time, satiation amount and correspondingly in the amount
consumed as percentage body weight whereas the amount of food consumed per
unit time shows a steady increase. From the two experiments the satiation time
ranges from 27-58 min and satiation amount from 391 -2mg-505 *3 mg for
group I5 15 -0-30 -0 min and 342 -0-456 'Omg for group II, 10 -5-28-0 min
and 295-2-393-6 mg for group III and 6-0-20-0 min and 350*8 mg for group
IV.
Taking into account only the light phase of the 24 hr day (the fish were found
to rest on the bottom individually and not to feed during the night and to reshoal
and start feeding at dawn), and the time for 100% stomach evacuation (9 hrs),
Table 1. Effect of teleostean prey size on the daily ration of C. thomassi
(immature adults) when fed at intervals and in bulk.
At
intervals
In bulk
Prey fish
size groups
Daily ration as
% wet body weight
of predator
Prey fish
size groups
Daily ration as
% wet body weight
of predator
Group I
22-76
Group I
19-18
Group II
18-78
Group II
17-88
Group III
16*98
Group III
16-98
Group IV
16-50
Group IV
16-50
• 'AFTER 2<.hrs
o- oAFTER 12hrs
AFTER 20 min
0*96
3.81
6-83 9-75
SALINITY. {%«)
12,69
15-62
Figure 3. Effect of six non lethal salinities on the food consumption (25 min and
12 hrs) and daily ration of C. thomassi.
Teleostean prey size and satiation in Chanda thomassi (Day) 511
it was found safe to assume that active feeding is restricted mainly to the dawn
and dusk (twice a day). Thus a rough estimate of the daily ration was made as
twice the satiation amount and presented as percentage of the predator body weight
in table 1.
The satiation amounts for the first 25 min, for 12 hrs and 24 hrs for the six
different salinities are shown in figure 3. The highest amount of food intake in
24 hrs is shown to be at6'83%0 S i.e., 24-14% of the predator body weight,
the lowest amount being at 15*62%0 S i.e., 13*41% of the predator body weight.
Thus in the 0 *9%0-15 *62%0 salinity range, a rough daily ration range of 13 *41%-
24-14% of the predator body weight is seen.
4. Discussion
The results suggest that the satiation time and satiation amount vary considerably
with the size of the fish prey. The satiation amount and time are inversely pro-
portional and the amount of food consumed per unit time is directly proportional
to the size of the fish prey. It would appear that the appetite of the fish is lost
on consuming relatively fewer number of larger fish prey, while the predator could
accommodate a much larger number of small fish prey of greater gross size.
An analysis of the available information would indicate that (1) Stretch receptors
in the stomach wall constitute one of the mechanisms controlling the appetite of
vertebrates (Lepkovsky 1948 ; Paintal 1954). Consequently the size of individual
particles would apparently determine the point at which further disteation is
declined. This will also be checked to some extent by the shape of the individual
particles, especially in predators where the prey is swallowed as a whole as in
the present study, so as to utilise the maximum space of the total available exten-
ded stomach volume. (2) Animals tend to eat to satisfy their energy demand so
that the calorific content of the food will also affect the size of daily ration (Rozin
and Mayer 1961).
The predator's maximum single intake (satiation amount) of the prey fish fry
and juveniles (Groups I and II) decreases when the prey is available in bulk
than at regular intervals (483*57 to 407- 5 mg and 399-0 to 380 -Omg). Thus
the predator may consume more food if the fry and juveniles of the prey fish form
scattered groups being available to the predator as individuals at short intervals
than when they are in abundance forming large tight-knit shoals.
Thus, it would appear that a 58 min feeding time with feeding at intervals,
and 35 min feeding time with feeding in bulk would be quite adequate to satiate
C. thomassi immature adults at 27 ± 1° C independent of the size of the fish
prey. The Jack mackerel (Trachurus japonicus) and the rainbow trout (Salmo
gairdneri) feeding on mackerel meat and * compound feed ' at 25° and 10° C,
respectively, required 60 and 65 min to reach satiation, whereas two other
species, the puffer (Fugi vermicularis) and the file fish (Stephanolepis cirrhifer)
were satiated within 6 and 13 min respectively, indicating a marked species
difference (Ishiwata 1968). Brett (1971) found the satiation time for three different
sizes of the sockeye salmon, Oncorhynchus nerka varied from 33 to 50 min while
feeding on 'Abernathy pellets' (Fowler and Banks 1969) at 15°CT
512 / Rajasekharan Nair and N Balakrishnan Nair
The maximum single intake (satiation amount) for C. thomassi (4-250 ± 0'250g)
ranged from 8-25% to 11*38% of the predator wet body weight. Brett (1971)
found that the amount of food in a full stomach of the sockeye salmon varied
from 3 to 13% among the small fish (3 to 6 g) and from 1 to 5% among the
larger fish (1 50-350 g).
The computed maximum daily intake (daily ration) from the two experiments
and for the different salinities shows a range of 16-50 to 22-76% (for different
prey size) and 13 -51% to 24-14% (for different salinities) of the predator wet
body weight at a water temperature of 27 ± 1°C. In terms of single and daily
maximum intake the smaller prey fish contributed to the maximum values and
vice versa. The daily rations of the Cuban predaceous fish from the family
Serranidae were 2 -41-5 -7% of the body weight during the summer at 28 to 29 ° C
(Reshetnikov and Popova 1975 ; Reshetnikov et al 1975). According to Brett
(1971), the total daily intake decreased from 16*9% of dry body weight for the
4g fish to 4-3% for the 216 g fish (sockeye salmon) when fed on pellets.
It is of interest to note that daily rations are highest in young fish during the
transition to predation, 9 to 40%, an average of 21-9% of the body weight in
new broods of 4 to 5 cms sheat fish, 9*50% in fry 5-7 cms and 7-7% in indivi-
duals 7 to 9 cm long (Popova 1978). The transition to a piscivorous feeding
habit is during the late juvenile and immature adult stages in glassy perchlets and
may be one of the reasons for the high values for daily ration obtained (22 -76%
and 24*14%) in the present study. Also in the extreme instance of a starving
predaceous fish (starving for 2 days prior to feeding during the experiments),
the pattern of feeding may lead to degrees of stomach distension that considerably
exceed that of the maximum capacity exhibited by the daily particulate feeder.
However, these results only give the maximum single and daily intake under labo-
ratory conditions whereas the daily ration and maximum single intake will be
different in the natural waters as the intensity of feeding of predaceous fish and
their daily ration will change with the seasonal changes in ecological conditions,
but it is important that during favourable periods they can attain these or higher
values.
The results of the present study thus show the destructive potential this predator
has in the form of high values of food intake (piscivorous). At the same time
the study also reveals how the size of the prey fish and mode of feeding can be
favourably manipulated in captivity to maximise the daily food intake and
thereby promote growth in the culture of other predaceous fish.
Acknowledgements
One of us (JRN) is thankful to the Council of Scientific and Industrial Research,
Government of India for a Research Fellowship during the tenure of which the
present work was carried out.
Refere»ces
Brett J R 1971 Satiation time, appetite and maximum food intake of sockeye salmon (Oncor-
hynchus nerka) ; /. Fish. Res. Bd. Canada 28 409-415
Teleostean prey size and satiation in Chanda thomassi (Day) 513
Brett J R, Shellbourn J E and Snoop C T 1969 Growth rate and body composition of
fingerlings sockeye salmon, Oncorhynchus nerka, in relation to temperatuie and
ration size ; /. Fish. Res. Bd. Can. 26 2363-2394
Brocksen R W, Davis G E and Warren C E 1968 Competition, food consumption and produc-
tion of sculpins and trouts in laboratory stream communities ; /. WildL Manage. 32 51-75
Fowler L G and Banks J L 1969 Tests of vitamin suppliments and formula changes in
Abernathy salmon diets, 1966-1967 ; U.S. Fish. WildL Serv. Tech. Pap. 26 1-19
Ishiwata N 1968 Ecological studies on the feeding of fishes IV. Satiation curve ; Bull. Jpn.
Soc. Sci. Fish. 34 691-693
Lepkovsky S 1948 The physiological basis of voluntary food intake (appetite) ; Advan. Food
Res. 1 105-148
Paintal A S 1954 A study of gastric stretch receptors. Their role in the peripheral mechanism
of satiation of hunger and thirst ; /. Physiol 126 271-285
Popova O A 1978 The role of predaceous fish in ecosystems ; in Ecology of freshwater Fish
Production (ed.) S D Gerking (Oxford : Blackwell Scientific Publications) pp. 215-249
Reshetnikov Yu S and Popova O A 1975 The daily rations and rate of food digestion in
tropical fish ; Sbornik ' Biologiya Shelfa ' , Vladyvostok, 144-145
Reshetnikov Yu S, Sylva A, Claro R and Popova O A 1975 Rates of food digestion in tropical
fishes ; Zool. Zh. 54 1506-1514
Rozin P and Mayer J 1961 Regulation of food intake in the gold fish ; Am. J. Physiol. 201
968-974
Swenson W A and Smith L L Jr 1973 Gastric digestion, food consumption, feeding periodicity
and food conversion efficiency in Walleye (Stizostedion vitreiim vitreum) ; /. Fish. Res. Bd.
Canada 3Q 1327-1336
Warren C E, Wales J H, Davis C E and Doudoroff P 1964 Trout production in an experi-
mental stream enriched with sucrose ; /. WildL Manage. 28 617-660
Windell J T 1966 Rates of digestion in the blue gill sunfish ; Invest. Indiana Lakes and Streams
7 185-214
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 6, November 1982, pp. 515-521.
© Printed in India.
Studies on some Tetmcotyle Fillipi (1859) inetacercariae from
fishes of Lucknow
NIRUPAMA AGRAWAL and SHAKILA KHAN
Zoology Department, Lucknow University, Lucknow 226007, India
MS received 16 November 1981 ; revised 10 August 1982
Abstract. Three unknown Tetmcotyle metacercariae, collected from piscine host,
have been described. Tetmcotyle pandei n.sp., Tetmcotyle srivastavai n.sp. and
Tetmcotyle ramalingi n.sp. were collected from the visceral organs and musculature
of Channel punctatus (BL). They are characterised by the shape and position of
pseudasuckers, shape of hold fast organ and hold fast gland, number and posi-
tion of genital rudiment and pattern of reserve excretory system.
Keywords. Tetmcotyle pandei n.sp.,; Tetmcotyle srivastavai n.sp.; Tetmcotyle
ramalingi n.sp. ; metacercariae ; Channa punctatus (BL).
Tetracotyle pandei* n. sp
Host : Channa punctatus (BL)
Location : mesenteries and liver of infected host
Locality : Lucknow
Number of host examined : 55
Number of host found infected : 3
Measurements in mm. Cyst— 1 -22-1 '24 x 0 -75—0 -16, outer layer— 1 -22-1 *24 x
0*75-0-76, middle layer— 0 • 66-0 • 69 and inner layer— 0' 44-0 '46 x 0-24-0-26.
Body— 1-80-1-82 X 1-32-1-33 (live) and 0-52-0-57 X 0-40-0-50 (fixed). Oral
sucker— 0-13-0-14 (live) and 0-67-0-08 (fixed). Ventral sucker 0-19-0-20 (live)
and 0-09-0-10 (fixed). Pharynx— 0-04-0-07 (live) and 0-02-0-03 (fixed).
Pseudosucker~-0 -15-0* 18 x 0-13-0-14 (fixed). Hold fast organ— 0-08-0-09 X
0-05-0-06 (fixed).
Oval cyst (figure 1) three layered. Outer layer thick, fibrous, tough and pig-
mented, middle and inner layers thin. Body (figure 2) aspinose, with broad
anterior and narrow posterior ends. Ventral sucker equatorial, larger. Large
triangular pseudosucker one pair, posterior to oral sucker. Host fast gland tri-
angular, deeply stained cell mass. Mouth terminal. Pharynx round and muscular.
* The species has been named in honour of Late Prof. B P Pande.
515
516
Nirupama Agrawal and Shakila Khan
Figures 1-3. Tetmcotyle pandei n.sp. 1. Encysted metacercaria (drawn from a
live specimen). 2. Metacercaria (drawn from a fixed specimen). 3. Metacer
caria showing reserve excretory system (drawn from a live specimen).
Oesophagus and intestinal caeca not visible. Genital rudiments two, anterior
rudiment at the posterior border of hold fast organ, posterior rudiment in the
posterior body region.
The excretory system (figure 3) of secondary reserve excretory system and a
primary system of flame cells. " V " shaped excretory bladder at posterior end
with terminal excretory pore giving rise to thrge pairs of canals, outer, middle and
inner longitudinal canals. Each inner and outer longitudinal canals joined in the
region of pseudosuckers, forming an isthmus of small canals. Median longitudinal
canal, running up to the region of ventral sucker. Seven transverse canaliculae to
inner longitudinal canal and eight bifurcated transverse canaliculae to outer
longitudinal canal. Whole reserve excretory system filled with freely moving,
round excretory corpuscles. Primary system of flame cells not observed.
Discussion
The present form chiefly differs from the other species in having three layered cyst
aad the pattern of reserve excretory system. It can be further differentiated from
Studies on Tetracotyle Fillipi 517
T. ranae (Kaw 1950) in having a cyst, from T. xenentodoni (Chakrabarti 1970b)
and T. muscularis (Chakrabarti 1970a) in the ratio of suckers, from T. sophoriensis
(Singh 1956), T. glossogobi (Chakrabarti 1970c) and T. tandoni (Pandey 1973) in
having an undivided body, from T. indicus (Singh 1956) by the number of genital
rudiments, from T. baughi (Pandey 1973), r. lymnaei (Pandey and Agrawal 1978),
T. lucknowensis, (Pandey 1971b) T. lali (Pandey 1971a), and T. szidati (Chakrabarti
and Baugh 1970) in the position of pseudosuckers and shape of hold fast organ
and T. bufoi (Agrawal 1975) in having a well-developed ventral sucker.
It, however, closely resembles with Tetracotyle of Apatemon pellucidus Yamaguti
1933 and Tetracotyle of Apatemon fuligulae Yamaguti 1933. It can be distinguished
from Tetracotyle of A. pellucidus by the number of genital rudiments and from
Tetracotyle of A. fuligulae in having two masses of genital rudiments and in the
absence of prepharynx. It differs from T. communis and 71. diminuta Hughes,
1928 in the pattern of excretory system and number of genital rudiments.
Tetracotyle srivastavai n. sp.
Host : Channa punctatus (Bl.)
Location : mesenteries
Locality : Lucknow
Number of hosts examined: 55
Number of hosts found infected: 2
Measurements in mm. Cyst— 3 -18-3 '19 x 1-57-1-59 (live) and 1*68-1-70 x
0-84-0-85 (fixed). Forebody— 2-01-2-03 x 1 -37-1-39 (live) and 1 -34-1 -36 x
1-12-1-14 (fixed). Hindbody— 0-44-0-45 x 0-77-0 -79 (live) and 0-34-0-36 x
0 • 55-0 • 57 (fixed). Oral sucker— 0 • 18-0 • 19 (live) and 0 -08-0 -09 (fixed). Ventral
sucker— 0-25-0-27 (live) and 0-16-0-18 (fixed). Pseudosuckers— 0-44-0-45 x
0-14-0-16 (live) and 0-23-0-24 x 0-13-0-14 (fixed). Pharynx— 0- 09-0 -10 x
0-07-0-08 (live) and 0-05-0*06 X 0-035-0-04 (fixed). Oesophagus— 0-57-0-58
(live) and 0-30-0-31 (fixed). Hold fast organ— 0*45-0*47 x 0-38-0-40 (live)
and 0-30-0-32x0-29-0-31 (fixed).
Oval cyst (figure 4) thin, transparent, single layered, with colourless fluid.
Aspinose body (figure 5) divided into large fore and small hind body. Ventral
sucker larger, equatorial. Pseudosuckers muscular, oval, in oesophageal region.
Mouth terminal. Pharynx oval and muscular. Intestinal caeca up to the hold
fast organ. Hold fast organ elongated, multilobed, with prominent cavity. Hold
fast gland "U "-shaped, posterior to hold fast organ. Small mass of genital
rudiment in posterior body region.
Small excretory bladder (figure 6) " V "-shaped, at hind end of body. Two
main longitudinal canals, from excretory bladder run anteriorly up to oral sucker.
Two transverse canals, anterior and posterior, joined by three lateral longitudinal
and one median longitudinal canal. Inner lateral longitudinal canals of two sides,
joined together in ventral sucker region by a short, median transverse canal. Main
longitudinal canal and three lateral longitudinal canals of each side, joined
together by 10-14 short transverse canaliculae.
P.(B)-4
518
Nirupama Agrawal and Shakila Khan
Figures 4-6. Tetracotyle srivastavai n.sp. 4. Encysted metacercaxia (drawn from
a live specimen). 5, Metacercaria (drawn from a fixed specimen). 6. Metacer
caria, showing reserve excretory system (drawn from a live specimen). £ :L ^t
Studies on Tetracotyle Fillipl 519
Discussion
The present larva shows close resemblance with T. ranae Kaw 1950 ; T. ujjainensis
Trivedi 1964 ; r. muscularis Chakrabarti 1970a ; T. baughi and T. tandoni
Pandey 1973 and T. lymnaei Pandey and Agrawal 1978 in having a divided body.
However, it differs from T. ranae and T. ujjainensis in the ratio of suckers, from
T. muscularis in the ratio of suckers and genital rudiment, from T. baughi and
T. tandoni in the number of genital rudiment and lobed hold fast organ and from
T. lymnaei in the number of genital rudiment. It also differs from all the above
species in having different pattern of reserve excretory system.
This form shows resemblance also with T. communis Hughes 1928 ; Tetracotyle
of A. pellucidus Yamaguti 1933 and Tetracotyle of A. fuligulae Yamaguti 1933.
However, it differs from T. communis in having lobed hold fast organ, from
Tetracotyle of A. pellucidus in the number of genital rudiment and from Tetracotyle
of A. fuligulae in having lobed hold fast organ and in the absence of a prepharynx.
Tetracotyle ramalingi n. sp.
Host : Channa punctatus (BL)
Location : muscle fibres of infected host
Locality : Lucknow
Number of hosts examined : 55
Number of hosts found infected : 2
Measurements in mm. Cyst— 0-74-0- 75 X 0 '56-0 -57 (live) and 0-40-0-43 x
0-37-0-38 (fixed). Forebody— 0-70-0-72 X 0-60-0-62 (live) and 0-50-0-52 X
0-30-0-33 (fixed). Hindbody— 0-30-0-32 X 0-58-0-60 (live) and 0-20-0-22 x
0-24-0-26 (fixed). Oral sucker— 0-06-0-07 (live) and 0-04-0-05 (fixed). Ventral
sucker— 0-04-0-05 (live) and 0*025-0-03 (fixed). Pseudosuckers— 0-08-0*09 X
Q'05-0-055 (live) and 0 '06-0 -07 X 0 '03-0 -04 (fixed). Pharynx— 0-04-0-05 x
0.03-0-04 (live) and 0-03-0-04 X 0-03-0-035 (fixed). Oesophagus— 0-05-0 -06
(live) and 0-03-0-04 (fixed). Hold fast organ— 0-08-0-09 x 0-07-0-075 (fixed).
Cyst (figure 7) oval, thick and double layered. Outer layer thicker. Body
(figure 8) oval, spinose and divided. Forebody larger. Oral sucker round,
terminal and larger. Ventral sucker equatorial in forebody. Pseudosuckers
lateral, muscular and kidney-shaped. Mouth leading to oval, muscular pharynx.
Intestinal caeca up to posterior body region. Hold fast organ round to oval
posterior to ventral sucker. Bilobed hold fast gland, close to hold fast organ.
Single mass of genital rudiment in posterior hind body region.
Four longitudinal excretory canals (figure 9) from cornu of "V "-shaped
excretory bladder, running anteriorly up to pharynx, joined anteriorly by anterior
transverse canal, and posteriorly, by posterior transverse canal. Median longi-
tudinal canal descending from anterior transverse canal up to posterior transverse
canal. Further, longitudinal canals joined together by 5-8 transverse canaliculae.
520
Nirupama Agrawal and Shakila Khan
Figures 7-9. Tetracotyle mmalingi n.sp. 7- Encysted metacercaria (drawn from
a live specimen). 8. Metacercaria (drawn from a fixed specimen). 9. Metacer-
caria, showing reserve excretory system (drawn from a live specimen).
Discussion
The present form shows resemblance with T. ranae Kaw, 1950 ; T. ujjainensis
Trivedi 1964 ; T. muscularis Chakrabarti 1970a; T. baughi Pandey 1973 ;
T. tandoni Pandey 1973 ; and T. lymnaei Pandey and Agrawal 1978 in having a
divided body. It differs from T. ranae in presence of oesophagus from T. ujjai-
nensis and T. lymnaei in the number of genital rudiment from T. tandoni and
T. baughi in the relative size of suckers and number of genital rudiments and
from T. muscularis in having a bilobed hold fast gland and poorly developed
genital rudiments.
It resembles also with Tetracotyle of A. pelluddus Yamaguti, 1933 and
Tetracotyle of A. fuligulae Yamaguti 1933 in having a divided body. However,
it differs from Tetracotyle of A. pelluddus in the ratio of suckers and number of
genital rudiments and from Tetracotyle of A. fuligulae in the ratio of suckers and
absence of prepharyax.
Studies on Tetmcotyle Fillipi 521
Acknowledgements
The authors are thankful to Prof. B K landau, former Head and Prof. B Dev,
Head, Zoology Department, Lucknow University, Lucknow, for the laboratory
facilities and to Prof. K C Pandey for helpful suggestions. They extend their
thanks to the authorities of SCST for the grant of a research scheme, under which
the work has been carried.
References
Agrawal N 1975 A new strigeid larva (Tetracotyle bufoi n.sp.) from a common toad ; Indian
J. Zoot. 16 187-188
Chakrabarti K K 1970a A double metacercarial infection in an Indian freshwater fish Hetero-
pneustes fossilis (Bl.) ; Rev. BioL Trop. 17 91-96
Chakrabarti K K 1970b Two new species of strigeid metacercaria from an Indian freshwater
fish , Xenentodon cancila (Ham.) ; Proc. Helm. Soc. Wash. 37 5-10
Chakrabarti K K 1970c A new strigeid metacercaria, Tetracotyle glossogobi sp.n., from an
Indian freshwater fish Glossogobius giuris (Ham.) ; Helminthologia 11 1-4
Chakrabarti K K and Baugh S C 1970 Tetracotyle szidaii n.sp. a metacercaria from the Indian
freshwater fish Channa punctaius (Bl.) ; Indian J. Zoot. 11 79-82
Hughes R C 1928 Studies on the trematode family Strigeidae (Holostomidae) No. XIII. Three
new species of Tetracotyle', Trans. Am. Micr. Soc. 47 414-433
Kaw B L 1950 Studies in helminthology. Helminth parasites of Kashmir. Part I. Trematode ;
Indian J. Helminth. 2 67-126
Pandey K C 1971 a Studies on the trematode parasites of fishes of Lucknow (India)-I ; Proc.
Nat. Acad. Sci. India 41 311-314
Pandey K C 1971b Studies on metacercariae of freshwater fishes of India. VII. On morphology
of Tetracotyle lucknowensis n.sp. from Channa striatus Bloch. ; Proc. Indian Acad. Sci.
(Anim. Sci.) 74 1-5
Pandey K C 1973 Studies on metacercariae of freshwater fishes of India. II. Description
of a known and five unknown strigeid metacercariae from Lucknow ; Indian J. Zoot,
14 155-166
Pandey K C and Agrawal N 1978 A new Tetracotyle larva, T. lymnaei n.sp. from a freshwater
mollusc Lymnaea auricularia ; Indian J. Parasit. 2 199-210
Singh K S 1956 On some strigeid from India ; J. Zool. Soc. India 8 47-56
Trivedi H S 1964 On two metacercariae from freshwater fishes in Ujjain ; /. Vk. Univ. 3 91-94
Yamaguti S 1933 Studies on the helminth fauna in Japan. Pt. I. Trematoda of birds, reptiles
and mammals ; Jpn. J. Zool. 5 1-134
Proc. Indian Acad. Sci. (Anini. Sci.), Vol. 91, Number 6, November 1982, pp. 523-532.
© Printed in India.
Toxic and sublethal effects of endosulfan tm.Barbus stigma
(Pisces : Cyprinidae)*
T MANOHARAN and G N SUBBIAH
Zoological Research Laboratory, Thiagarajar College, Madurai 625009, India
MS received 4 December 1981 ; revised 12 July 19S2
Abstract. Toxicity and the effect of sub-lethal concentrations of endosulfan on a
fresh water fish Barbus stigma had been studied. Endosulfan proved to be lethal
to JB. stigma at a concentration of 0*01 ppm and above. The LC50 was 0*0043 ppm
and the sub-lethal concentration was 0*1)03 ppm and below. At sub-lethal concen-
trations the fish exhibited erratic swimming activity and at lethal concentrations
it lost the sense of balance. The rate of feeding was reduced by 5-94% to 9*02%
and assimilation by 6-44 to 9 -60% in different sub-lethal concentrations. Growth
(weight) retarded from H'*6mg/day in the control fish to 7*3, 6-0 and 5-1 mg/
day in the endosulfan treated fishes. Respiratory rate of the pesticide treated fish
also dropped by 10 to 16*6%. Due to the over all effect of the toxicity, the fish.
B. stigma comparatively showed a poor nutritive value by displaying a drop of
nearly 35% in the organic constituents.
Keywords. Endosulfan ; pesticide ; lethal concentration ; sublethal concentrations
1. Introduction
Although pesticides produce good many results in the control of pests, their harm-
ful effects on the non-target animals are also not ruled out. Pesticides leave
residues in water and mud even several days after their spray in the adjacent
crop fields. This poses a constant threat to the non target organisms, especially
to the fishes, because pesticides are known to alter their behaviour pattern (Ander-
son 1971), growth and nutritional value (Korschgen and Murphy 1967; Aruna-
chalam et al 1980 ; Yaganobano et al 1981), reproductive potential (Johnson 1967),
cellular morphology (Mukhopadhyay and Dehadrai 1980) and Physiology (Baskaran
1980 ; Natarajan 1981). Though a good number of literatures are available
on the toxicity of pesticides in fishes, studies on the sublethal concentrations of
toxicants are meagre. The objective of the present study is to find out the effect
of sublethal concentrations of endosulfan (the most effective and widely used
pesticide in the field) on survival, behaviour, energy budget, respiratory pattern
and the organic constituents of B. stigma.
* A part of the M.Phil, dissertation submitted by T. Manoharan to the Madurai Kamaraj
University.
524 T Manoharan and G N Subbiah
2, Materials and methods
The fish B. stigma, used in the present experiment, is edible, commercially valu-
able, and distributed all over India. The fishes were obtained from the Public
Works Department and stocked in glass aquaria, after dipping in a 3 -5% salt
solution to prevent any parasitic attack. They were acclimatized to the laboratory
condition for ten days and were fed on Oligochaete worm Tubifex tubifex. Prelimi-
nary tests were conducted in five aquaria containing five individuals each, to find
out the toxicity range of the toxicant. The mortality range was assessed by using
five arbitrarily chosen concentrations of endosulfan. For pesticide dilutions the
static bioassay method (APHA 1971) was employed. After determining the mortality
range (100% mortality) of the pesticide, desired concentrations down to the sublethal
dose were prepared by diluting 35% EC endosulfan.
To find out the LCSO of 1-0 g unit weight of B. stigma, mortality rate was
observed for 96 hrs at different arbitrarily chosen concentrations of endosulfan.
At a concentration of 0*0043 ppm 50% of the fishes died at the end of 96 hrs.
At 0 • 003 ppm and below, all fishes survived over a period of 30 days. Thus, 0 * 0043
ppm and 0*003 ppm of endosulfan were taken as LCSQ and sublethal concen-
trations respectively. Following the method of Sprague (1973), the LC50 curve was
drawn and mortality rate and concentration were expressed in probit and log
values.
2.1. Experiments in sublethal concentrations
Experiments were conducted in three different concentrations (0-003, 0-002 and
0-001 ppm) of endosulfan. In each concentration three replicates and one control
(without insecticide) were used simultaneously and the experiment was carried out
for 20 days at 28 ± 1° C. The fishes were fed on freshworms of T. tubifex ad
libitum for 3 hrs/ day. The unfed food was collected carefully by a pipette and the
faeces by filtering the water daily. Water was changed once in a day. Both
the left over food and the excreta were dried to constant weight at 90°C. Water
content of the fish and the worms were determined by using the sacrifice method
(Maynard and Loosli 1962). The scheme of energy balance was expressed
by IBP formula (Petrusewicz and MacFadyen 1970), i.e.9
C=P+ R+ F + U
Where C = Food consumed ; P = Production (i.e. difference between the initial
dry weight and the final dry weight) ; R = Respiration ; F= Faeces and
U = Nitrogenous excretory products.
Assimilation was estimated by substracting "F" from "C". Assimilation
efficiency was calculated as the percentage of food assimilated in relation to food
consumed, gross (JKj.) and net (K2) conversion efficiencies were represented as
percentage of food converted in relation to food consumed and assimilated res-
pectively. Rates of feeding, assimilation and production were calculated to the
respective quantities of food consumed, assimilated and converted relating to per
unit live weight (g) of the fish per unit time (day).
2. la. Statistical analysis : Different sublethal concentrations of endosulfan were
correlated with rates of feeding, assimilation and conversion.
Effects of endosulfan on Barbus stigma 525
2.1b. Specific growth rate: Specific growth rate (mg/day) was calculated using the
method adopted by Kosi Onodera (1962).
2.2. Respiratory studies
Control and experimental fishes were introduced into separate troughs containing
two litres of water. A thin layer of Kerosene was layered carefully on the surface
of the water to avoid the diffusion of atmospheric oxygen. After 30 min, 200 ml
of water was siphoned out from each of the troughs and the oxygen content was
estimated (Winkler 1948). The oxygen consumed by the experimental and control
fishes were calculated by subtracting the value from the initial oxygen content
of water.
2.3. Bio-chemical analysis
At the end of experiment (after 20 days), 5 mg of dried powder of total homogenate
of control and experimental fishes were used and the total protein (Lowry et al
1951), the total lipid (Bragdon 1951) and the total sugar (Seifter et al 1950) were
colorimetrically estimated.
3. Results
Endosulfan caused 100% mortality within 24 hrs of exposure at a concentration
of 0-01 ppm (lethal). The LC50 (figure 1) was 0-0043 ppm during the 96 hrs of
exposure. At the concentration of 0-003 ppm and below no mortality was
observed (sublethal).
3.1. Behaviour
There was a marked increase in the swimming activity of the fishes immediately
after they were transferred to lethal and sublethal concentrations.
3.2. Feeding rate (table 1)
Sublethal concentrations of endosulfan affected almost equally all the intermediary
processes connected to food utilization (figure 2). The average feeding rate of
test fish reared in fresh water (control) was 18*84mg dryfood/g live fish/day. This
value decreased to 17-72 (5-9%), 17-52 (7%) and 17-14 (9-02%) mg dry food/g
live fish/day, when they were reared in 0-001, 0-002 and 0-003 ppm concentration
of endosulfan respectively.
3.3. Assimilation rate (table 1)
Assimilation rate also decreased from 17'07mg dry food/g live fish/day (control)
to 15-97 (6-44%), 15-89 (6*91%) and 15*43 (9-60%) in the experimental fishes
(figure 3).
3.4. Production rate (table 1)
Fish growth was found to have retarded with increased concentrations of endo-
sulfan in the medium (figure 4). The average production rate of B. stigma was
P.(B)-5
526
T Manoharan and G N Subbiah
Table 1. Effects of different sublethal concentrations of Endosulfan on food
utilization and efficiencies in Barbus stigma.
Parameters —
Concentration
o-ooo
o-ooi
PPm
0
•002 ppm
0-003
ppm
Feeding rate (Cr)
18-
84±0-98
17-
72±1-
10
17-
52dbO'
33
17
•14±0
•85
Assimilation rate (Ar)
17
•07±0'65
15-
97 ±0-
•98
15-
89±0-
99
15
•43±0
•54
Production rate (Pr)
2-
47±0'17
1-
41 ±0-
40
1-
23 ±0-
33
1
•14±0
•50
Assimilation efficiency
90
•61 ±0-74
90
•07 ±0
•39
90
•65±0
•51
89
•76±0
-31
Gross conversion efficiency
13
•09 ±0-96
7
•97±0
•35
7
•07±0
•89
5
•88±0
•85
Net conversion efficiency
14
•54±0-95
8
•81 ±0
•33
7
•80dzO
•82
7
•37±0
•49
Each value represents the average performance of three (mean ± SD) individuals observed
for 20 days at 28° C ± 1° C. Rates are expressed in mg dry weight/g live fish/day and the
efficiencies are expressed in percentage.
2*47 mg dry substance/g live fish/day. The growth rate dropped to 1 -41 (42-90%)
in 0-001 ppm, 1-23 (50-20%) in 0-002ppm and 1-14 (53-84%) in 0-003 ppm
concentration of endosulfan.
Correlation coefficient values obtained between different sublethal concentrations
of endosulfan and feeding (r = — 0-95), assimilation (r — — 0-93) and conversion
(r = — 0*&8) rates were negatively correlated and the values were significant at
0-1% level.
90
JO
o
a.
o
10
0-001
OJ01
Figure 1. Dotted line indicates the LC^ value at 96 hr exposure
Effects of endosulfan on Barbus stigma
527
20
19
2 18
r 17
u
16
4 8 12
N o. of Days
Q.OOO
1,003
Figure 2. Effects of different sublethal concentrations of Endosulfan on the
feeding rate of B. stigma.
18
17
* 16
6 15
V.
<
0.000
0.003
4 8 12
No. of Days
16
20
Figures. Effects of different sublethal concentrations of Endosulfan on the
assimilation rate of B. stigma.
3.5. Efficiencies of Assimilation, gross (Ki) and net (K^) conversion (table 1)
Endosulfan did not affect assimilation efficiency which averaged as 90%. The
gross conversion efficiency (K:) of the fish reared in fresh water was 13-09%, while
the value was decreased in different sublethal concentrations (7-97%, 7*07%
and 5*88% in 0-001, 0-002 and 0-003 ppm respectively). Net conversion effici-
ency (JSy of the control fish was 14*54%, while the value was reduced to 8*81%
in 0-001 ppm, 7-89% in 0-002 ppm and 7-39% in 0*003 ppm (figure 5).
3 . 5a. Specific growth rate : Specific growth rate of the control fish was 1 1 • 60 mg/
day. This value decreased to 7-3, 6*0 and 5-lmg/day in 0-001, 0*002 and 0*003
ppm of endosulfan media respectively (figure 6).
528
T Manoharan and G N Subbiah
1 *
I
0.000
8 12
No. of Days
18
Figure 4. Effects of different sublethal concentrations of Endosulfan on the
production rate of B. stigma.
12
OOOO 0.001 0.002 0.003
Conc,(ppm)
Figure 5. Effects of different sublethal concentrations of Endosulfan on the
gross (Ki) and net (JQ conversion efficiencies of B. stigma.
3.6. Respiration
The respiratory rate of the fish reared in pesticide-free water (control) was 1-08
mlo2/g live fish/hr. In 0*001 ppm of endosulfan, the fish maintained more or less
identical value. However, when the concentration was increased to 0*002 and
0-003 ppm the respiratory rate declined by 10-8% and 16-6%.
3.7. Organic constituents
The average values of protein, lipid and sugar content in the control fish were
280, 118-30 and 50*50 mg/g dry weight of the fish. The experimental fishes
Effects of endosulfan on Barbus stigma
529
12
10
« s
Si
4- tfl
1 =
o
o cr»
Qj £•
Q. *-*
CO
0.000 0.001 0.002
Cone. (
0.003
Figure 6. Effects of different sublethal concentrations of Endosulfan on the specific
growth rate of B. stigma.
exhibited a significant drop in their organic constituents. Protein content was
found to have dropped to 233,221 and 180mg/g in 0-001, 0-002 and 0-003 ppm
endosulfan treated fishes. The value of lipid also declined from 118-30 to 97*56,
91*40 and 77-70mg/g. Similarly sugar content of the experimental fishes also
reduced from 50-50 to 45-00 in 0-001 ppm, 43-50 in 0-002 ppm and 40-00 in
0-003 ppm concentration.
4. Discussion
e
4.1 LCgo and sublethal concentration
The pesticide endosulfan has produced lethal effect at 0*01 ppm in B. stigma.
The LC50 is 0*0043 ppm, at which 50% of fishes died within 96 hrs of exposure.
Fishes survived at 0-003 ppm and below, indicating that 0-003 ppm is the sublethal
level. In their toxicity studies, using endosulfan, Amminikutty and Rege (1971)
observed that 0-00 16 ppm is the LCSO for the fish Gymnocorymbus ternetzi. The
data obtained in the present work indicates that different fishes have different
tolerance range against the toxic effects of the same pesticide. In this way B.
stigma seems to be more tolerant (about three times) to eridpsulfan than Q, ternetzi.
530 T Manoharan and G N Subbiah
4. la. Behaviour : At sublethal concentrations the fish becomes restless, exhi-
biting erratic swimming activity and at lethal doses it loses its balance. A similar
observation has been made in Ictalurus punctatus and Channa punctatus by Carter
(1971) and Munawar Ahmed Anees (1975) respectively. Desi et al (1974) and
Kingsley (1973), from their neurotoxicological studies, concluded that cholin-
esterase activity of various parts of nervous system is affected by the pesticide
leading to the imbalance of the animal. In the present case also the disturbed
swimming activity of the fish may be explained in the same line.
4.2. Rates of feeding, assimilation and production
Rate of feeding, assimilation and growth declined in B. stigma with increased
concentrations of endosulfan in the medium. This observation falls in line with
the findings made by Arunachalam et al (1980) and Baskaran (1980), in Mystus
vittatus and Channa striatus. The decrease in growth and conversion efficiency
may be due to the diversion of more energy for the stress put up against the
toxic effect of the pesticide, as suggested by Arunachalam et al (1980).
4.3. Respiration
Respiratory rate decreases up to 16*7% in endosulfan treated fishes. This result
is in conformation with the earlier reports by Baskaran (1980) in C. striatus using
DDT and methyl parathion and by Carolyn et al (1976) using Carbaryl and Dieldrin
on Rainbow trout. Blood smear studies made in the present experiments have
revealed the severe damage caused to the red blood corpuscles in the endosulfan
treated fishes. A reduction in the RBC count in C. striatus following exposure
to Metasystox (Demeton) has been reported by Natarajan (1981). Therefore,
it is presumed that due to the injury caused to the red blood corpuscles by the
pesticides, the efficiency of the fish to trap the- dissolved oxygen is considerably
reduced resulting in the reduced rate of respiration.
4.4. Nutritive value of the fish
Finally the data obtained in the present experiments have shown that the nutritive
value of the fish treated with pesticide is significantly reduced (16 to 35% drop
in protein, 17 to 34% in lipid and 9 to 20% drop in sugar level). A similar pheno-
menon has been demonstrated byglamana Rao and Ramamurthi (1980) and Kabeer
Ahmed et al (1978) in Sumithion treated Pila globosa and Malathion treated
Lamellidens marginalis. Although the above references are from invertebrates,
they prompt one to think that such a phenomenon (reduced level of organic consti-
tuents) is common to all animals irrespective of species.
5. Conclusion
From this investigation it is obvious that the toxic nature of pesticides produces
lethal effect in fishes at higher doses. JSveq. in s^bl^tkal poncentjrations, it r
Effects of endosulfan on Barbus stigma 53 1
in degraded metabolic changes, affecting the nutritive value of the animal. Therefore
it may be suggested that necessary care may be taken to avoid contamination of
fresh water bodies while spraying pesticides.
Acknowledgement
Facilities provided for this work by the Management, Principal and the Professors
are gratefully acknowledged.
References
Amminikutty C K and Rege M S 1977 Effects of acute and chronic exposure to pesticides,
Thiodan BC 35 and Agallol ' 3 ' on the liver of Widowtetra Cymnocorymbus ternetzi ;
Indian J. Expl BioL 15 197-200
Anderson J M 1971 Sublethal effects and changes in ecosystems. Assessment of the pollu-
tants o.n physiology and behaviour ; Proc. R.Soc. London 177 307-320
APHA 1971 American Public Health Association New York 1971 In Standard methods for the
examination of water and sewage 13th edition.
Arunachalam S, Jeyalakshmi K and Aboobucker S 1980 Toxic and sublethal effects of Carbaryl
on a fresh water catfish Mystus vittatus ; Arch. Environ. Contam. Toxicol. 9 307-316
Baskaran R 1980 Biological studies on a chosen thermo conformer (Channa striatus) ; Ph.D.
thesis submitted to Madurai Kamaraj University, Madurai
Bragdo-n J H 1951 Colorimetric determination of blood lipids ; /. BioL Chem. 190 513
Carolyn R Lunn, Daniel P Toews and David J Pree 1976 Effects of three pesticides on
respiration, coughing and heart rates of Rainbow trout ; Can. J. ZooL 54 214-219
Carter F L 1971 In vitro studies of brain cholinesterase inhibition by oxgano phosphate and
carbamate insecticides in fish ; Ph.D. thesis, University of Louisiana, Baton rouge,
Louisiana (Diss. Abstr. Int.) 13 2772-73
Desi Ilees, Lili Genczi Gyorgy Simon, Ildiko Farkas and Zsuzsa Kneffel 1974 Neurotoxicological
studies of two carbomate pesticides in sub-acute animal experiments ; Toxicol. AppL
PharmacoL 27 465
* Johnson H E 1967 The effects of Endrin in the reproduction of a freshwater fish, Cryzias
latipes ; Ph.D. thesis, Univ. Washington, Seattle, Wash.
Kabeer Ahamed I, Begum Md R, Sivaiah S and Ramana Rao K V 1978 Effect of Malathion
on free amino acids, total proteins, glycogen and some enzymes of Pelecypod Lamellidens
marginalis ; Proc. Indian Acad. Sci. (Anim. Sci.) 87 377-380
Kingsley Kay 1973 Toxicity of pesticides ; Recent advances, Environ. Res. 6 202
Korschgen L J and Murphy D A 1967 Missouri federal aid project progress report
Kosi Onodera 1962 Some data on Eel culture in Japan ; Indo-Pac. Fish. Counc.9 Occasional
Pap. 6216 4
Lowry O H, Rosebrough N J, Farr A L and Randal R J 1951 Protein measurement with
Folin phenol reagent ; /. BioL Chem. 193 265-275
Maynard A L and Loosli K J 1962 Animal nutrition 5th ed. (New York : McGraw-Hill)
Mukhopadhyay R K and Dehadrai P V 1980 Studies on air breathing catfish Clarias batrachus
under Malathion sublethal exposure ; Indian J. Exp. BioL 18 348-352
Munawar Ahmed Anees 1975 Acute toxicity of our organo phosphorous insecticides to a fresh
water teleost Channa punctatus ; Pakistan J. ZooL 7 135
Natarajan G M 1981 Changes in the bimodel gas exchange and some blood parameters in
the air breathing fish, Channa striatus following lethal (LC50y48hrs) exposure to Metasystox
(Demeton) ; Current Sci. 50 40-41
Petrusewicz and MacFadyen A 1970 Productivity of terrestrial animals ; IBP handbook No, 13
(Oxford : Blackwell)
Ramanarao M V and Ramamurthi R 1980 Effect of sublethal concentration of sumithion on
some biochemical constituents of the freshwater snail Pila globosa Geobios 7 247-250
532 f Manoharm and G N Subbiah
Seifter S and Dayton S 1950 The estimation of glycogen with anthrone reagent ; Arch. Biochem.
Biophys. 25 191-200
Sprague J B 1973 The ABC's of pollutant Bioassay using Fish ; American Society for Testing
and Materials 6-30
Winkler 1948 In Welch P S Limnological methods (New York : McGraw-Hill)
Yaganobano, Seikh Amjad Ali and Tariq Hameed 1981 Effects of sublethal concentration of
DDT on muscle constituents of an air breathing catfish , Clarias batrachus ; Proc. Indian
Acad. ScL(Anim.ScL), 90 33-37
* Originals not referred.
Froc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 6, November 1982, pp- 533-538.
© Printed in India.
Interruption of pregnancy by barbiturates in albino rats
SARASWATI B PATIL and M APPASWAMY RAO*
Department of Zoology, Gulbarga University, Gulbarga 585 106, Karnataka, India
* Retired Professor of Zoology, 5th Main, Yadavagiri, Mysore 570020, India
MS received 18 May 1982 ; revised 7 August 1982
Abstract. Barbiturates inhibit the LH surge and release of ganadotrophins (UH
and FSH) and prolactin from the pituitary in rats and hamsters. In the present
study administration of phenobarbital (7'5mg) or barbital sodium (20 mg) twice
a day from day 8-11 interrupts the pregnancy in rats with little or no foetal
survival. The. ovaries and the uterus of these rats resemble those of non-pregnant
rats when autopsied on day 20 of pregnancy. These results suggest that the
failure of maintenance of pregnancy by barbiturate treatment may be due to the
inhibition of luteotrophic hormones from the pituitary during the crucial period
of pregnancy, resulting in the insufficient secretion and release of ovarian proges-
terone and also estrogen.
Keywords. Gonadotrophins ; pituitary ; phenobarbital ; barbital sodium ; luteo
trophins ; corpora lute a ; foetuses.
1. Introduction
Maintenance of pregnancy is the consorted efforts of all endocrine glands mediated
through the hypothalamo-hypophyseal-ovarian and placental axes. Hypophysis
is indispensable during first half of pregnancy, but the ovaries are essential through-
out the gestation, as the placental gonadotrophins take over the functions of the
pituitary gonadotrophins during later part of pregnancy in rats (Lyons and Ahmad
1973 ; Rothchild et al 1974). Maintenance of pregnancy by exogenous LH or
other luteotrophins in hypophysectomized rats, and by proper doses of progesterone
and estrogens in ovariectomized rats and hamsters is achieved by several investigators:
(Jagannadha Rao et al 1972 ; Yoshinaga et al 1972),
Studies with pheno- and pentobarbital indicate that these drugs inhibit the
pituitary LH surge and tonic release of FSH, LH and prolactin, and also interfere with
the ovarian steroidogenesis directly (Gupta and Karavolas 1973 ; Norman et al
1973 ; Blake 1974). As barbiturates interfere with much of the endocrine activities
of pituitary and ovary that are essential for the maintenance of pregnancy, the
aim of the present investigation was to study the effects of phenobarbital and
barbital sodium on pregnancy in rats.
533
P.(B)-6
534 Saraswati B Patil and M Appaswamy Rao
2. Material and methods
Normal cycling, nulliparous rats of Holtzman strain, weighing 140-1 50 g, 80-90
days old were mated with fertile males at proestrus or early estrus. The rats showing
spermatozoa in the vaginal smears on the subsequent day were selected for experi-
mentation and that day was designated as day 1 of pregnancy. The selected rats
were laparotomized under mild ether anaesthesia on day 8 of pregnancy to note
the number of implantations and those having normal implantations were taken
for the further experimentation,
2.1.. Experiment I
To study the different dose effect of phenobarbital ; 2'5mg, 5*0mg or 7 -5m,
phenobarbital/100 g body weight, in 0-5 ml saline was injected subcutaneously
twice a day from day 8 through day 19 of pregnancy.
2.2. Experiment II
To study the different dose effect of barbital sodium 10 mg, 15 mg or 20 mg bar-
bital sodium/100 g body weight in 0*5 ml saline was injected subcutaneously,
twice a day from day 8 through day 19 of pregnancy.
For the above experiments, saline treated controls were maintained. The drug
treatment was continued until profuse vaginal bleeding was observed. All the
rats were autopsied on day 20. The number of foetuses, placentomas, placental
scars and placentae were recorded. Ovaries were weighed, fixed in Bouin's fluid,
embedded in paraffin, sectioned and stained in haematoxylin-eosin for histological
observations. The rats were maintained in individual cages with Hindustan Lever
rat feed and water ad libitum at a room temperature of 27 ± 1 °C with 12hrs of
lighting schedule.
3. Results
3.1. Interruption of pregnancy (tables 1 and 2)
Administration of different doses of phenobarbital or barbital sodium,twice a day
from day 8 through day 19 interrupts the pregnancy in rats to various levels, Low
doses of phenobarbital i.e. 2*5 mg/lOOg body weight interrupts pregnancy in
1/5 rats, while 5 mg of the same drug causes partial maintenance in 2/5 rats and
the remaining rats in these groups exhibit successful maintenance of pregnancy to
full term.
Similarly 10 or 15mg barbital sodium is not effective in interrupting the preg-
nancy wherein 5/6 rats or 5/5 rats maintain the pregnancy completely up to day 20.
Only one rat with 10 mg barbital sodium exhibits partial maintenance.
These results indicate that the phenobarbital is more potent than barbital sodium
in affecting the pregnancy even in low doses. The effective dose of phenobarbital
or barbjtal sodium in interrupting the pregnancy is 7*5mg or 20 mg respectively
wherein ?/9 rats or 8/9 rats show complete abortion with profuse vaginal bleeding
on day 12 or 13 of pregnancy. In saline treated controls, the pregnancy is main-
tained successfully in almost all rats.
Interruption of pregnancy by barbiturates in albino rats 535
Table 1. Effect of graded doses of phenobarbital on pregnancy in rats.
Treatment— day 8-19 Dose/100 gms body wt. 2 doses/day
Mean in relation to pregnant at laparotomy % foetal Ovarian
M ± S.E. survival wt. mg/
Treatment 10° Sms
body wt.
Implan- Placen- Placentoraas Live M ± S.E.
tations tal scars foetuses
Control 7-60 0-2
7-40 97-4 39-89
(5) ± ±
± ±
1-51 0-2
1-51 4-19
Pfcenobarbitat 7-40 1-40
6-20 S3- 8 40-61
2*5mg =b ±
± ±
(5) 0-67 1-39
1-70 7-75
5'Orng 8-60 0-8
7-60 88-4 40*23
(5) i ±
± ±
0-51 0-37
0-56 1-37
7- 5m* 7-50
0-00 31-17*
(9) ±
±
0-59
1-68
Laparotomy is done on day 8 and autopsy on day 20 of pregnancy.
Number in paranthesis denotes the number of rats-
M ± S. E.= Mean ± Std. error. * P < 0- 05.
3.2. Foetal survival
The percent foetal survival is calculated in relation to number of live foetuses on
day 20, with reference to the number of implantations observed on day 8, at laparo-
tomy. In saline treated controls 37 foetuses were found out of 38 implantations
indicating 97*4% foetal survival. With 2-5 mg or 5-0 mg phenobarbital treatment
83*8% or 88*4% respective foetal survival is observed. But with 10 mg or 15mg
barbital sodium administration the respective foetal survival is 97-9 or 100-0%
which is almost similar compared to that of controls. However, 7*5mg pheno-
barbital or 20 mg barbital sodium administration, the implantation loss is consi-
derable, wherein the number of foetuses vs implantation sites is 0/60 or 7/63
respectively, thereby indicating that foetal survival is nil or 11*1% with respective
to drug treatment.
The above results indicate that phenobarbital is more potent in its litter destroying
effect than barbital sodium, which may be due to its prolonged action on the central
nervous system-
536 Saraswati B Patil and M Appaswamy Rao
Table 2. Effect of graded doses of barbital sodium on pregnancy in rats.
Treatment-day 8-19 Dose/100 gms body wt. 2 doses/day
Mean in relation to pregnant at laparotomy
M ± S.E.
Ovarian
% foetal wt. mg/
TVf* qtmf»nt
survival 100 gms
JLICdllUCUl
Implan-
Placental Placen- Live
body wt.
tations
scars tomas foetuses
M ± S.E.
Control
7-60
0-2 ... 7-40
97-4 39*89
(5)
±
± ±
±
1-51
0-2 1-51
4-19
Barbital sodium
7-83
0-17 ... 7-66
97-9 36-60
10 mg
±
± ±
±
(6)
0-48
0-17 0-37
1-50
15 mg
8-20
8*20
100-0 42-2
(5)
d=
±
dz
0-36
0-36
3-66
20 mg
7*00
0-78
11-1 25-59**
00)
±
±
±
0-41
0-78
i-4e
Laparotomy is done on day 8 and autopsy, on day 20 Of pregnancy.
Number in paranthesis denotes the number of rats. . .
M ± S.E. = Mean ± Std. error. ** P < 0- 01.
3.3. Gravimetric and histological changes of the ovary
In the controls, where the pregnancy is maintained to full term, the ovaries .are
large with well developed corpora lutea, weighing 38 * 89 mg. With 2 • 5 mg or 5 • 0 mg
phenobarbital treatment, wherein pregnancy is not much affected, the ovary exhi-
bits large well developed corpora lutea similar to those of controls. However in
the rats treated with 7'5mg phenobarbital or 20 mg barbital sodium, where the
complete abortion has occurred, with almost nil foetal survival, the ovaries are
small with moderate sized corpora lutea and ovulated follicles. The ovaries are
reduced significantly weighing 37-17mg (P <0'05) or 25*59 mg (P < O'Ol) with
the administration of phenobarbital or barbital sodium respectively. These
aborted rats come to estrus within 3-4 days after profuse vaginal bleeding and hence
the ovaries resemble those of [nonpregnant rats. These results indicate a good
correlation between the percent foetal survival, ovarian weight and its histology.
The adverse effect of barbiturates on pregnancy seems to be due to blockade of
pituitary gbnadotrophins release during the critical period of gestation (day 8-11),
interruption of pregnancy by barbiturates in albino rats 537
wherein the pituitary hormone balance is essential for the normal functioning of
the ovaries which are responsible for the maintenance of gestation during its first
half.
4. Discussion
The apparent neutralization of Lii during days 7-11 of pregnancy in rats results
in the termination of gestation by foetal resorption (Rothchild et dl 1974). initi-
ation of a rise in the progesterone synthesis and pituitary LH release coincides with
an increased follicular growth and hypertrophy of corpora lutea between day 9-12
of pregnancy in rats and hamsters (Greenwald 1973 ; Rothchild et al 1974). There-
fore it is evident that pituitary LH is essential for the maintenance of corpora lutea
in the functional state as to produce progesterone, sufficient to maintain the preg-
nancy during the early half. In the present investigation, phenobarbital (7*5mg)
or barbital sodium (20 mg) causss profuse vaginal bleeding with foetal loss in 8/8
or 8/9 rats respectively when treated from day 8-11. The ovaries of these rats are
significantly reduced with very small corpora lutea and resemble to those of non-
pregnant rats when observed after autopsy on day 20. The probable modus
operandi is the continued inhibition of pituitary LH release during the crucial period
of pregnancy by the chronic treatment of barbiturates, as these drugs are known to
inhibit the LH surge and release in rats and hamsters (Norman et al 1973 ; Blake
1974 ; McCormack 1974). Therefore for all probabilities, the ovaries of barbi-
turate treated rats may not be functional due to LH inhibition, as LH stimulates the
production of progesterone from corpus luteum, and Yoshinaga et al (1972) and
Jagannadha Rao et al (1972) have observed a decrease in the progesterone and
20 a — OH — P by neutralizing endogenous LH by LH antiserum treatment.
Therefore the corpora lutea of pregnant rats seem to be dependent upon LH to
maintain the high progesterone levels during gestation. It can be postulated that
interruption of pregnancy by chronic treatment of barbiturates is due to continued
blockade or lowering of LH, resulting in subnormal production of progesterone.
Besides, it has been reported that barbiturates interfere directly with the ovarian
steroidogenesis by decreasing the 3/?~hydroxy steroid dehydrogenase activities
(Gupta and Karavolas 1973).
It is also stated by Greenwald and co-workers (1973, 1974) that prolactin with
FSH or estrone forms the luteotrophic complex during the early part of pregnancy.
These luteotrophins might have been decreased in barbiturate treated rats, as bar-
biturates inhibit both gonadotrophins (FSH and LH) and prolactin release (Ajika et al
1972 ; Beatti et al 1973). The ineffectiveness of low doses of these drugs causing
abortion or foetal resorption may be due to their failure in inhibiting the pituitary
gonadotrophins and prolactin effectively. Therefore it can be concluded that
the interruption of pregnancy in barbiturate treated rats is not only because of
the decreased pituitary gonadotrophins and prolactin release during the crucial
period, but also due to the direct interference of these drugs in the ovarian
steroidogenesis.
Ackno wle dgemenf s
We are thankful to the Department of Zoology, Karnataka University, Dharwad,
P.(B)-7
538 Sarasmti B fatil and M Appaswamy Rad
for providing the necessary facilities and one of us (SBP) is gratefui to UGC for the
award of Jr Research Fellowship during the tenure of this investigation.
References
Ajika K, Kalra S P, Fawcett C P, Krulich I and McCann S M 1972 The effect of stress and
nembutal on plasma levels of gonadotrophins and prolactin in ovariectomized rats;
Endocrinology 90 707-715
Beattie C W, Campbell C S, Nequin L Q, Soyker L F and Schwartz N B 1973 Barbiturate
blockade of tonic LH secretion in the male and female rats ; Endocrinology 92 1634-1638
Blake C A 1974 Localization of the inhibitory action of the ovulation blocking drugs on
release of luteinizing hormone in ovariectomized rat ; Endocrinology 95 999-1004
Greenwald G S 1974 Modification in ovarian and pituitary function in the hypophysectomized
pregnant hamster ; /. EndocrinoL 61 35-51
Greenwald G S 1973 Further evidence for a luteotrophic complex in hamster ; Progesterone
determination of plasma and corpora lutea ; Endocrinology 92 235-242
Gupta C and Karavolas R J 1973 Lowered ovarian conversion of 14 C-pregnenolone and other
metabolites during phenobarbital (PB) block of pMS-induced ovulation in immature rats ;
inhibition of 3^-hydro.xy steroid dehydrogenase ; Endocrinology 92 117-124
Jagannadha Rao A, Madhwa Raj H G and Moudugal N R 1972 Effect of LH, FSH and their
antisera on gestation in hamster ; (Mescricetus auratus) ; /. Reprod. Pert. 29 239-249
Lyons W R and Ahmad N 1973 Hormonal maintenance of pregnancy in hypophysectomized
rats ; Proc. Sec. Exp. Biol. Med. 142 198-202
McCormack C E 1974 Reversal by progesterone of barbiturate blockade of ovulations : effect of
concentration of serum LH (38098) ; Proc. Soc. Exp. Biol. Med. 146 329-332
Norman R L, Blake C A and Sawyer C H 1973 Evidence for neural sites, of action of pheno.
barbital and progesterone on LH release in the hamster ; Biol. Reprod. 8 83-86
Rothchild I, Pepe G J and Morishige W K 1974 Factors affecting the dependency on LH
in the regulation of carpus luteum progesterone secretion in the rat ; Endocrinology 95
280-238
Yoshinaga K, McCdonald G J and Greep R O 1972 Influence of various doses of LH on
foetal survival in hypophysectomized rats ; Proc. Soc. Exp. BioL Med. 140 193-195
Proc. Indian Acad, Sci. (Anim. Sci.), Vol. 91, Number 6, November 1982, pp. 539-552.
© Printed in India.
Observations on the natural history and population ecology of the
social wasp Ropalidia mavginata (Lep.) from Peninsular India
(Hymenoptera: Vespidae)
RAGHAVENDRA GADAGKAR, MADHAV GADGIL,
N V JOSHI and A S MAHABAL*
Centre for Theoretical Studies, Indian Institute of Science, Bangalore 560012, India
* Zoological Survey of India, Pune 411005, India
MS received 23 June 1982
Abstract. Ropalidia marginata, the most common Indian social wasp, belongs to a
crucial stage of social evolution showing no obvious morphological caste differentia
tion but a behavioural caste differentiation and a dominance hierarchy that appears
to influence division of labour. The nests consist of a single open comb that can some-
times have up to 500 cells and 10 pedicels. Nests are initiated and abandoned all
round the year. Initiation is by 1-20 foundresses, 1-4 being the most common
number. There is a great deal of variation in brood developmental times both within
and between nests. Male progeny disappear from the nest soon after emergence
while daughters stay on at the parent nest for a mean period of about a month.
Small nests have a single egg layer while large nests have two or more females with
well developed ovaries that presumably lay eggs. Most nests are short-lived, small
nests being highly susceptible to failure. Large nests are less susceptible to failure
but the emergence of multiple egg layers reduces the average relatedness of workers
to the brood which presumably is the cause for large scale emigrations from these
nests. An interaction of ecological and soical factors therefore appears to. determine
the growth of a nest.
Keywords. Social wasp ; Ropalidia marginata ; natural history ; population ecology;
hymenoptera ; caste differentiation.
1. Introduction
Recent years have witnessed a great surge of interest in social hymenoptera because
the emergence of a considerable body of theoretical ideas (Hamilton 1964 a,b ;
Lin and Michener 1972 ; Alexander 1974) have raised hopes that herein lies the
key to understanding the evolution of social behaviour (West-Eberhard 1969,
1975 ; Wilson 1971, 1975 ; Jeanne 1972, 1980 ; Michener 1974 ; Trivers and Hare
1976 ; Litte 1977, 1979, 1981 ; Starr 1979). Bees and wasps are of special interest
in this connection because they exemplify a series of stages in the evolution of
539
540 Raghavendra Gadagkar et al
sociality from the completely solitary to the highly advanced eusocial species (see
Evans and West-Eberhard 1970 ; Michener 1974 ; Wilson 1971).
Ropalidia marginata is the commonest social wasp of Peninsular India (Van
der Vecht 1962). This species shows cooperative brood care, reproductive caste
differentiation and overlap of generations (Gadgil and Mahabal 1974 ; Gadagkar
1980 ; Gadagkar and Joshi 1982b, 1983a ; Gadagkar unpublished observations)
and hence can be called eusocial according to the classification of Michener (1969).
There is no obvious morphological differentiation between egg layers and non
egg layers (Gadgil and Mahabal 1974) and division of labour is brought about
by a dominance hierarchy among the females belonging to a nest (Gadagkar
1980). Analysis of the time-activity budgets of adults on R. marginata nests
has in fact revealed the presence of a behavioural caste differentiation, in this
primitively eusocial wasp (Gadagkar and Joshi 1982b, 1983a).
Apart from these few recent studies there is very little information in the litera-
ture about this interesting genus (Roubaud 1916 ; Carl 1934 ; Darchen 1976 ;
Belvadi and Govindanl981 ; Gadagkar and Joshi 1982a,c, 1983b). Moreover, in
addition to understanding reproductive differentiation and social organization, infor-
mation on the dynamics of initiation, growth and extinction of colonies is essential
before we even begin to speculate about the factors that might be responsible for
the origin and maintenance of sociality. We present in this paper the results of
our observations on the natural history as well as population ecology of Ropalidia
marginata in Peninsular India.
2. Materials and methods
2.1. Study sites
In all we have observed 125 nests of Ropalidia marginata from the cities of Pune
(18°30'N and 73°53'E) (45 nests) and Bangalore (13°00'N and 77° 32' E)
(80 nests) at various times over a period of nine years from October 1971 to
October 1980.
2.2. Population fluctuations
Our population observations include records of the numbers of pupae and adults
in a nest maintained at roughly 8-10 day intervals. Such observations were main-
tained on three nests in Pune from October 1971 to May 1973 and for 35 nests in
Bangalore from October 1974 to October 1976. The 35 nests in Bangalore were
all located on the windows of one building about 20,000 sq.ft. in area and a height
of about 40 ft. Our records of the population in this site also provide information
on (i) seasonal variations in numbers of adult wasps, pupae and nests, (ii) seasonality
of initiation and abandoning of nests and (iii) life spans of nests.
2. 3. Brood developmental times
For one nest in Pune and two nests in Bangalore the contents of each cell in the
nest were noted to provide estimates of developmental times of the eggs, larvae
and pupae.
A social wasp from peninsular India 541
2-4. Period of residence of adults on nests
Every adult on two nests in Bangalore was marked with a unique spot of quick
drying paint immediately upon emergence without removing it from the nest.
A census of all ths adult; present on the nests was taken on alternate days
from November 1979 to June 1980 to provide records of the total period of
residence of 60 females and 3 males.
2 . 5. Collection of nests
28 nests in Pune and 31 nests in Bangalore were collected taking precaution not
to bias the sampling in favour of any particular size class of nests aod to collect
entire combs along with all the adults and immature stages. The numbers of
pedicels, cells, eggs, larvae, pupae and adults were determined. The adults were
sexed and the females were dissected to determine the state of development of
their ovaries. The females were classified arbitrarily into 3 catagories : those
with undeveloped ovaries, those with moderately developed ovaries and those with
well developed ovaries on the basis of maximum ovariole width. Those classi-
fied as * with well-developed ovaries ' appeared to have mature eggs and were
probably laying eggs. These females are designated as egg layers. We do not
however know if all females with well-developed ovaries actually laid eggs.
3. Results
3.1. The nest and its structure
R. marginata builds nests with simple, open (Gymnodomous according to the
classification of de Saussure (1853-59) and Richards and Richards (1951) ) combs
the construction of which begins with the laying down of the pedicel which is
usually 5-10 mm long and about 1 mm thick. The first cell is constructed at the
tip of this pedicel and the subsequent cells are added either all round the first
cell or only on one side so that in larger combs the initial pedicel may either end
up being approximately in the centre of a layer of cells or at one extreme end. As
the comb grows in size the initial pedicel is enlarged in width and may gtow up to
about 5-6 mm in diameter in large combs. In addition to enlarging the original
pedicel, new thin pedicels (about 1 mm in diameter) that reinforce the attachment
of the comb to the substratum are added at several points. Most small combs
(< 100 cells) have a single pedicel while large combs (> 100 cells) often have
more than one pedicel (table 1). The largest comb we have recorded had about
500 cells and 10 pedicels, the latter also being the largest number of pedicels
recorded on a comb.
All but one of the nests recorded, has a single comb per nest. In one case
however, there were two combs within about 20 mm of each other and the
adults clearly moved between these two combs.
3-2. Initiation of nests
Nests of jR. marginata are initiated and abandoned all round the year (table 2) .
nests are initiated by 1-20 females, 1-9 being the commonest number (figure 1) ,
542 Raghavendra Gadagkar et al
Table 1. Nests with different number of pedicels
Number of
cells in
nest
Frequency of nests with different number of pedicels
12 3 4 7 10
Pedicel Pedicels Pedicels Pedicels Pedicels Pedicels
1-100
17 3
101-200
1 ... 2
201-300
...
301-400
1 1 ... 1
401-500
1 2 ... 1 1
Table 2. Year-round initiation and abandoning of nests*
Month
Number
of nests
initiated
Number
of nests
abandoned
January
4
3
February
0
3
March
0
1
April
3
2
May
5
2
June
1
8
July
1
2
August
8
7
September
3
1
October
2
1
November
4
2
December
0
0
Data pooled from observations throughout the study period both in Bangalore and Pune.
In many cases the initial single foundress appears to be joined by other females
within a few days of initiation of the nest. When newly emerging females were
marked with spots of paint, it was noticed that some of the newly emerged indi-
viduals were not spending every night on the parent nest but were occasionally
missing for 2-3 days before returning to it. It is possible that these individuals
had been visiting other newly founded nests on the nights they were absent. There
were emigrations of large number of adults from nests which had grown to more
than 40-50 adults in size. Groups of individuals from these exoduses probably
constitute the initial set of foundresses for many nests.
A social wasp from peninsular India
543
if)
Ss
UJ
z
fc*
ct
UJ
CD
53
nn
1 2 3 4 5 6 7 8 9 10 11 12 13 U 15 16 17 IS 19 20
NUMBER OF FOUNDRESSES
Figure 1. Frequency distribution of the number of nests with different numbers of
foundresses.
3- 3. Brood developmental times
The accurate determination of brood developmental times is beset with a number
of problems and the estimates given here are only to be treated as first approxi-
mations. The duration of the egg, larval and pupal stages both in Pune and
Bangalore are given in table 3. In each stage the duration in Pune is much less
than in Bangalore. This difference could either be a genuine [difference due
to different environmental conditions in Pune and Bangalore. However, it cannot
be ruled out that the differences are simply a result of small sample sizes in terms
of the number of nests studied. The data in Pune in fact represent a single nest
and that in Bangalore two nests. The difference could therefore be simply a
manifestation of different stages in the nest cycle or of different local conditions.
The nest in Pune, may have been located close to a good food source and therefore
the difference may not even reflect differences batween Pune and Bangalore as such.
The data both from Pune and Bangalore show a very great degree of variation.
The standard deviations are close to half or sometimes more than half of the mean.
The wide variation in egg developmental times is primarily because there is a signi-
ficant degree of egg cannibalism which remains undetected. Eggs are eaten and
replaced by new ones and several consecutive replacements may occur before
an egg successfully hatches into a larva. The variation in larval developmental
times almost certainly reflects differences in food supply. A larva can complete
development and pupate in as little as 7 days in Bangalore under laboiatory condi-
tions when the adults feeding the larva are provided with an ad libitum food
supply (Gadagkar, unpublished observations). The variations in pupal develop-
544
Raghavendra Gadagkar et al
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1
(N O 2
•<— I T— | *~^
I
I
1
A social wasp from peninsular India
545
mental times are the hardest to understand. The hypothesis that a strong correla-
tion between larval and pupal developmental times is the cause of this variation
is not borne out because we find that the correlation coefficients between larval
and pupal developmental times are not significantly different from zero at 5%
level. This is true even in the large sample size from weekly observations in Pune.
3-4. Duration of residence of adults on the nest
In all nests in which the newly emerging adults were marked it was observed that
the males always disappeared within two to four days of emergence. While some
females disappeared very soon after emergence, others stayed on at the parent
nest for long periods of time. On two nests all the emerging adults (a total of 75
females and 3 males) were marked. The 3 males disappeared from the nest within
2, 3 and 4 days respectively of emergence. Of the 75 females we have information
on the duration of residence on the nest for 60 females that disappeared before
the end of our study. The frequency distribution of residence times for these 60
females is shown in figure 2. This corresponds to a (mean ± SD) residence time
of 27 ± 23 days and a range from 1-160 days. When a wasp disappears from
one nest it may either have died (mortality) or initiated or joined another nest
(emigration). In our records these two components cannot be distinguished
directly. The (mean ± SD) age-specific day to day probability of remaining
at the same nest (inset, figure 3) has a value of 0*95 ± 0-04 which is nearly
constant with age. This seems to suggest that mortality as opposed to emigra-
tion forms a very large component of our estimates. The reasoning behind this
is that mortality seems to occur during the foraging trips because the wasps simply
do not return to the nest at the end of the day. Perhaps they are lost or preyed
upon. It is reasonable to assume that the probability of these events would be
OL
DURATION OF RESIDENCE ON NEST IN DAYS
Figure 2. Frequency distribution of residence times on a given nest of 60 female
wasps of R. marginata. The age-specific day-to-day probability of remaining at
the same nest (inset) of 0-95 iO' 04 is nearly constant with age. Note that most
of the points lie between 0'90 and I'O.
546 Raghavendra Gadagkar et al
Independent of the age of the animal but that the probability of emigration to
found or join another nest would show some age dependence.
3-5. Reproductive differentiation
Our dissections from the harvested colonies indicate that although there is no
morphological differentiation amongst the females, there is a marked differentia-
tion amongst them in terms of development of ovaries. In all the nests, a
majority of the females possessed rudimentary, completely undeveloped ovaries,
while only 1 to 6 females possessed moderately or well developed ovaries. In
most cases, the females with well-developed ovaries tended to be heavier in weight
than the other females (Gadgil and Mahabal 1974). In addition there is a domi-
nance hierarchy amongst the females at a nest with the dominant females doing
less foraging (Gadagkar 1980). There is extensive food sharing at the nests of
R. marginata, and since frequency of dominance behaviour and snatching food are
significantly correlated (Gadagkar and Joshi 1983a) it is quite plausible that
the dominant individuals get a disproportionately greater share of the food, while
expending less energy on foraging. They may thus be able to grow heavier and
develop their ovaries, while the less dominant individuals, the workers, suffer
from 'nutritional castration'.
The number of females with developed ovaries does not bear any clear relation
to the total number of females on the colony ; while it shows evidence of an
increase with the number of cells in the comb (table 4). Thirty out of 32 nests
with less than 100 cells had a single egg-layer, while 14 out of 17 larger nests had 2
or more. The number of cells in a comb is a good indicator of the age of the
colony, while the number of females in a colony keeps constantly fluctuating
because of periodic large scale emergence and emigrations. We may therefore
conclude that the number of egg-layers in a colony increases with the age of the
colony. Initially, at the founding, a single female dominates and monopolises
Table 4. Nests with different numbers of egg layers
Frequency of nests with different numbers of females with well
developed ovaries (egg-layers)
rxUJLUUCi v^i
cells in nest
1
egg-layer
2
egg-layers
3
egg-layers
4
egg-layers
5
egg-layers
6
egg-layers
1-100
30
2
1
0
0
0
101-200
2
1
1
1
1
2
201-300
1
0
0
1
0
0
301-400
2
1
0
1
0
0
401-500
0
1
1
0
0
1
A social wasp from peninsular India 547
all egg-laying ; as the colony develops, this monopoly is broken and other females
again the heavier, more dominant ones, also begin to lay eggs.
3 - 6. Population fluctuations
Nests of R. marginata that have long life spans are characterised by continuous
fluctuations in the number of adults. Figures 3 and 4 represent the population
changes at two nests which grew to a considerable size and lasted for
two years or more. In both cases the number of adults on the nests increased
initially and following one or more mass emigrations, remained fluctuating for
several months at less than 20 adults. In the case of the first nest (figure 3) there
were four clear cut instances of mass emigrations. These involved 30 or more
adults leaving the nest perhaps to initiate other nests nearby. In the case of the
second nest (figure 4), there was a single major exodus, apparently in direct res-
ponse to predation on the nest by Vespa tropica. This large wasp feeds on eggs,
larvae and pupae of R. marginata. The particular nest depicted in figure 4 was
under continual observation, and it is known that the mass exodus followed the
first ever visit of the predator to the nest. The predator continued to regularly
visit this nest thereafter, and apparently kept the population in check for a year
or so. Beyond this period, the nest failed to grow further, although the visits
of the predator apparently ceased.
We have rather complete information on population fluctuations at one site in
Bangalore where we observed all the nests present at that site for a period of 104
weeks. In all, 35 nests were observed at this study site. The number of nests and
the total population of adults and pupae present in all the nests at different times
at this site are shown in figure 5. The total population of adults varied between
70 and 400, the population of pupae between 0 and 340 and the total number of
nests present at any given time varied between 8 and 16. The largest number of
adults were present during January to April in both years. However, the number
of pupae and that of the nests seemed to fluctuate rather widely.
1 Oct '74
1 Apr 75
1 Oct '75
TIME
1 Apr '76
1 Oct '76
Figure 3. Number of adults at a Ropalidia marginata nest in Bangalore.
Arrows indicate mass exoduses.
548
Raghavendra Gadagkar et al
' PERIODS OF PREDATION
BY VESPA TROPICA
1 Oct '71 1 Apr '72 T Oct '72 1 Apr '73 1 Oct '73 1 Apr '74
TIME
Figure 4. Number of adults at an R. marginata nest in Pane. There was a single
exodus following the first instance of predation on the nest by Vespa tropica. Arrows
indicate periods of regular predation by this wasp.
1 Oct '74
1 Apr '75
1 Oct '75
TIME
t Apr 76
Figure 5. The total number of adults (left ordinate and solid line) and pupae
(left ordinate, broken line) and tho total number of nests (right ordinate and dotted
line) present at different times at a single study site in Bangalore over the period
of 104 weeks.
The long lived nests represented in figures 3 and 4 are only a small propor-
tion of the total nests. Most of the nests in fact have a shorter life span. The
total life span of 18 nests is known because both the initiation and abandoning of
these nests occurred during the period of study. The frequency distribution of
total life span of these nests (figure 6A) shows that most nests (70 %) have a live
span of 10 weeks or less. There was only one nest among these that survived for
longer than 30 weeks. However, estimates of the total life spans obtained from
any finite period of study is likely to be biased in favour of short lived nest^. The
distribution of minimum life spans, i.e.9 where either initiation or abandoning alone
were observed (figure 6B) reveals that 10 out of 16 additional nests survived for
longer than 30 weeks. Moreover, for one nest neither initiation nor abandoning
observed ; in other words, it survived for longer than 104 weeks (the
A social wasp from peninsular India
549
10 20 30 40 50 60 70 80 90 100 >100
LIFE SPAN (WEEKS)
D
JLXL
J » i
„„ To 20 30 40 50 60 70 80 90 100 > 100
MINIMUM LIFE SPAN (WEEKS)
Figure 6. Frequency distribution of total life spans (A) and minimum life spans
(B) of R. marginata nests. Total life span is defined as the time interval between
initiation and abandoning of a nest and is therefore known only for those nests for
which both initiation and abandoning occurred during the period of study. Minimum
life span is given only for those nests for which either the initiation or abandoning
alone is known.
10
20
30 40 50 6O 7O
NUMBER OF ADULTS ON NEST
100
Figure 7. Probability of increase in adult numbers as a function of number of
adults already present. The arrows indicate the expected change on the mean in
the number of adults in colonies of various sizes.
tion of observation). Thus, although most nests are short lived, some do survive
for very long periods of time.
Figure 7 presents further analysis of the population fluctuations. Here we
present the probability of increase in the number of adults at a nest as a function
of the number of adults already present. These probabilities have been computed
by pooling together our data for the 8 nests monitored for over 2 years. As can
550
Raghavendra Gadagkar et at
60
50
40
g 30
UJ
o
UJ
Q.
10
10 20 30 "40 50 60 70
NUMBER OF ADULTS ON A NEST
80
9O
100
Figure 8. Frequency distribution of the total population of R. marginata nests i a
terms of the number of adults present on the nest.
be seen, the smallest nests have the lowest probability (only 0 • 3) of further increase
in number. They are thus nests most susceptible to extinction. The only sizes
at which the nests have near even or better than even chance of increase are
between 10 to 40. Thus, a nest which has increased to this level may further
increase rapidly till it crosses 40 adults. Beyond this, the nests tend to have a
high probability of decrease (due to mass exoduses). The resultant size frequency
distribution of nests is presented in figure 8. The vast majority of the nests have
less than 10 adults, most go extinct without getting beyond this stage.
4. Discussion
According to the theory of kin selection (Hamilton 1964a, b ; 1972 ; West-
Eberhard 1975) the rationale for the development of sociality in ants, bees jand
wasps lies in their haplodiploid system of sex determination. Because of this, a
female wasp is genetically more closely related to her sister than she is to her
daughter, and it is therefore more ' advantageous ' for a female wasp to help her
mother raise daughters which would be her sisters, than to attempt to raise
daughters by herself. It is believed that this is why females are selectively
favoured to stay on with their mother and help her with the colony labour. At
the same time, sons are more closely related to females than brothers are ; hence
the workers would have a tendency to lay male eggs, and the males themselves
would not share in the colony labour (Hamilton 1964a, b ; Wilson 1971 ;
West-Eberhard 1975 ;-Trivers and Hare 1976).
Wasp nests with multiple foundresses and multiple egg layers do not fall neatly
in this scheme, particularly if the egg-laying females themselves were not close
relatives. We however know that in the case of Polistes the foundresses do in
fact tend to be sisters (West-Eberhard 1969 ; Ross and Gamboa 1981). This
system of multiple foundresses can evolve if the nests are highly susceptible to
failure in the early stages of growth. Then, if the coming together of several
A social wasp from peninsular India 551
females increases the probability of success of a nest by a factor of 1 • 5 or more,
sisters may band together, and relinquish reproduction to the most dominant
female as the female brood they are raising will be related to them as nieces with
coefficient of relatedness = 0*375. If a single female remains reproductive, the
workers of the later brood will be raising their sisters with coefficient of
relatedness =0-75.
If, however, more than one of the founding sisters starts to lay, the workers will
now be raising a brood related at least in part to them as first cousins coefficient
of relatedness =0-19. At this point the workers may find it more advantageous to
leave the nest and attempt to initiate one on their own. This tendency will increase
with an increase in the number of egg-layers in the nest.
As discussed earlier, the small nests of R. marginata are in fact highly suscep-
tible to failure, hence the banding together of several foundresses is expected. We
have no evidence that these are sisters, though this is plausible as new nests are
very often founded close to old ones and the foundresses are likely to be sisters
who leave together in an exodus from a nest.
We have also shown that there is a tendency for mass exoduses from nests with
over 40 adults. This may be related to these being older nests with multiple egg-
layers in which the average degree of relationship between the workers and the
brood would tend to be low, making it less advantageous for the workers to stay
on at nest. Difficulties of sustaining a larger number of adults on the food
resources of the home range could be ruled out as a major factor since the new
nests are often founded next to the parental nest and must therefore utilize much
the same food resources.
In conclusion it appears that ecological pressures render small nests highly
susceptible to failure and therefore necessitate the banding together of several
females. As the nest grows in size, a single female can no longer dominate it to
the level of exclusively monopolizing all egg-laying. With the emergence of
multiple egg-layers the workers are at less of an advantage in remaining on the
nest and hence begin to leave in significant numbers producing large population
fluctuations. An interaction of ecological and social pressures thus determine
the course of growth of a nest.
Acknowledgements
We are grateful to O W Richards for his kind help in identifying the R. marginata
and V. tropica material.
References
Alexander R D 1974 The evolution of social behaviour ; Ann. Rev. Ecol. Syst. 5 325-383
Belvadi V V and Govindan R 1981 Nesting habits and behaviour of Ropalidia (Icariola)
marginata (Hymenoptera : Vespidae) in South India ; Colemania 1 95-101
Carl J 1934 Ropalidia montana sp, et son nid. Un type nouveau d' architecture vespienne;
Rev. Suisse. Zool. 41 677-691
Darchen R 1976 Ropalidia cincta, guepe sociale de la savane de Lamto (Cote-D'Ivoire) ; Ann.
Soc. Entomol. Fr. (NS) 12 579-601
Evans H E and West-Eberhard M J 1970 The wasps Ann Arbor, The Univ. of Michigan press ;
pp. 265
552 Raghavendra Gadagkar et al
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 and Joshi N V 1982a Behaviour of the Indian Social Wasp Ropalidla cyathfformis
(Fab.) on a nest of separate combs (Hymenoptera : Vespidae) ; /. Zool 198 27-37
Gadagkar R and Joshi N Y 1982b A Comparative study of social structure in colonies of
Ropalidia, in The Biology of the Social Insects, (eds) MD Breed, C D Michener and H E
Evans, Proc. IX congress of the International Union for the study of social Insects, Boulder,
Colorado USA pp. 187-191
Gadagkar R and Joshi N V 1982c When a wasp colony splits In The Biology of the Social
Insects, (eds) M D Breed, C D Michener and H E Evans Proc. IX congress of the
International union for the Study of Social Insects, Boulder Colorado USA pp. 217
Gadagkar R and Joshi N V 1983 a Quantitative ethology of social wasps : Time-activity
budgets and caste differentiation in Ropalidia marginata (Lep.) (Hymenoptera : Vespide) ;
Animal Behaviour 31 26-31
Gadagkar R and Joshi N V 1983b Sociai organization in the Indian wasp Ropalidia
cyathiformis (Fab.) (Hymenoptera : Vespidae) ; Z. Tierpsychol (in Press)
Gadgil M and Mahabal A S 1974 Caste differentiation in the paper wasp Ropalidia marginata
(Lep.) ; Curr. Sci. 43 482
Hamilton W D 1964a The genetical evolution of social behaviour I ; /. Theor. Biol. 7 1-16
Hamilton W D 1964b The genetical evolution of social behaviour II ; /. Theor. Biol. 7 17-52
Hamilton W D 1972 Altruism and related phenomena mainly in the social insects ; Ann. Rev.
Ecol. Syst. 3 193-232
Jeanne R L 1972 Social biology of the Neo-tropical wasp Mischocyttarus drewseni ; Bull. Mus.
Comp. Zool. Harv. Univ. 144 63-150
Jeanne R L 1980 Evolution of social behaviour in the Vespidae ; Ann. Rev. Entomol. 25 371-396
Lin N and Michener C D 1972 Evolution of sociality in insects ; Q.Rev. Biol. 47 131-159
Litte M 1977 Behavioural ecology of the social wasp, Mischocyttarus mexicanus; Beh. Ecol
Sociobiol 2229-246
Litte M 1979 Mischocyttarus flavitarsis in Arizona : social and nesting biology of a polistine
wasp ; Z. Tierpsychol. 50 282-312
Litte M 1981 Social biology of the Polistine wasp Mischocyttarus labiatus : survival in a
Colombian rain forest ; Smiths. Contr. Zool. No. 327
Michener C D 1969 Comparative social behaviour of bees ; Ann. Rev. Entomol. 14 299-342
Michener C D 1974 The social behaviour of the bees ; Massachusetts : Harvard University
Press xii + 404 pp.
Richards O W and Richards M J 1951 Observations on the social wasps of South America
(Hymenoptera : Vespidae) ; Trans. Roy. Entomol. Soc. Lond. 102 1-170
Ross N M and Gamboa G J 1981 Nestmate discrimination in social wasps (Pollsters metricus*
Hymenoptera :. Vespidae) ; Beh. Ecol. Sociobiol. 9 163-165
Roubaud E 1916 Recherches biologiques sur les guepes sociales et cosales d'Afrique. La
guese de la vie social et T evolution de Tinstinct maternal chez les vespides ; Ann. Sci. Nat.
Zool. 1 1-160
Saussure H de 1853-59 Monographie de quepes sociales, ou de la tribu des Vespiens9 onvrage
faisant suite a la monogrophie des guiepes solitaires V. Masson Paris
Starr C K 1979 Origin and evolution of insect sociality ; A review of modern theory ; In
Social Insects9 (ed) H R Hermann (New York : Academic Press) Vol. I pp. 35-79
Trivers R L and Hare H 1976 Haplodiploidy and the evolution of the social insects ; Science
191 249-263
Vecht J Van der 1962 The Indo-Australian species of the genus Ropalidia (Icaria) (Second part);
Zool. Verhand. Leiden 57 1-72
West-Eberhard M J 1969 Social biology of polistine wasps ; Misc. PubL Mus. Zool. Univ.
Mich. 140 1-101
West-Eberhard M J 1975 The evolution of social behaviour by kin selection: Q. Rev. Biol
50 1-33
Wilson E O 1971 The insect societies ; Massachusetts : Harvard University Press x + 543 pp.
Wilson E O 1975 Sociobiology ; Massachusetts : Harvard University Press ix + 697 pp.
Proc. Indian Acad. Sci. (Ante*. ScL), Vol. fill, Number 6, November 1082, pp. 553-562.
© Printed in India.
Ecobiology of Corvospongilla lapidosa (Annandale 1908) (Porifera :
Spougillidae) in the Manjira reservoir, Sangareddy, Andhra Pradesh
I SESHAGIRI RAO and M A KHAN*
Department of Zoology, N.B. Science College, Hyderabad 500002, India
* Department of Zoology, Osmania University, Hyderabad 500 007, India
MS received 2$ July 1981 ; revised 2 April 1982
Abstract The Spongillid Corvospongilla lapidosa (Annandale 1908) (Porifera :
Spongillidae) is reported for the first time from the Manjira reservoir in Andhra
Pradesh. Its sausage shaped spicules have adaptive value to thrive in low silica
environments. The species is tolerant to high turbidity. High calcium and bicarbo-
nate may be unfavourable and the sponge has not been found either on molluscan
shells or on aquatic vegetation. It can thrive in waters more than 4m deep.
The range and mean values of twenty-seven physico-chemical parameters of the
habitat of C. lapidosa are given as the base data for the species ecology.
Keywords. Ecology ; Porifera ; Corvospongilla lapidosa ; Manjira reservoir ; silica ;
turbidity ; spicules.
1, Introduction
In, the aquatic ecosystems poriferan fauna occupying the benthic habitat constantly
circulate water in their elaborate and complex canal system through their multi-
porous body surface. So it is reasonable to expect that in the lentic and lotic
bodies of fresh-water the spongillid species play an important but yet unrecognized
role in the cycling of abiotic and biotic substances. Apart from the pioneering
work and important contributions on the taxonomy and geographical distribution
by Annandale (1907, 1908, 1909, 1909a, 1911, 1912, 1913 and 1915) and sub-
sequent supplemental work of Gist (1930, 1932) the Indian spongillids had to
remain neglected until Tonapi (1964) added to their list of habitats of earlier
known species. There is no record of the environmental parameters of the fresh-
water bodies in which the Indian spongillids colonized except for the ecologically
poor descriptions of their habitats as clear, dirty, turbid, polluted waters etc. In
the course of a two year study from February 1979 through January 1981, on
the ecology of the Manjira reservoir formed by a man-made barrage (17° 39' N,
78° 04' E) located near Sangareddy, Andhra Pradesh, when spicules were frequently
encountered in the samples of plankton, search was undertaken to locate
the habitats of the sponges. During these studies a number of specimens of
553
554 / Seshagiri Rao and M A Khan
the spongillid species, Corvospongilla lapidosa (Annandale 1908) (family : Spongilli-
dae) were discovered and observations made on certain aspects of its ecobiology
are discussed in this paper.
2. The river Manjira and its reservoir
The river Manjira, a tributary of the Godavari in South India has a barrage
constructed across it near Sangareddy (17°37'N, 78°06'E) to form a potable
water reservoir (figure 1) to supply the city of Hyderabad. The reservoir has a
catchment area of about 16,780 km2 and a maximum storage capacity of 73 -63
Mm3. Due to heavy siltation the maximum depth of the reservoir near the
barrage is now about 9 * 5 m. The river bed is dotted with boulders of various
sizes some of them buried deep in the silty clay while a few lie exposed during the
depletion of water level in summer.
3. Materials and methods
Surfacial water samples (2-5 1) from four stations (I, II, III and IV— figure 1)
bathymetric water samples (2.4 1) from 3m, 6m and 0*5m near the bottom of
the reservoir (V, VI and VII stations — figure 1) operating a closing type of bottle
sampler (1*2 1 capacity) twice near station III were collected monthly for two
years from February 1979. Temperature of water was read in the field by using
a mercury thermometer (0°-110°C). Secchi disc (Welch 1948) was used near
station III for determining the Zsd. In a field laboratory near the barrage pH and
conductivity were determined by using conductivity-pH meter (CLO1/03
Toshniwal). Carbonic species of water were estimated by titrimetry and the
samples were then brought to the Department of Zoology, Osmania University,
Hyderabad for further analyses of various chemical parameters according to APHA
AWWA and WPCF (1971).
4. Observations
4.1 Habitat
During the summer months of May and June of 1979 and 1980 the water level
in the reservoir receded considerably exposing a number of boulders, cobbles and
pebbles in the reservoir bed in the vicinity of the sampling stations I and IV.
These provided the substratum for the encrustation of the sponge, Corvospongilla
lapidosa. In summer the exposed reservoir bed presented dry caked mud that
is extensively fissured to a depth of 0*4 to 0'5m. During winter and rainy sea-
sons, the littoral region of the reservoir supports fairly rich emergent growth of
aquatic plants Typha angustata and Scirpus littoralis and submerged species of
Potamogeton perfoliatus and Vallisneria spiralis. The depth of the littoral varies
from 0*5 to 1 • 5 m. There are no trees on the margins of the reservoir to impart "
any shade.
Ecobiology of Corvospongilla lapidosa
555
bfl
•S
I
s
5
e
o
^ B
556 I Seshagiri Rao and M A Khan
4.2. Ecomorphic characters
Corvospongilla grows on large boulders covering their exposed surfaces as a more
or less flat sheet of 0*6 to 2* Ocm thickaess (figure 2a). On small cobbles and
pebbles (figure 3a) the thickness of the sponge is reduced to 0*3 to 1 • 0 cm. The
sponge body is steel grey or bluish black in colour. Near Station III the barrage
wall of the north flank was also found encrusted with this sponge which is
yellowish brown in colour. The water at this station is 6 to 9- 5m but usually
8 m deep and devoid of angiospermic vegetation.
Microscopic examination of the sponge body (figure 2) reveals the appearance
of a corrugated body surface with sausage shaped amphistrongylous spicules
lying embedded on the surface in different directions. Oscula are inconspicuous,
dispersed at random, some raised on irregular eminences. The thick chitinous
membrane at the base of the sponge body is tough and shows a few furrows
probably made by worms. The structure, shape and size of the spicules (figure
3b, c) conform to the descriptions of Annandale (1911) for the species,
4.3. Distribution
According to Khera and Chaturvedi (1976) the distribution of Corvospongilla
lapidosa is Maharashtra — Igatpuri lake, the river Godavari at Nasik ; Karnataka
— Bangalore ; Bihar — Santhal Paragana ; West Bengal — Barrackpore. Tonapi
(1964) reported it from small rivers near Poona. The occurrence of this species
of spongillid in the Manjira reservoir is a new record for this river and also for
Andhra Pradesh. Schizotype of this species was sent to Zoological Survey of
India, Calcutta and the identification was confirmed.
"5. Discussion
From our observations Corvospongilla is able to thrive in water more than 4 m deep
and can encrust masonary constructions like the walls of a barrage. It was not
found either on molluscan shells or on aquatic plants. In the Manjira reservoir
it has not yet established itself near the south flank of the barrage. The reasons
may be lack of suitable substrata, dense growth of floating and submerged vege-
tation, epiphytic algae, feeble currents in the water, decaying organic matter at
the bottom that can clog the canal system of the sponge and relatively .higher
concentrations of calcium and bicarbonates. Station II showed the highest mean
values of 22 -675 mg/1, 22.735mg/l for calcium and high mean values of 217-36
mg/1, 193-71 mg/1 for bicarbonate in the first and second years of our study
(table 1). Jewell (1939) stated that some sponges are sensitive to calcium bicarbo-
nate concentration. The concentration of silica in the Manjira reservoir is low,
the mean value for all stations ranging from 0-983 to 2 -229 mg/1 (table 1) and
the maximum value of 6 mg/1 was recorded only once in February 1979 when
grouting work of the north flank bund was in progress. Allee et al (1955) stated
that silica content of lakes may be a limiting factor in the growth and distribu-
tion of fresh water sponges and their skeletal development may be much affected
Ecobiology of Corvospongilla lapidosa
557
Figure 2. a. Dried sheet of Corvospongilla from a boulder (surface view).
b. Magnified surface view showing Oscula (os). c. Undersurface of the
Sponge showing chitinous membrane and worm furrows.
558 / Seshagiri Rao and M A Khan
Proc. tndiari Acad. Slci. (Anto. Scl.), Vol. 01, lumber 6, November ij$l, pp. 553-561
© Printed in India.
Ecobiology of Corvospongilla lapidosa (Annandale 1908) (Porifera :
Spongillidae) in the Manjira reservoir, Sangareddy, Andhra Pradesh
I SESHAGIRI RAO and M A KHAN*
Department of Zoology, N.B. Science College, Hyderabad 500 002, India
* Department of Zoology, Osmania University, Hyderabad 500 007, India
MS received 28 July 1981 ; revised 2 April 1982
Abstract The Spongillid Corvospongilla lapidosa (Annandale 1908) (Porifera :
Spongillidae) is reported for the first time from the Manjira reservoir in Andhra
Pradesh. Its sausage shaped spicules have adaptive value to thrive in low silica
environments. The species is tolerant to high turbidity. High calcium and bicarbo-
nate may be unfavourable and the sponge has not been found either on molluscan
shells or on aq'uatic vegetation. It can thrive in waters more than 4 m deep.
The range and mean values of twenty-seven physico-chemical parameters of the
habitat of C. lapidosa are given as the base data for the species ecology.
Keywords. Ecology ; Porifera ; Corvospongilla lapidosa ; Manjira reservoir ; silica ;
turbidity ; spicules.
1, Introduction
In the aquatic ecosystems poriferan fauna occupying the benthic habitat constantly
circulate water in their elaborate and complex canal system through their multi-
porous body surface. So it is reasonable to expect that in the lentic and lotic
bodies of fresh-water the spongillid species play an important but yet unrecognized
role in the cycling of abiotic and biotic substances. Apart from the pioneering
work and important contributions on the taxonomy and geographical distribution
by Annandale (1907, 1908, 1909, 1909a, 1911, 1912, 1913 and 1915) and sub-
sequent supplemental work of Gist (1930, 1932) the Indian spongillids had to
remain neglected until Tonapi (1964) added to their list of habitats of earlier
known species. There is no record of the environmental parameters of the fresh-
water bodies in which the Indian spongillids colonized except for the ecologically
poor descriptions of their habitats as clear, dirty, turbid, polluted waters etc. In
the course of a two year study from February 1979 through January 1981, on
the ecology of the Manjira reservoir formed by a man-made barrage (17° 39' N,
78° 04' E) located near Sangareddy, Andhra Pradesh, when spicules were frequently
encountered in the samples of plankton, search was undertaken to locate
the habitats of the sponges. During these studies a number of specimens of
553
P.<B)-8
554 / Seshagiri Rao and M A Khan
the spongillid species, Corvospongilla lapidosa (Annandale 1908) (family : Spongilli-
dae) were discovered and observations made on certain aspects of its ecobiology
are discussed in this paper.
2. The river Manjira and its reservoir
The river Manjira, a tributary of the Godavari in South India has a barrage
constructed across it near Sangareddy (17°37'N, 78°06'E) to form a potable
water reservoir (figure 1) to supply the city of Hyderabad. The reservoir has a
catchment area of about 16,780 km2 and a maximum storage capacity of 73*63
Mm3. Due to heavy siltation the maximum depth of the reservoir near the
barrage is now about 9 • 5 m. The river bed is dotted with boulders of various
sizes some of them buried deep in the silty clay while a few lie exposed during the
depletion of water level in summer.
3. Materials and methods
Surfacial water samples (2*5 1) from four stations (I, II, III and IV — 'figure 1)
bathymetric water samples (2.4 1) from 3m, 6m and 0*5 m near the bottom of
the reservoir (V, VI and VII stations — figure 1) operating a closing type of bottle
sampler (1 • 2 1 capacity) twice near station III were collected monthly for two
years from February 1979. Temperature of water was read in the field by using
a mercury thermometer (0°-110°C). Secchi disc (Welch 1948) was used near
station III for determining the Zsd. In a field laboratory near the barrage pH and
conductivity were determined by using conductivity-pH meter (CLO1/03
Toshniwal). Carbonic species of water were estimated by titrimetry and the
samples were then brought to the Department of Zoology, Osmania University,
Hyderabad for further analyses of various chemical parameters according to APHA
AWWA and WPCF (1971).
4. Observations
4 . 1 Habitat
During the summer months of May and June of 1979 and 1980 the water level
in the reservoir receded considerably exposing a number of boulders, cobbles and
pebbles in the reservoir bed in the vicinity of the sampling stations I and IV.
These provided the substratum for the encrustation of the sponge, Corvospongilla
lapidosa. In summer the exposed reservoir bed presented dry caked mud that
is extensively fissured to a depth of 0 ' 4 to 0 • 5 m. During winter and rainy sea-
sons, the littoral region of the reservoir supports fairly rich emergent growth of
aquatic plants Typha angustata and Scirpus littoralis and submerged species of
Potamogeton perfoliatus and Vallisneria spiralis. The depth of the littoral varies
from 0*5 to 1 • 5 m. There are no trees on the margins of the reservoir to impart
any shade.
Ecobiology of Corvospongilla lapidosa
555
S
I
es
s
a
CM
o
o
>
,
H
S 8
556 I Seshagirl Rao and M A Khan
4.2. Ecomorphic characters
Corvospongilla grows on large boulders covering their exposed surfaces as a more
or less flat sheet of 0-6 to 2 -Ocm thickness (figure 2a). On small cobbles and
pebbles (figure 3a) the thickness of the sponge is reduced to 0-3 to 1 -0 cm. The
sponge body is steel grey or bluish black in colour. Near Station III the barrage
wall of the north flank was also found encrusted with this sponge which is
yellowish brown in colour. The water at this station is 6 to 9 • 5 m but usually
8 m deep and devoid of angiospermic vegetation.
Microscopic examination of the sponge body (figure 2) reveals the appearance
of a corrugated body surface with sausage shaped amphistrongylous spicules
lying embedded on the surface in different directions. Oscula are inconspicuous,
dispersed at random, some raised on irregular eminences. The thick chitinous
membrane at the base of the sponge body is tough and shows a few furrows
probably made by worms. The structure, shape and size of the spicules (figure
3b, c) conform to the descriptions of Annandale (1911) for the spepies,
4.3. Distribution
According to Khera and Chaturvedi (1976) the distribution of Corvospongilla
lapidosa is Maharashtra — Igatpuri lake, the river Godavari at Nasik ; Karnataka
— Bangalore ; Bihar — Santhal Paragana ; West Bengal — Barrackpore. Tonapi
(1964) reported it from small rivers near Poona. The occurrence of this species
of spongillid in the Manjira reservoir is a new record for this river and also for
Andhra Pradesh. Schizotype of this species was sent to Zoological Survey of
India, Calcutta and the identification was confirmed.
5. Discussion
From our observations Corvospongilla is able to thrive in water more than 4 m deep
and can encrust masonary constructions like the walls of a barrage. It was not
found either on molluscan shells or on aquatic plants. In the Manjira reservoir
it has not yet established itself near the south flank of the barrage. The reasons
may be lack of suitable substrata, dense growth of floating and submerged vege-
tation, epiphytic algae, feeble currents in the water, decaying organic matter at
the bottom that can clog the canal system of the sponge and relatively higher
concentrations of calcium and bicarbonates. Station II showed the highest mean
values of 22 -675 mg/1, 22.735mg/l for calcium and high mean values of 217-36
mg/1, 193 -71 mg/1 for bicarbonate in the first and second years of our study
(table 1). Jewell (1939) stated that some sponges are sensitive to calcium bicarbo-
nate concentration. The concentration of silica in the Manjira reservoir is low,
the mean value for all stations ranging from 0-983 to 2 -229 mg/1 (table 1) and
the maximum value of 6 mg/1 was recorded only once in February 1979 when
grouting work of the north flank bund was in progress. Allee et al (1955) stated
that silica content of lakes may be a limiting factor in the growth and distribu-
tion of fresh water sponges and their skeletal development may be much affected
Ecobiology of Corwspongilla lapidosa 559
Table 1. Stationwise and yearwise mean values of certain physico-chemical para-
meters— Manjira reservoir.
Parameters
I
II
III
Stations
IV
V
VI
VII
Calcium (mg/1)
17-302
22-675
16-
498
17-402
15
-164
15
•115
14-563
20-842
22-735
17-
568
17-
269
15
'431
13
-780
14-569
Bicarbonate (mg/1) 203 '07
217-36
201-
75
190'
18
198
•01
202
•10
209-71
180*52
193-71
178-
69
161-
80
174
•77
179
•60
177-33
Silica (mg/1)
2*042
0-983
1-
854
1-
813
1
•833
1-
833
2-229
1-813
1-718
1-
917
1-
792
1'
•750
1-
771
1-850
Turbidity
41
6-75
47
50
82
101
111
(Hydrazine units)
71
35
43
79
58
84
70
(Values in the upper row are for 1979-80 while those of lower row are for 1980-81)
by the quantity of available silica in waters. Jewell (1935) reported that Spongilla
lacustris living in water of silica content below 0-4 mg/1 and low conductivity
and solids shows progressive attenuation of its spicules, while marked variations
occurred in another species, Tubella pennsylvanica. But Corvospongilla shows
the preponderance of thin, hollow, sausage-shaped spicules over the relatively few
small isolated groups of birotulate and amphioxi spicules in its body. Apparently
these spicules have adaptive value to exploit the low silica environment of the
Manjira reservoir and interestingly it is the only species occurring in this reservoir.
The sausage shaped spicules offered greater surface area per unit mass of available
body silica and thereby enhanced the surface area of the body to increase the
ability to absorb the scarce silica from the ambient environment. The silica-lemma
of the developing spicule is known to be the site of inward transport of silica
for polymerizing it on its inner surface (Harrison and Cowden 1976). The
competition of C. lapidosa for the leachable silica is one of the factors for the
poor standing crop of planktonic diatoms in this reservoir (Rao 1982).
The water of the Manjira reservoir is prone to sudden, short but fairly frequent
spells of moderately high turbidity even though the mean values in table 1 suggest
that the water is optically less turbid. Excluding the II station, 33% of our data
points, showed more than 90 Hydrazine units of turbidity. Import of silt from
the catchment during floods, wind generated turbulence, water discharge across
the barrage cause high turbidity in this shallow reservoir. Abnormal values of
660, 3080, 2640 and 3800 units of turbidity were recorded at stations III, V, VI
and VII respectively on 14 June 19SO when four out of eleven flood gates were
raised to flush a part of the silt in the reservoir and even then this species was
found to survive.
As this species is found covering the entire exposed surface of the boulders and
even small stones, backwashing (Storr 1976) and amoebocyte scavenging acti-
vity (Harrison 1974) must be at work in this spongillid to prevent the silt fronj
560
I Seshagiri Rao and M A Khan
covering the body surface and clog the canal system. The low profile of the body
and the loosely architectured form in this species and the apparent flexibility of
the spicules are no doubt helpful in such an endeavour of the animal in tolera-
ting a wide range of turbidity.
The species ecology of North American spongillids (Harrison 1974, 1977 ;
Harrison et al 1977 ; Harrison and Harrison 1979) provided the basis for possi-
ble use of sponges as indicators of pollution. The paucity of data on the eco-
logical parameters of the Indian spongillids precludes, for the present, comparative
evaluation of the species ecology of C. lapidosa with its other, habitats and with
other spongillids of the Indian subcontinent. Meanwhile in the absence of any
point source of anthropogenic pollution at the Manjira reservoir, the physico-
chemical conditions of the habitat of C. lapidosa (table 2) provide the base data.
Table 2. Physico-chemical parameters of the Manjira reservoir habitat of
Corvospongilla lapidosa
Parameter
Data
points
Min.
Max.
Mean
Water temperature ( °C)
165
19-10
34-00
27- "73
pH
165
7-60
9*35
8-40
Conductivity (wS/cm at 25° C)
102
90
390
193-55
Secchi disc depth (Zsdm)
48
0*030
1-065
0-472
Turbidity (Hydrazine units)
161
1-6
386*
63
Suspended solids (mg/1)
161
o-o
456-0*
71-388
Dissolved solids (mg/1)
165
96-0
417- 0
236-756
Oxidizable organic matter as Oo
absorbed from KMnO4
(incubated for 3 hours at 37 °C)
165
0.00
12-72
2*801
Dissolved oxygen (mg/1)
165
2'45
18- 59
8*774
Carbon dioxide (mg/1)
165
0*00
14*52
1-02
Carbonate alkalinity (mg/1)
165
0"00
57-00
14* 375
Bicarbonate alkalinity (mg/1)
165
88-48
314*25
190-528
Chlorides (mg/1)
165
9-96
54'95
22-418
Sulphates (mg/1)
165
O'OO
39-25
18*896
Silica (mg/1)
165
0-40
6"00
1-74
Ammonia nitrogen (mg/1)
165
0*000
3'944
0-4915
Albuminoid nitrogen (mg/1)
165
0*000
3-697
0-6775
Nitrite nitrogen (mg/'i)
165
0' 0000
0-0186
0-0026
Nitrate nitrogen (mg/1)
165
O'OOO
0-750
0-0945
Total nitrogen (mg/1)
165
0*0022
7- 1271
1-2647
Orthophosphate phosphorus (mg/1)
165
0*000
0*90
0-0176
Total phosphorus (mg/1)
165
0*000
0*190
0*0394
Sodium (mg/1)
165
6'5
135*0
50-595
Potassium (mg/1)
165
1-7
4*9
2-85
Calcium (mg/1)
165
3-21
32-06
17-212
Magnesium (mg/1)
165
0*2
54-0
16-232
Total iron (mg/1)
165
O'OO
1*32
0-1465
(* Abnormal values when two or more flood gates are temporarily lifted are ignored)
Ecobiology of Corvospongilla lapidosa 561
On this evidence, it can be broadly concluded that C. lapidosa can inhabit rocky
substrata in alkaline, and turbid waters with low dissolved silica.
Acknowledgements
One of us (ISR) is thankful to the University Grants Commission, New Delhi,
for award of fellowship under Faculty Improvement Programme and to the Head
of the Department of Zoology, Osmania University for laboratory facilities.
Thanks are due to Dr G C Rao, zsi, Calcutta, for confirming the identifica-
tion and to DrM Babu Rao, zsi, Hyderabad, for useful suggestions and library
facilities. Grateful acknowledgements are to the Superintending Engineer,
Manjira water works circle for relevant hydrographic data and permission to
collect water samples and to Sri G Goutham Reddy, Assistant Engineer, for
excellent cooperation and help in the field work. Thanks are to Dr M Mahmood
for photography.
References
Allee W C, Emerson A E, Park O, Park T and Schmidt K P 1955 Principles of Animal
Ecology (London : W B Saunders Co.) p. 204
Annandale N 1907 On fresh water sponges from Calcutta and the Himalayas ; /. Asiatic Soc.
Bengal (n.s.) 3 15-26
Annandale N 1908 Preliminary notice of a collection of sponges from West India with
descriptions of two new species ; Rec. Indian Mus. 2 25-28
Annandale N 1909 Report on a small collection of sponges from Travancore ; Rec. Indian
Mus. 3 101-104
Annandale N 1909a Description of a new species of Spongilla from Orissa ; Rec. Indian Mus.
3 275
Annandale N 1911 The Fauna of British India including Ceylon and Burma Freshwater
sponges, Hydroids and Polyzoa (London : Taylor and Francis) 1-251
Annandale N 1912 Fresh- water sponges of Malabar zone ; Rec. Indian Mus. 7 3S3-398
Annandale N 1913 Notes on freshwater sponges No. XI Sponges from the shells of Aetheria;
Rec. Indian Mus. 9 237-240
Annandale N 1915 Notes on freshwater sponges No. XVI. The genus Pectispongilla and its
allies; Rec. Indian Mus. II 171-178
APHA, AWWA and WPCF 1971 Standard methods for the examination of water and waste
water, 13 edition, (eds.) M J Taras, A E Greenberg, R E> Hoak and M C Rand American
Public Health Association, Washington
Gist G N 1930 Notes on the Freshwater sponge Trochospongilla phillottiana and its varieties ;
Rec. Indian Mus. 32 491-495
Gist G N 1932 Spongilla carteri and its varieties ; Rec. Indian Mus. 34 185-195
Harrison F W 1974 Sponges (Porifera : Spongillidae). In Pollution ecology of freshwater
invertebrates, (eds) C W Hart, S L H Fuller (New York and London : Academic Press)
29-66
Harrison F W 1977 The taxonomic and ecological status of the environmentally restricted
spongillid species of North America. III. Corvomeyenia carolinensis Harrison 1971 ; Hydro-
biologia 56 187-190
Harrison F W and Cowden R R 1976 (eds) Aspects of sponge biology (New York and
London : Academic Press) 40-43
Harrison F W and Harrison M B 1979 Taxonomic and ecological status of environmentally
lestricted spongillid species of North America. IV. Spongilla heterosclerifera Smith 1918 ;
Hydrobiologia 62 107-111
562 / Seshagiri Rao and M A
Harrison F W, Johnston L, Stansell KB and McAndrew W 1977 The taxonomic and ecological
status of the environmentally restricted spongillid species of North America. 1. Spongilla
sponginosa Penny 1957 ; Hydrobiologia 53 199-202
Jewell M E 1935 An ecological study of the fresh-water sponges of Northern Wisconsin ; Eco .
Monogr. 5 461-504
Jewell M E 1939 An ecological study of the freshwater sponges of Wisconsin. H. The influence
of Calcium ; Ecology 20 11-28
Khera S and Chaturvedi Y 1976 Check-list of Indian freshwater sponges ; Rec. ZooL Surv.
India Occ. paper No. 4 pp. 29
Rao I S 1982 Ecology of the Manjira reservoir ; Ph.D. thesis (submitted)
Storr J F 1976 Field observations of sponge reactions as related to their ecology. In Aspects
of Sponge biology (eds.) F W Harrison and R R Cowden (New York : Academic Press)
277-282
Tonapi G T 1964 A note on the freshwater sponges of Poona ; Curr. Sd. 33 372-373
Welch P S 1948 Limnological methods (London : McGraw-Hill Book Co. Inc.) 159-160
Ecobiology of Corvospongilla lapidosa 559
Table 1. Stationwise and year wise mean values of certain physico-chemical para-
meters— Manjira reservoir.
Parameters
I
ir
III
Stations
IV
V
VI
VII
Calcium (mg/1)
17-302
22-675
16-
498
17"
402
15
•164
15
•115
14-563
20-842
22*735
17*
568
17-
269
15
-431
13
•780
14-569
Bicarbonate (mg/1) 203* 07
217-36
201-
75
190'
18
198
•01
202
•10
209*71
180-52
193-71
178-
69
161-
80
174
•77
179
•60
177-33
Silica (mg/1)
2-042
0-983
1-
854
1-
813
1
•833
1-
833
2-229
1-813
1-718
1-
917
1-
792
!•
•750
1-
771
1-850
Turbidity
41
6-75
47
50
82
101
111
(Hydrazine units)
71
35
43
79
58
84
70
(Values in the upper row are for 1979-80 while those of lower row are for 1980-81)
by the quantity of available silica in waters. Jewell (1935) reported that Spongilla
lacustris living in water of silica content below 0*4 mg/1 and low conductivity
and solids shows progressive attenuation of its spicules, while marked variations
occurred in another species, Tubella pennsylvanica. But Corvospongilla shows
the preponderance of thin, hollow, sausage-shaped spicules over the relatively few
small isolated groups of birotulate and amphioxi spicules in its body. Apparently
these spicules have adaptive value to exploit the low silica environment of the
Manjira reservoir and interestingly it is the only species occurring in this reservoir.
The sausage shaped spicules offered greater surface area per unit mass of available
body silica and thereby enhanced the surface area of the body to increase the
ability to absorb the scarce silica from the ambient environment. The silica-lemma
of the developing spicule is known to be the site of inward transport of silica
for polymerizing it on its inner surface (Harrison and Cowden 1976). The
competition of C. lapidosa for the leachable silica is one of the factors for the
poor standing crop of planktonic diatoms in this reservoir (Rao 1982).
The water of the Manjira reservoir is prone to sudden, short but fairly frequent
spells of moderately high turbidity even though the mean values in table 1 suggest
that the water is optically less turbid. Excluding the II station, 33% of our data
points, showed more than 90 Hydrazine units of turbidity. Import of silt from
the catchment during floods, wind generated turbulence, water discharge across
the barrage cause high turbidity in this shallow reservoir. Abnormal values of
660, 3080, 2640 and 3800 units of turbidity were recorded at stations III, V, VI
and VII respectively on 14 June 1980 when four out of eleven flood gates were
raised to flush a part of the silt in the reservoir and even then this species was
found to survive.
As this species is found covering the entire exposed surface of the boulders and
even small stones, backwashing (Storr 1976) and amoebocyte scavenging acti-
vity (Harrison 1974) m\ist be at work in this spongillid to prevent the silt from
560
I Seshagiri Rao and M A Khan
covering the body surface and clog the canal system. The low profile of the body
and the loosely architectured form in this species and the apparent flexibility of
the spicules are no doubt helpful in such an endeavour of the animal in tolera-
ting a wide range of turbidity.
The species ecology of North American spongillids (Harrison 1974, 1977 ;
Harrison et al 1977 ; Harrison and Harrison 1979) provided the basis for possi-
ble use of sponges as indicators of pollution. The paucity of data on the eco-
logical parameters of the Indian spongillids precludes, for the present, comparative
evaluation of the species ecology of C. lapidosa with its other habitats and with
other spongillids of the Indian subcontinent. Meanwhile ia the absence of any
point source of anthropogenic pollution at the Manjira reservoir, the physico-
chemical conditions of the habitat of C. lapidosa (table 2) provide the base data.
Table 2. Physico-chemical parameters of the Manjira reservoir habitat of
Corvospongilla lapidosa
Parameter
Data
points
Min.
Max.
Mean
Water temperature (°C)
165
19-10
34-00
27-73
pH
165
7-60
9*35
8'40
Conductivity (wS/cm at 25° C)
102
90
390
193-55
Secchi disc depth (Z8dm)
48
0-030
1-065
0-472
Turbidity (Hydrazine units)
161
1-6
386*
63
Suspended solids (mg/1)
161
o-o
456-0*
71-388
Dissolved solids (mg/1)
165
96-0
417' 0
236-756
Oxidizable organic matter as Oi>
absorbed from KMnO4
(incubated for 3 hours at 37 CC)
165
0.00
12-72
2-801
Dissolved oxygen (mg/1)
165
2*45
18-59
8*774
Carbon dioxide (mg/1)
165
O'OO
14-52
1-02
Carbonate alkalinity (mg/1)
165
o-oo
57-00
14*375
Bicarbonate alkalinity (mg/1)
165
88-48
314-25
190* 528
Chlorides (mg/1)
165
9-96
54-95
22-418
Sulphates (mg/1)
165
o-oo
39-25
lg-896
Silica (mg/1)
165
0-40
6-00
1'74
Ammonia nitrogen (mg/1)
165
O'OOO
3-944
0-4915
Albuminoid nitrogen (mg/1)
165
0*000
3-697
0-6775
Nitrite nitrogen (mg/l)
165
o-oooo
0-0186
0-0026
Nitrate nitrogen (mg/1)
165
o-ooo
0*750
0-0945
Total nitrogen (mg/1)
165
0-0022
7- 1271
1-2647
Orthophosphate phosphorus (mg/1)
165
0*000
0'90
0*0176
Total phosphorus (mg/1)
165
O'OOO
0-190
0*0394
Sodium (mg/1)
165
6*5
135-0
50-595
Potassium (mg/1)
165
1-7
4-9
2-85
Calcium (mg/1)
165
3-21
32-06
17-212
Magnesium (mg/1)
165
0*2
54*0
16-232
Total iron (mg/1)
165
o-oo
1*32
0-1465
(* -Abnormal values when two. or more flood gates are temporarily lifted are ignored)
Ecobiology of Corvospongilla lapidosa 36i
On this evidence, it can be broadly concluded that C. lapidosa can inhabit rocky
substrata in alkaline, and turbid waters with low dissolved silica.
Acknowledgements
One of us (ISR) is thankful to the University Grants Commission, New Delhis
for award of fellowship under Faculty Improvement Programme and to the Head
of the Department of Zoology, Osmania University for laboratory facilities.
Thanks are due to Dr G C Rao, zsi, Calcutta, for confirming the identifica-
tion and to Dr M Babu Rao, zsi, Hyderabad, for useful suggestions and library
facilities. Grateful acknowledgements are to the Superintending Engineer,
Manjira water works circle for relevant hydrographic data and permission to
collect water samples and to Sri G Goutham Reddy, Assistant Engineer, for
excellent cooperation and help in the field work. Thanks are to Dr M Mahmood
for photography.
References
Allee W C, Emerson A E, Park O, Park T and Schmidt K P 1955 Principles of Animal
Ecology (London : W B Saunders Co.) p. 204
Annandale N 1907 On fresh water sponges from Calcutta and the Himalayas ; /. Asiatic Soc.
Bengal (n.s.) 3 15-26
Annandale N 1908 Preliminary notice of a collection of sponges from West India with
descriptions of two new species ; Rec. Indian Mus. 2 25-28
Annandale N 1909 Report on a small collection of sponges from Travancore ; Rec. Indian
Mus. 3 101-104
Annandale N 1909a Description of a new species of Spongilla from Orissa ; Rec. Indian Mus.
3275
Annandale N 1911 The Fauna of British India including Ceylon and Burma Freshwater
sponges, Hydroids and Polyzoa (London : Taylor and Francis) 1-251
Annandale N 1912 Fresh- water sponges of Malabar zone ; Rec. Indian Mus. 7 383-398
Annandale N 1913 Notes on freshwater sponges No. XI Sponges from the shells of Aetheria;
Rec. Indian Mus. 9 237-240
Annandale N 1915 Notes on freshwater sponges No. XVI. The genus Pectispongilla and its
allies; Rec. Indian Mus. II 171-17$
APHA, AWWA and WPCF 1971 Standard methods for the examination of water and waste
water, 13 edition, (eds.) M J Taras, A E Greenberg, R D Hoak and M C Rand American
Public Health Association, Washington
Gist G N 1930 Notes on the Freshwater sponge Trochospongilla phillottiana and its varieties ;
Rec. Indian Mus. 32 491-495
Gist G N 1932 Spongilla carteri and its varieties ; Rec. Indian Mus. 34 185-195
Harrison F W 1974 Sponges (Porifera : Spongillidae). In Pollution ecology of freshwater
invertebrates, (eds) C W Hart, S L H Fuller (New York and London : Academic Press)
29-66
Harrison F W 1977 The taxo-nomic and ecological status of the environmentally restricted
spongillid species of North America. III. Corvomeyenia carolinensis Harrison 1971 ; Hydro-
biologia 56 187-190
Harrison F W and Cowden R R 1976 (eds) Aspects of sponge biology (New York and
London : Academic Press) 40-43
Harrison F W and Harrison M B 1979 Taxonomic and ecological status of environmentally
restricted spongillid species of North America. IV. Spongilla heterosclerifera Smith 1918 ;
Hydrobiologia 62 107-111
562 / Seshagiri Rao and M A
Harrison F W, Johnston L, Stansell K B and McAndrew W 1977 the taxonomic and ecological
status of the environmentally restricted spongillid species of North America. 1. Spongilla
sponginosa Penny 1957 ; Hydrobiologia 53 199-202
JeweU M E 1935 An ecological study of the fresh-water sponges of Northern Wisconsin ; Eco .
Monogr. 5 461-504
Jewell M E 1939 An ecological study of the freshwater sponges of Wisconsin, n. The influence
of Calcium ; Ecology 20 11-28
Khera S and Chaturvedi Y 1976 Check-list of Indian freshwater sponges ; Rec. Zool. Surv.
India Occ. paper No. 4 pp. 29
Rao. I S 1982 Ecology of the Manjira reservoir ; Ph.D. thesis (submitted)
Storr J F 1976 Field observations of sponge reactions as related to their ecology. In Aspects
of Sponge biology (eds.) F W Harrison and R R Cowden (New York : Academic Press)
277-282
Tonapi G T 1964 A note on the freshwater sponges of Poona ; Curr. Sci. 33 372-373
Welch P S 1948 Limnological methods (London : McGraw-Hill Book Co. Inc.) 159-160
ftroc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 6, November 1982, pp. 563-561
© Printed in India.
Seasonal fluctuations in the diet composition of Rhinopoma
hardmckei in the Rajasthan desert
RANJAN ADVANI
Rodent Research Centre, Central Plantation Crops Research Institute,
Kasaragod 670 124, India
MS received 30 January 19&2 ; revised 27 July 1982
Abstract. The small mouse-tailed bat, Rhinopoma hardwickei, collected from various
districts of Rajasthan, is primarily an insectivorous species. Orthoptera, Dictyoptera,
Lepidoptera, Hymenoptera, Coleoptera and Diptera are preferred in all main four
seasons in varying amounts, while Isoptera are consumed in all but the winter
season. Occurrence of ground dwelling insects, caterpillars, spiders and water
beetles in the stomachs of bats have been discussed in the light of behavioural
adaptations of this species. Presence of fur of same bat species in stomachs coincides
with its breeding season. Presence of various polyphagous insect pest species
of crops in feeding menu of bats shows that this species plays an important role in
biological management of harmful insects.
Keywords. Rhinopoma ; diet composition ; biological management; insect pest.
I. Introduction
The small mouse-tailed bat, Rhinopoma hardwickei Gray, 1831 (Chiroptera : Rhino-
pomatidae) is a fairly well distributed species in Rajasthan which is part of the
Great Indian Thar desert (24 -5-30- 5° N ; 60-70° E). Associated with arid and
semi arid regions, of which it is adapted ecophysiologically, this species is confined
to subtropical latitudes. In the Indian subcontinent this bat is absent from forested
regions of Ghats. In its diurnal roost, it coexists with other bat species of
Rhinopoma microphyllum kinneari and Taphozous spp. inhabiting natural caves,
man made cellars, and underground irrigation tunnels.
In spite of the occurrence of R. hardwickei in abundance, constituting of 9*63%
of the total bat fauna of desert biome of Rajasthan (Advani 198 la), except some
reports (Advani and Vazirani 1981 ; Prakash 1963 ; Sinha and Advani 1976),
little is known about the ecology, biology and behaviour of this species. The present
studies were undertaken to investigate the food composition and seasonal variation
in the feeding pattern of this species.
563
P.(B)-9
564 Ran/an Advani
2. Study area
The Indian desert has four distinct seasons in one year, receiving different magni-
tudes of rainfall, temperature fluctuations, relative humidity and sun shine hours.
These factors individually and/or combinely have an impact on reproduction, abun-
dance and activity of animal life in the desert including insects and bats. In winter,
the mean maximum and minimum temperatures are 25*4 and 9 -5° C respectively,
with a mean rainfall of 2 • 8 mm and a mean relative humidity of 22% (Prakash et al
1971). In summer, the temperature is very high (mean max t = 39-8° C, mean min
t = 27'9°C), with 18 -2 mm mean rainfall and 20*1 mean relative humidity. In
the monsoon months, the mean rainfall is 110 -5 mm, relative humidity being
47 '5% and the temperature (mean max t =J34B5° C, mean min. t = 25'1°C)
lesser than that of summer. In post-monsoon season (October and November)
the temperature fluctuations are 28'5°C (max.) to 10-8°C (min). The relative
humidity is 20*5%.
3. Materials and methods
The bats were collected during various seasonal and periodical faunistic surveys
conducted by Desert Regional Station, Zoological Survey of India of twelve
districts— Jodhpur, Barmer, Nagaur, Pali, Dungarpur, Banswara, Jhalawar, Tonk,
Boondi, Ajmer, Sawai Madhopur and Kota, well distributed in arid and semi arid
parts of Rajasthan State. 171 individuals were collected and examined. For
each season, the break up of the sample size (N) is shown in table 1. After
anaesthesia the bats were dissected and their alimentary canals cut open. The
stomach contents were taken out with a brush and forceps and then dried on filter
paper at room temperature. After sorting, stomach items were identified to the
lowest taxonomic level feasible (Order-Family) through the aid of microscope
later these items were weighed on the balance to calculate their percent frequency
of occurrence in the stomach contents following Murton et al (1964).
The seasonal fluctuations in the feeding pattern were determined by pooling
data among four main seasons occurring in the Indian desert.
4. Results
The examination and analysis of the stomach contents revealed that R. hard-
wickei is primarily an insectivorous species, though some traces of vegetable matter
were also observed in summer and monsoon (rainy) seasons (table 1). Fur of
the same bat species occurred during summer and monsoon, whereas, it was com-
pletely absent during post-monsoon and winter. There were no remains of other
animals except insects and spiders.
In winter December to February, Orthoptera (gryllids, house crickets) and
Coleoptera (beetles) constitute more than 45% of the total diet. However,
Hymenoptera (ants) Lepidoptera (moths), Dictyoptera (Cockroaches) and
diptera (flies, mosquitoes) are also preferred in appreciable amounts in decreasing
order. Araneida (spiders) were noted in the stomach in moderate proportions
during this season.
Diet composition of JR. hardwickei
565
Table 1. Seasonal fluctuations in stomach contents of Rhinopoma h. hardwickei,
expressed in percent of total dry mass.
Stomach items
Winter
(Dec.-Feb.)
JV= 36
Seasons
Sumrnei Monsoon
(Mar. -June) (July-Sept.)
JV= 43 N= 45
Post-monsoon
(Oct.-Nov.)
N= 47
Orthoptera
GrylUdae
22^
15-1
12-1
10-4
Acrididae
4-0
8-5
5-5
2-9
Isoptera
Tennitidae
...
10-0
28-2
6-8
Dictyoptera
10-9
11-8
1-3
4-7
Lepidoptera
Noctuidae
4-5
8-1
3-8
4-4
Arctidae
6-1
8-2
10-1
8-2
Unidentified
1-2
...
1-0
0-5
Caterpillars
...
1*2
3-2
1-1
Hymenoptera
Vespidae
1-2
5-0
5-8
8-8
Formicidae
14-2
4-3
4-5
7-4
Neuroptera
Mantispidae
2-4
...
1-4
1-3
Diptera
Chironomidae
3-7
...
...
2-2
Culicidae
4-5
1-2
1-3
«*•
Unidentified
1-0
...
1-1
0-5
Coleoptera
Scarabaeidae
8-5
4.9
8-3
14-1
Curcutionidae
3-4
7-3
3-2
11-5
Carabidae
4-1
2-0
2-C
12-2
Bruchidae
2-0
1-0
...
...
Dytiscidae
1-2
4-0
...
...
Unidentified
...
1-3
2-2
3-0
Araneida (Spideis)
4-9
1-2
...
...
Bafs own fur
...
4.9
3-8
...
Plant parts
...
0*5
1-2
...
566 Ranjan Advani
Table 2. *jp* values indicating seasonal difference in diet composition of
JR. hardwickei.
Major stomach
items
*W-S
N-79
6P' values
S-M
N=8£
between successive seasons
M-PM PM-W
N= 92 N== 83
Orthoptera
0-05
0-05
NS
0-05
Isoptera
0-001
0-001
0-001
0-001
Dictyoptera
NS
0-001
0-05
0-001
Lepidoptera
0-01
NS
NS
NS
Hymenoptera
0-05
NS
0-05
NS
Neuroptera
0-001
0-01
NS
NS
Diptera
0-001
NS
0-05
0-001
Coleoptera
0-05
0-05
0-001
0-001
Araneida
0-05
0-01
0-05
0-001
Bat fur
0-001
NS
o-ooi
NS
Plant parts
0-05
0-05
0-05
NS
*W— Winter, S— Summer, M— Monsoon, PM— Post-Monsoon, NS— Non Significant,
N— Total number of bats observed after dissection.
In summer there is an. increase in the relative occurrence of Lepidoptera (P < 0 *01)
while Isoptera, (termites, Odontotermes obessus, Anacanthotermes sp.) which are
absent in winter, form about 11% of the total diet (table 2). However, Hymenop-
tera, Diptera and spiders reduce considerably (P ± 0*05, O'OOl and 0"05
respectively). Preference for Coleoptera (P <0'05), Orthoptera (P <0-05) and
Dictyoptera remains more or less same as in winter season.
During monsoon months, when there is abundant insect life in nature, there
is a significant rise in the consumption of winged soft-bodied termites (Micro-
termes obesii, O. obessus, Anacanthotermes sp.), slightly less than three times
(28 *2%) of the summer season (P < O'OOl). Relative percent frequency of Coleop-
tera (P < 0-01) and Orthoptera (P < O'Ol) declines further, while that of Hymenop-
tera, Diptera and Lepidoptera increases slightly. However, drastic reduction is
observed with regard to relative occurrences of Dictyoptera and Spiders in the
diet (P < 0-001 and 0-01).
In the two months of post-monsoon season, October and November, beetles
mainly belonging to families Scarabaeidae (while grubs, Holotrichia spp.),
Curciiionidae and Carabidae constitute major proportion of the diet of bats.
Occurrence of ants also increases considerably in the stomachs. Isoptera
reduces abruptly, whereas, moderate decline is found regarding consumption of
Orthoptera and Lepidoptera. Spiders, bat's own fur and plant parts do not
figure at all in this season.
Diet composition of R. hardwickei . $67
4. Discussion
The small mouse-tailed bat, R. hardwickei is primarily an inhabitant of the cave
and rocky habitat while about 16% population roosts near or in the midst of human
settlement (Advani 1981a). Its roosting habitats have certainly an impact on its
feeding behaviour particularly in deciding the composition and seasonal relative
occurrence of various insect orders like Diptera, Dictyoptera, Hymenoptera and
Orthoptera which are available in and around human environment and Coleoptera
(bruchids, scarabaeids, carabidds), Isoptera and Lepidoptera which occur in abun-
dance in agro-ecosystems and forested rocky habitat. However, it appears that the
feeding habits of this species are also probably a combination of opportunism and
selective predation, varying with local ecobiotic conditions such as relative
abundance of different kinds of vegetation patterns on which the insect fauna exists.
Occurrence of traces of plant parts in the stomachs of bats during summer and
monsoon is perhaps due to the remains of undigested gut contents of insects eaten
by bats. The presence of orthopterans, caterpillers of Lepidoptera, spiders and some
ground beetles suggests that this species also feeds by picking these animals
from the ground or other surfaces. Likewise, as observed the drinking behaviour
of R. hardwickei of skimming over the water surfaces is also very similar to those
of allied species R. microphyllum (Advani 1981b). However, the requirement of
water is also compensated in the desert by the almost exclusive diet of the insects
which contain 80-90% water (Robinson 1928). The presence of water beetles
(dytiscids : Lacconectus sp., Agabus sp. Rhantus sp.) in the stomachs indicate the
ability of bats to swoop over the water surfaces and pick up the most active insects.
Regarding composition of food items, R. hardwickei markedly differs from the
Indian false vampire, Megaderma lyra lym which depends upon an equal propor-
tion of insect and the vertebrate (lizards, fishes, birds etc.) animal diet (Advani
198 lc) on an annual basis.
Seasonwise, during winter when temperature falls to about 4 -5° C in the Rajas-
than desert, the bats are relatively inactive and they thrive upon insects available
in their vicinity or home ranges. These include mosquitoes, flies, gryllids, house
crickets, cockroaches, ants and beetles, forming major portion of their diet. In
this season bats under extreme climatic conditions can also subsist upon their own
fat reserves which they accumulate after the monsoon season. During summer
and monsoon months preference for termites is quite obvious, as this period coin-
cides with emergence of winged, soft bodies, slow flying termites after the first
few showers (From mid June onwards) in Rajasthan. Likewise, in post-monsoon
season, occurrence of winged ants and wasps and abundant beetles determine the
diet composition of this species. However, the climatological differences among
four main seasons, mainly temperature and rainfall variations, govern reproduction,
metamorphosis and abundance patterns of various insect orders which act as food
for insectivorous bats. These parameters also cause changes in activity and
behaviour of bats, as in winter being relatively less active their foraging range is
confined to places nearby roosting habitat.
The survival of bats upon some of the most prominent and polyphagous insect
species of summer as well as winter crops in Rajasthan desert like 0, obessus^
568 Ranjan Advani
M. obesii (termites) ; White grubs (Holotrichia spp.) and Cuculionids (Coleoptera)
and several grasshopper species, evidently show that this species plays an important
role in the ecological balance of the population of these harmful insects in natural
crop ecosystem. On the other hand, occurrence of predatory insects like Neurop-
tera in stomachs, though in low relative percentages, points out towards non-
beneficial aspect of feeding ecology of this species.
Acknowledgements
The author expresses his thanks to the Director, Zoological Survey of India,
Calcutta, for providing facilities, to Dr T. G. Vazirani, Sr. Entomologist, C.I.E.,
British Museum (Nat. Hist.), London for identification of Coleoptera. He is also
very grateful to Dr Ishwar Prakash, Principal Animal Ecologist at
Central Arid Zone Research Institute who encouraged me to take bat ecology as
Ph.D. problem.
References
Advani R 1981 a Biaecological evaluation of the Chiroptera fauna of desert biome of Rajasthan ;
Zeit. angew. Zool 68 281-305
Advani R 1981b Food and feeding ecology of the Rat-tailed Bat in the Rajasthan desert ; Acta
Theriol 26 269-272
Advani R 1981 c Seasonal fluctuations in the feeding ecology of the Indian false vampire,
Megaderma lyra lyra (Chiroptera : Megadermatidae) in Rajasthan ; Zeit. jur Saugetierk.
46 90-93
Advani R and Vazirani T G 1981 Studies on ectoparasites of bats of Rajasthan and Gujarat ;
Rec. Zool. Surv. India Delhi, Occasional Paper No. 22 1-155
Murton R K, Westwood M J and Isaacson A J 1964 Feeding habits of the Wood pigeon,
Columba palambus, Stockdove, Columba cenas and Turtle dove, Streptopelia turtur. Ibis,
106 177-188
Prakash I 1963 Taxonomic and biological observations on the bats of the Rajasthan Desert ;
Rec. Ind. Mus. 59 149-170
Prakash I, Gupta R K, Jain A P, Rana B D and Dutta B K 1971 Ecological evaluation of
rodent populations in the desert biome of Rajasthan; Mammalia 35 384-407
Robinson W 1928 Water conservation in insects ; /. Econ. Ent. 21 897-902
Sinha Y P and Advani R 1976 Notes on food and reproduction of some Rajasthan bats ;
Geobios, 3 37-40
Prac. Indian Acad- Sci. (Anim. Sci.), Vol. 91, Number 6, November 1982, pp. 569-576.
© Printed in India.
The annual reproductive cycle of Achaetobonellia maculata Fisher
(Echiura : Bonellidae)
R N SINGHAL
Department of Zoology, Kurukshetra University, Kurukshetra 132 119, India
MS received 10 April 19S2 ; revised 23 September 1982
Abstract. This study is the first to detail the annual reproductive cycle of any
echiuran. Here the annual reproductive cycle of Achaetobonellia maculata Fisher
is described. Oocytes first appear in the coelomic fluid in late spring or early
summer. During fall and winter, gametes production and differentiation continue.
Differentiation of gametes lasts four to six months. Spawning occurs in spring.
Since the males are the permanent residents in the gone duct of the female, the
fertilization is internal in Bonellidae. Temperature of the sea water probably is
the mast important exogenous factor controlling the reproductive cycle. Indi-
viduals reach sexual maturity when they are o-ne year old.
Keywords. Reproductive cycle ; spawning ; gonoduct ; accessory cells ; oocyte.
1. Introduction
Little is known about the annual reproductive cycle in Echiura. Although some
investigators (Hiraiwa and Kawamura 1936 ; Newby 1940) have reported Urechis
caupo to be fertile throughout the year (with the exception of one or two months
in the summer) this appears to be based solely on general observations but no
firm data exist.
A general description of reproduction of Echiura has been provided by Gould-
Somero (1975), and Singhal and DattaGupta (1982). Gould-Somero (1975)
mentioned that the fertilization is internal in Bonellidae., but the males are already
permanent residents in the male-sac of the female gonoduct. In other Echiurans
fertilization is external but we do not know what (if any) factors ensure simul-
taneous spawning by males and females in neighbouring burrows. Partially or
completely spawned-out U. caupo has been collected in late summer (Ricketts
and Calvin 1962 ; Gould 1967), and animals will sometimes spawn in the labo-
ratory if the water temperature is raised above 15° C. Therefore, temperature
may be a factor. Pilger (1977) studied the annual reproductive cycle in Listriolobus
pelodes. He found that ovulation lasts three to five months and spawning takes
place in spring. Singhal and DattaGupta (1982) reported that oocytes were present
569
570 R N Singhal
in the coelom of Achaetobonellia maculata and Acanthobonellia vulgans for about
nine months in a year. This study included a determination of the size at which
A. maculata becomes sexually mature, the structure of the gonads, the development
of the reproductive cells in the coelom and the time, durations and geographical
variation in spawning.
2. Materials and methods
A. maculata is a common species of the Pirotan Island, Gulf of Kutch (DattaGupta
and Singhal 1978). Since its discovery by Fisher (1953) from the central lagoon
of Onotoa, Gilbert Island, the species has not been reported from anywhere except
from the aforesaid locality. The population of A. maculata from Pirotan Island
was studied for its reproductive cycle during 1976 and 1978. Specimens were
collected and fixed every month using the method described by Singhal and
DattaGupta (1980). Gonads were fixed in Gilson's fluid sectioned at 8^ and
stained with Delafield's hematoxylin and eosin.
In A. maculata the young oocytes are associated with a complex of accessory
cells during the first stage of development. Later they lose these cells and continue
to develop without them. Oocytes with accessory cells will be referred to as
" stage I " and those without them as " stage II ". To determine the cycle, a
sample of coelomic fluid was withdrawn with a syringe from each of five females
for each month. Twenty oocytes of each stage were measured for their diameter in
each female making a total of 100 for each stage per month. The means of stage
I and stage II were adjusted according to the percentage contribution of each
stage to the coelomic gamete population. This was accomplished by calculating
the mean weighted diameter for each month using the formula :
PSIn X Sin -f PSIIn X Slln = mean, weighted diameter where PSIn and PSIIn
are the percentage of stage I and stage II oocytes respectively in the coelom of
five female A. maculata, during the month n. The values XSIn and XSIIn are
the mean diameters for the stage I and stage II oocytes during the same month.
As another measure of reproductive periodicity, the concentration of stage I
and stage II oocytes within the coelom was determined. To accomplish this, a
small sample of coelomic fluid was removed. After fixation and during storage,
individual A. maculata tend to lose coelomic fluid by diffusion through the body
wall and appear deflated although the actual volume it can contain remains un-
changed. The net result of this is that the coelomic cells are more concentrated
than under normal conditions. To remedy this situation, the individuals were
" inflated " with 70% isopropyl alcohol until they reached a subjectively determined
uniform tension. Each specimen was shaken to mix the coelomic cells before
removal of the sample. The sample then was diluted by an equal volume of
70% isopropyl alcohol and the concentration of each gamete type was determined
using a hemocytometer. Ten values were obtained from each of the five females
every month from September 1976 to December 1978.
The reproductive cycles were analyzed with several environmental parameters
using a multiple regression analysis program. This program is part of the Statis-
tical Analysis System (SAS) and was developed by Barr et al (1976). Also from
Reproductive cycle of A. maculata Fisher 571
SAS, Backward Elimination and Maximum JR2 Improvement variable selection
procedures were used to determine which, if any, of the parameters contribute
significantly to the cycle. •< Coelomic gamete concentration and weighted gamete
diameter are handled separately as the dependent variables. The independent
variables include DDT, cadmium, organic nitrogen, sulphide, and bottom tempe-
rature.
3. Results
The differentiation of gametes consists of three distinct phases. The gametes begin
their development while attached to the gonads. They are in the second phase
when they break loose and continue their growth floating freely within the coelom.
Finally mature gametes are collected and stored in the gonoducts until spawning.
The structure of the gonads of A. maculata has already been described (Singhal
and DattaGupta 1982).
3.1. Size at sexual maturity
The smallest sexually mature female specimen found weighted 2*0g and measured
25 mm long from mouth to anus. In a sample of 100 female individuals, none
weighing less than 2 -0 g was sexually mature. Since the male is a permanent resident
of the male-sac of the gonoduct of the female, the weight of the female specimen
also includes the weight of the male and it cannot be determined as to what is the
smallest size and weight of the male for sexual maturity.
3.2. Coelomic oocyte diameters
The mean diameters of stage I and stage II coelomic oocytes are shown in figure 1.
Each point represents the mean of 100 measurements and the solid bar equals one
standard deviation. The smallest stage I oocytes are present in the coelom during
the summer months. These cells are 5-7 ^m in diameter. The mean diameter of
these oocytes begins to increase during the early fall and by November has reached
22 fan. Since this is a mean value, it does not indicate the upper size limit of stage I
oocytes. The actual size of an oocyte when it loses its accessory cells is 40 to 42 jura
in live material. A mean diameter of app. 22/mi is maintained until late spring
when it begins to decrease. This decrease is due to the transformation of large
stage I oocytes into stage II.
Stage II oocytes appear in November at the time when the stage I oocytes first
reach their maximum mean diameter. The mean size of stage II oocytes increases
through the spring. By June, all of the stage II oocytes have been collected from the
coelom by the gonostome and accumulated in the egg-sac of the gonoduct. The
actual size of an oocyte when it is removed from the coelom is 60-62 jam stage II
oocytes are not present in the coelom again until fall.
Also shown in figure 1 is the weighted average diameter of stage I and stage II
oocytes combined. An annual cycle clearly is seen in this representation. Small
oocytes first appear in the summer, Most of their growth takes place during the
fall and winter months.
572
R N Singhal
80
70
60
'50
0
UJ40
o
§30
20-
JFMAMUJASONO
Figure 1. The mean diameter of coelomic oocytesin^t. maculata. The bar equals
±1 standard deviation. •= Stage I oocytes ; A= Stage II oocytes ; O =
weighted mean diameter of stage I and stage II oocytes combined.
The diameters of fixed oocytes differ from those of live oocytes. This was tested
by measuring the diameters of 100 live oocytes and 100 fixed oocytes from the same
individual and comparing their means. The results show that there is less than a
3% increase in the average diameter after fixation. Since relative values are more
important than absolute values, the increase is considered insignificant.
3.3. Coelomic oocyte concentration
Figure 2 indicates the concentration of coelomic oocytes. During spring and first
half of the summer stage I oocytes are at their lowest concentration (2-2-3'5/mm3).
In specimens collected in July and August, stage I oocytes concentration begins to
increase. By September, the concentration has increased four-fold. By November,
stage I oocytes reach their highest concentration and become stage II oocytes
by the loss of the accessory cells. As more and more stage I oocytes reach this
point their concentration slowly decreases, reaching the lowest level again in summer.
Oocytes first appear in November and rapidly reach maximum concentration.
From December through spring their concentration declines steadily as they are
accumulated in the gonoduct. By June, stage II oocytes are not present in the
coelomic phase at all.
These data illustrate the same reproductive cycle as do the oocyte diameter data.
During the summer, few coelomic gametes are present. Their number increase
through the fall and early winter. During late winter and spring they are collected
in the storage organ until spawning. Spawning apparently extended from spring
until the end of winter.
Reproductive cycle of A. maculata Fisher
573
20
8
o
o"
10
STAGE I
,0 30
e
N.
cn
LU
i- 20
>
o
O
o
STAGE II
FMAMJJ ASON 0
Figure 2. The mean concentration of stage I and stage II coelomic oocytes in
A. maculata. The bar equals 95% confidence interval.
3.4. Regression analysis of abiotic parameters with the reproductive cycle
By regressing the abiotic data from Pirotan Island (DDT, cadmium, organic
nitrogen, sulphide, and bottom temperature) against the weighted mean oocyte
diameter, it was determined that the environmental parameters did not account
significantly for the variation of oocyte size (P < 0-05). However, temperature,
nickle and sulphides accounted for a significant amount of the oocyte size variation
during the study period (R2 = 0'78, P<0'01). Both Backward Elimination
and Maximum R* Improvement techniques generated the same three-variable
model that accounts for 92% of the variation in oocyte diameter over time (R* =
0-92, P < 0-001). The independent variables selected by these procedures are in
order of decreasing importance, bottom water temperature (P <0'01), concen-
trations of nickle (P < O'Ol) and sulphides (P < 0*05) in the sediment. Adding
the remaining independent variables does not significantly improve the predicta-
bility of the model.
Regressing all of the independent variables (environmental parameters) against
the mean coelomic oocyte concentrations showed no significant contribution
(R*= 0-85, P < 0-05). By variable selection methods, however, it was found
that two parameters contribute nearly 81% of the variation (R2 = 0-79, P < O'Ol).
These are in order of decreasing importance, the concentrations of sulphide
(P < 0-01) and DDT (P < 0-05). Thus, only a few of the parameters measured are
important in determining the number of oocytes produced.
574 R N Singhal
4. Discussion
Unfortunately there is still insufficient information available on the annual repro-
ductive cycles of Echiura to determine how typical the cycle of A. maculata is.
The smallest sexually mature A. maculata encountered in this study is one year
old female, 25mm long, weighing 2-Og. This is reasonably consistent with the
observation of Fisher (1946) who found a 7 mm mature specimen. Baltzer (1931)
reported that females of Bonellia viridis require two years to reach sexual maturity
while the males mature in one or two weeks. U. caupo also requires one year to
reach sexual maturity.
No studies of annual reproductive cycles in echiurans are available for comparison.
However, seasonal gamete production has been reported in the echiurans Ikedosoma
gogshimense (May and June) (Sawada and Ochi 1962) and U.-unicinctus (Winter)
(Hiraiwa and Kawarnura 1936). In direct contrast to this, U. caupo produces
gametes continuously and contains all oocyte sizes in the coelom simultaneously
(Gould-Somero 1975).
The dynamics of oocyte development including the transition from stage I to
stage II have been illustrated in diameter frequency polygons. Because A. maculata
does not produce gametes continuously throughout the year, the frequency of the
various oocyte size classes is not proportional to the amount of time the oocyte
spends in a particular size class as has been suggested for U. caupo (Gould-Somero
1975).
Although the frequency polygons do not provide direct information as to the
time course of oogenesis, a rough estimate can be made based on the distribution
of the mean diameter of stage I and stage II oocytes ov^r time. The duration
of stage I can be estimated by determining the time interval between the onset of
increase of stage I mean diameter, which occurs in summer, and the first appearance
of stage II oocytes. For instance, at Pirotan Island, after summer the mean diameter
of stage I oocyte of A. maculata began to increase one month later, stage I oocytes
had grown to 22 pm and had become stage II. Thus, it is predicted that stage I
lasts from one to two months.
The period of time from the initial appearance of stage II oocytes in the coelom
until they reach their maximum diameter provides an estimate of the duration of
this phase of growth. Stage II oocytes appeared in coelom after being absent
over the summer and reach their maximum diameter after two months, indicating
a two-month period of differentiation. Based on the data available for summer,
the duration of stage II differentiation is estimated to be 1^ months (± \ month).
These data predict, therefore, that stage II lasts from one to two months.
Combining the estimate for stage I and stage II oocyte differentiation gives a
range -for the time course of oogenesis of three to five months.
Das (1976) has studied the cytochemical and biochemical processes of oogenesis
in Urechis. By radioactive labelling he has determined that the duration of the
period of oocyte differentiation is 135 days. This closely resembles the estimate
for A. maculata derived from the reproductive cycle data.
Based on the oocyte diameter data, spawning among A. maculata population
during spring. The data show that the exact time of spawning and the
Reproductive cycle of A. maculata fisher 575
length of the period preceding resumption of oocyte growth can vary from year
to year (Giese 1959a).
The regression analysis demonstrates that temperature plays an important part
in determining the gametogenic cycle. Orion's Rule first proposed by Thorson
(1946) states that sea temperature is related to the reproductive cycles of marine
organisms. While this is important to many animals, other exogenous and endo-
genous factors may also play vital roles (Giese 1959b ; Giese and Pearse 1974).
Controlled laboratory experiments are necessary to define the environmental
components essential for determining any gametogenic cycle (Giese and Pearse
1974).
Acknowledgements
The author is grateful to Prof. A K DattaGupta, Professor of Zoology, Kuru-
kshetra University, Kurukshetra, for guidance and laboratory facilities.
References
Baltzer F 1931 Eatwicklungsmeclianische Untersuchungen an Bonellia viridis. Die Abhaugigkeit
der Entwicklungsgeschwindigkeit und des Entwicklungsorades der mannlichen larvae von
der dauer der Russelparasitismus ; Rev. Suisso ZooL 38 361-371
Barr A J, Goodnight J H, Sail J P and Helwig J T 1976 A User's Guide to SAS (Raleigh :
Sparks Press) 1-329
Das N K 1976 Cytochemical and biochemical analysis of development of Urechis oocytes ;
Am. ZooL 16 345-362
DattaGupta A K and Singhal R N 1978 Morphology and histology of the males of Acantho-
bonellia ; Proc. Royal Soc. Edin. 68 267-276
Fisher W K 1946 Echiuroid worms of the North Pacific Ocean ; Proc. U.S. nat. Mus. 96
215-292
Fisher W K 1953 A new genus of bonellid worms (Echiura) ; /. Wash. Acad. Sci. 43 258-259
Giese A C 1959a Comparative Physiology : Annual reproductive cycles of marine invertebrates ;
A Rev. PhysioL 21 547-576
Giese A C 1959b Reproductive cycles of some West Coast Invertebrates. In Photoperiodism
and Related Phenomena in Plants and Animals ; (ed.) Robert B Withrow (Washington
D.C. : Am. Assoc. Adv. Sc.) 1-55
Giese A C and Pearse J 1974 Introduction : General principles of reproduction of marine inverte-
brates (San Francisco ; Academic Press) 1 501-546
Gould M C 1967 Methods in Developmental Biology (New York : Crowell) 1-163
Gould-Somero M 1975 Reproduction in marine invertebrates (San Francisco : Academic Press
Inc.) 3 201-277
Hiraiwa Y and Kawamura T 1936 Relation between maturation division and cleavage in arti-
ficially activated eggs of Urechis unicinctus ; Biol. Bull. 70 344-351
Newby W W 1940 The embryology of the echiuraid worm Urechis caupo ; Mem. Am. Phil.
Soc. 16 1-213
Pilger J F 1977 Biology and gametogenesis of Listriolobus pelodes \ Ph.D. Thesis, University
of California 1-205
Ricketts E and Calvin J 1962 Between pacific tides (California ; Stanford Univ. Press) 1-308
R N Singhat
Singhal R N and DattaGupta A K 1980 Ecological notes an a few Echiutan Animals ;
Indian J. Marine Set. 9 139-140
Singhal R N and DattaGupta A K 1982 Studies on some aspects of reproduction in Achaeto-
bonellia maculata Fisher (Echiura : Bonellidae) ; N.Z. J. of Zool. 9 215-231
Sawada N and Ochi O 1962 Studies on the fertilization in eggs of the echiuroid, Ikedosoma
gogoshimense (Ikeda) ; Mem. Echime Univ. 4 437-444
Thorson G 1946 Reproduction and larval development of Danish marine bottom inverte-
brates, with special reference to the planktonic larvae of the Sound (Oresund) ; Medd.
Konmn. Danm. Fisk. Havunders. Plankton 4 1-523
£roc. Indian Acad. Sci. (Atiini. !>ci.), Vol. 91, Number 6, November 1982, pp. 577-58^.
© Printed in India.
Synthesis of 4-metfayl (657-6-tetrahydrobenzofurano) coumarin and its
contraception like properties in male rabbits (Oryctolagus cuniculus)
RAKESH SINHA, V P DIXIT and MEERA AGRAWAL
Reproduction Physiology Section, Department of Zoology, University of Rajasthan,
Jaipur 302 004, India
MS received 11 December 1981 ; revised 19 July 1982
Abstract. Administration of 4-methyl (6,7-6-tetrahydrobenzofurano) coumarin,
20 mg/kg/alternate day, for a period of 40 days caused degenerative changes in the
testes of male rabbits. Inhibition of spermatogenesis was achieved at primary
spermatocyte stage level. Total protein, sialic acid and glycogen contents of the
testes, epididymis and seminal vesicle were significantly reduced while the testicular
cholesterol was elevated in the 4-methyl coumarin treated animals. Serum choles-
terol, phospholipid, triglyceride, NEFA, were elevated. Antispermatogenic activity
of 4-methyl coumarin is discussed.
Keywords. 4-methyl coumarin ; inhibition of spermatogenesis ; sialic acid ; anti-
androgenicity.
1. Introduction
Simple aliphatic compounds like triethylene melamine exhibit antifertility pro-
perties (Jackson 1964), characterized by damage of spermatogonia and germinal
epithelium (Steinberger 1962). Lednicer et al (1965) prepared a number of 3,4-
diaryl coumarins sterically related to 1,2-diaryl indene and showed that some of
these possess antifertility activity.
Marked antifertility activity was also observed in the compounds incorporating
triarylethylene and also in 3,4-diphenyl, 1,2,3,4-tetranaph.thalene when the hydroxy
or alkoxy group was introduced. Mishra and Agrawal (1977) synthesized several
new bis and di (or 4'-coumarynil-oxyalkanes) coumarins and later tested them for
possible antifertility activity.
Realising the importance of benzofuran, coumarin and cyclohexanol derivatives
(Tyagi et al 1979) as antifertility agents, it was considered worthwhile to design
molecule incorporating benzofuran, coumarin and cyclohexanol moieties.
577
578 Rakesh Sinha, V P Dixit and Meera Agrawat
1. Experimental
In this direction 2-halo-cyclohexane was condensed with 7-hydroxy, 4-methyl
coumarin. 4-methyl-7-hydroxy coumarin (4 -4 g, 0*025M), 2-broniocyclohexa-
none (5'31g; 0-03M), anhydrous K2CO3 (8*0 g) and dry acetone (80 '0
ml) were taken in a round bottom flask, fitted with a refluxed condensor. The
reaction mixture, after refluxing for 60 hr, was cooled and filtered.
The solvent was distilled off under vacuum. The crude product was crystallized
with 95% ethanol. A white crystalline solid compound was obtained. The
purity was ascertained by TLC (m.p. 160° C ; yield 3'50g, 55%, Rf 0-89). Its
derivative with 2,4-dinitrophenyl hydrazine was prepared (m.p. 169-170° C).
NMR spectrum was obtained in TFA using TMS as internal standard. NMR spectrum
indicated the presence of singlet for 3 protons at 8 2-4. In the spectrum compli-
cated pattern for 8 proton in the range of S 1*1 to 2'1, a broad singlet for a
proton at <> 6*2 and 2-protons in the aromatic region (d 6 '15 to 7*45) were
observed. The NMR spectrum accounted well for the presence of 14 protons.
The presence of a. singlet at d 2' 4 was due to the C-methyl (C-CH3) groups
attached to position 4 in coumarin system. Methyl group being attached to an
aromatic ring and the olifinic bond, conjugated to a carbonyl group alongwith its
presence in a pyron ring gave a broad singlet to the down field at <5 2*4. The
presence of a broad singlet at § 6*2 for a proton accounted for the presence of
olifinic proton. The presence of only two aromatic protons indicated fusion of
cyclohexane ring to the ortho position of -OH group. The presence of 8-protons
at much higher field (§ 1 *1 to 2*1) indicated the presence of 4-methylene groups
and the absence of methane protons at the same time. This clearly indicated the
presence of cyclohexane system.
Based on the observations IR/NMR the structure of the compound I was assigned
as 4-methyl (6,7-6-tetrahydrobenzofurano) coumarin (Tyagi et al 1980).
4-methyl (6, 7-Z>-tetrahydrobenzofurano) coumarin
3. Material and methods
Twenty healthy adult male rabbits were used in the experiment and were divided
into groups as outlined in table 1. Ten rabbits comprising group 2 were treated
with 4-methyl (6,7-Metrahydrobenzofurano) coumarin 20 mg/kg/each alternate day
s.c. for 40 days. An equal number of rabbits received the vehicle alone and
served as control. After the completion of the final dose of 4-methyl coumarin,
rabbits were sacrificed with nembutal anaesthesia. Blood was withdrawn through
cardiac puncture and serum analysed.
\
. 'tefa.it. - —
Contraception like properties of ^-methyl coumarin
579
Table 1. Changes in the weight of testis, epididymis and adrenal glands together
with seminiferous tubule and Leydig cell nuclear diameter of rabbit after 4-methyl
(6,7-6 -tetrahydrobenzofurano) coumarin treatment.
Body weight Testes
(kg)
Ep:didymis Adrenal Seminiferous Leydig cell
tubule dia- nuclear diameter
nig/kg
meter
Om)
Control 1-4 ±0*3 1931 ± 108 780 ± 64 210 ± 35 175 ±2' 4 6' 00 ±0' 17
(10)
4-methyl 1-3 ±0-2* 1336 ± 146* 470 ± 43* 278 ± 26t 114 ± I'O** 5*16 ±0'14S*
coumarin
(10)
4-methyl coumarin versus control: ** .?<()• 001 *P<0'02 fNS (Not significant) ; All
figures ± S.E.M. Figures in parenthesis represent the number of animals examined.
Final body weight of each animal from both groups were recorded.. Testoes,
epididymis, seminal vesicle and adrenal glands were dissected free of fat. Right
testis and epididymis were fixed in Bouin's fluid. 6 /roi sections were prepared
and stained with haematoxylin and eosin. Left testis, epididymis, seminal vesicle
and adrenal glands were frozen and the total protein, sialic acid, testicular choles-
terol, glycogen, acid phosphatase and adrenal ascorbic acid were later determined
(Lowry et al 1951 ; Warren 1959 ; Montgommery 1957 ; Fiske and Subbarow
1925 ; Roe and Kuether 1943). Quantitative estimation of cholesterol was made
according to the Libermann-Burchard method (Oser 1965). Serum was analysed
for cholesterol, phospholipids, triglyceride, non-esterified free fatty acid and serum
proteins (Varley 1969). The transaminase enzyme activity (SGPT) was determined
according to Mohun and Cook (1957).
One hundred seminiferous tubules appearing circular in sections were traced with
camera lucida at x 80. Two perpendicular diameters of each group tracing were
measured and expressed in terms of mean tubular diameters. Student's ' t ' test
was applied for comparing means. The measurements of the diameters of 100
Leydig cell nuclei were carried out from the sections of testes with camera lucida
drawings at x800.
The LD50 of 4-methyl (6,7-6-tetrahydrobenzofurano) coumarin worked out in
white albino rat comes out to be 200 mg/kg.
Dizziness and paralytic conditions were the main symptoms observed
4. Results
4.1. Body weight and organ weight (table 1)
The body weight of the rabbits treated with 4-methyl coumarin was insignificantly
reduced. The testicular weight and epididymal weight exhibit significant reduc-
tion in the 4-methyl coumarin-treated animals when compared with controls.
P.(B)-10
580
Rakesh Sinha, V P Dixft and Meera Agmwat
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Contraception like properties of 4-methyl coumarin
581
Figures 1-2. 1. Testis of a control rabbit showing various stages of spermato
genesis x 160 HE. 2. After 4-methyl coumarin treatment. Note the loss of
various cell stages x 160 HE,
Contraception like properties of 4-methyl coumarin 583
4.2. Histological changes
4.2a. Testes : In the rabbits treated with 4-methyl coumarin, the seminiferous
tubule diameter and Leydig cell nuclear diameter decreased significantly (table 1).
Spermatogenesis was arrested at primary spermatocyte stage. The changes
consisted of loss of spermatids and spermatozoa (figures 1,2). Sertoli cells were
normal.
4.2b. Epididymis : Histological examination of the epididymis of 4-methyl
coumarin-treated rabbits showed that the epithelium was normal and the lumen of
caput epididymis was filled with debris. Cauda epididymis and ductus deferens
were devoid of spermatozoa.
4.3. Biochemical changes
4.3a. Protein : The total protein contents of testis, epididymis and seminal vesicle
were significantly lower in the rabbits treated with 4-methyl coumarin in com-
parison with controls (table 2).
4.3b. Sialic acid : The level of sialic acid was significantly decreased in the testis,
epididymis and seminal vesicle of 4-methyl coumarin-treated rabbits (table 2).
4.3c. Acid phosphatase : Acid phosphatase enzyme activity of the testis, epidi-
dymis and seminal vesicle was reduced significantly after 4-methyl coumarin
treatment (table 2).
4.3d. Glycogen : The glycogen level of testes decreased significantly (table 2).
4.3e. Cholesterol : The total cholesterol of testis increased in treated animals
(table 2).
4.3f. Ascorbic acid : The ascorbic acid contents of adrenal glands were low
(table 2).
4.3g. Serum analysis : The decrease in serum protein of coumarin-treated
animals was highly significant (P <0*01). No significant change was observed
Table 3. Serum analysis of rabbit after 4»methyl (6,7-£-tetrahydrobenzofurano)
coumarin treatment.
Protein Cholesterol Phospholipid Triglyceride NEFA SGPT
(mg/100 m) mEq/L Reitman Frankel
units
Control 11420 ±180 121- 4 ±10- 2 130 ±1' 24 13 ±4 0'244i0'014 99 ± 10
4-methyl- 8030 ± 213* 152 ± 12f 1?6 ± 9** 116 ±3* 0'370±0'02* 109 ± 23f
coumarin
' 4-methyl coumarin versa* control: *P<0*01 **P<0'05 IT < NS (Not significant).
All figures ± SEM. Biochemical estimations : Means of six determinations,
584 Rakesh Sinha, V P Dixft and Meera Agrawat
in pyruvate transaminase activity, however an increase was recorded in the total
cholesterol, phospholipid, triglyceride and non-esterified fatty acids (table 3).
5. Discussion
Compounds which suppress spermatogenesis include many chemical classes and
modes of action (Jackson 1970). Little information is available concerning struc-
ture activity relationships and metabolism of these interesting compounds.
After 40 days of 4-methyl (6,7-fe-tetrahydrobenzofurano) coumarin treatment
resulted in disappearance of mature sperms and spermatids. These results are
similar as observed after ethylene dimethane sulphonate (Jackson 1970), nitrofuran
and a-chlorohydrin (Patanelli 1975). Decrease level of protein in the testes,
epididymis and seminal vesicle of rabbits treated with 4-methyl (6,7-6-tetrahydro-
benzofurano) coumarin suggest an inhibition of speramatogenesis and suppressed
Leydig cell function. Podesta et al (1975) describe a relationship between the
androgen sensitivity and protein synthesis, contents and concentration, in the
epididymis. A decrease in the level of protein of epididymis reflects the antiandro-
genic nature of the compound. Significant decrease in glycogen may affect protein
synthesis and thus subsequently inhibit spermatogenesis.
Reduced acid phosphatase enzyme/sialic acid activity confirms the inhibitory
role of 4-methyl coumarin on spermatogenesis in rabbit. Blackshow and Massey
(1978) showed that the total and free biochemical acid phosphatase decreased during
cryptorchidism. Peyre and Laporte (1966), Rajalakshmi and Prasad (1968)
reported a fall in the sialic acid contents of cryptorchid testes/epididymis of castrated
rats and intact langur monkeys (Braz et al 1979).
A significant increase in testicular/serum cholesterol after 4-methyl coumarin
treatment have been considered physiologically important, since testicular chole-
sterol derived from blood cholesterol is used for testosterone production (Anderson
and Dietschy 1977) and is the primary substrate for androgen biosynthesis (Dorf-
man et al 1963 ; Eik-Nes and Kekre 1963).
Serum protein was reduced while cholesterol was elevated. The phospholipids,
triglycerides and non-esterified fatty acids were also increased in the rabbits
following 4-methyl (6,7-6-tetrahydrobenzofurano) coumarin treatment, reflects
an interference in the lipid metabolism. However, more work is in progress for
the reversible action of the compound and shall be reported elsewhere.
Acknowledgements
The study was supported by ICMR, New Delhi.
References
Anderson J M and Dietschy J M 1977 Regulation of steroid synthesis in 15 tissues of rat.
U. Role of rat and human high and low density plasma lipoproteins and of rat chvlomicron
remnants;/. Biol Chem. 252 3652-3659
Contraception like properties of 4-methyl coumarin 585
Blackshow A N and Massey P F 1978 The effect of cryptorchidism on the quantitative histo-
logy, histochemistry and hydrolytic enzyme activity of the rat testis ; Aust. J. Biol. SCSI
53-64
Braz I, Shandilya L N and Ramaswami L S 1979 Effect of a-chlorohydrin on the male repro-
ductive organs of the Indian Langur (Presbytis entellus entellus Dufresne) ; Andrologia 8
290-296
Dorfman R I, Forchielli E and Gut M 1963 Androgen biosj-nthesis and related studies ;
Recent Prog. Horm. Res. 19 251-273
Eik-Nes K B and Kekre M 1963 Metabolism in vivo of steroids by the Canine testis ; Biochem.
Biophys. Acta 78 449-456
Fiske C M and Subbarow Y 1925 Colorimetric determination of phosphorus ; /. Biochem.
66 375-400
Jackson H 1964 The effect of alkylating agents on fertility ; Sri. Med. Bull. 20 107-114
Jackson H 1970 Antispermatogenic agents ; Bri. Med. Bull. 26 79-86
Lednicer D, Babeock J C, Marlatt P E, Lyster S C and Duncan G W 1965 Mammalian anti-
fertility agents. I. Derivatives of 2-3 diphenylindene ; /. Med. Chem. 8 52-57
Lowry O H, Rosenbrough M J, Farr A L and Randall R J 1951 Protein measurement with
the folin phenol reagent ; /. Biol Chem. 193 265-275
Mishra V S and Agrawal S 1977 Synthesis of possible antifertility compounds. Synthesis of
bis and di derivatives of coumarins ; /. Indian Chem. Soc. L IV12 1124-1126
Mohun A F and Cook I J Y 1957 Simple method for measuring serum levels of glutamic
oxaloacetic and glutamic pyruvic transaminases in routine laboratory ; J. Clin. Path. 10
349-399
Montgommery R 1957 Determination of glycogen ; Arch Biochem. 67 378-379
Oser B L 1965 In Hawk's Physiological chemistry 14th ed. (New York : McGraw-Hill) p. 246
Patanelli D J 1975 Suppression of fertility in the male. In Handbook of physiology edited by
D W Hamilton, R O Greep, Sect 7 : Endocrinology, Vol. 5 : Male Reproductive system
(Washington D C : American Physiological Society) p. 245
Peyre A and Laporte P 1966 Action de la testosterone et de Toestradiol sur les sialo proteins
de la queque de 1'epididyme de rat impubere castre ; C R Seanc. Soc. Biol 160 2178-2180
Podesta E J, Calendra R S, Rivarola M A and Blaquier J A 1975 The effect of castration
and testosterone replacement on specific protein and androgen levels of rat epididymis ;
Endocrinology 97 399-405
Rajalakshmi M and Prasad M R N 1968 Changes in the sialic acid content of the accessory
glands ; /. Endocrinol. 41 471-476
Roe J H and Kuether C A 1943 The determination of ascorbic acid in whole blood and urine
through the 2,4-dinitro phenyl hydrazine derivative of dehydro ascorbic acid ; / Biol. Chem.
147 399-407
Steinberger E 1962 A quantitative study of the effect of an alkylating agent (Tiiethylene meta-
mine) on the seminiferous epithelium of rats ; /. Reprod. Fertil. 3 250-259
Tyagi A, Joshi B C, Kumar S and Dixit V P 1979 Spermatogenic activity of cyclohexanol in
geibil (Meriones hurrianae Jerdon) and House rat (Rattus rattus Rufescens) ; Indian J. Exp.
Biol. 17 1305-1307
Tyagi A, Dixit V P and Joshi B C 1980 New novel non-steroidal antifertility agents ;
Naturwissemchaften 67 104
Varley H 1969 Practical clinical biochemistry, 4th ed. (London : White Friers Press Ltd.) p. 126
Warren L 1959 The thiobarbituric acid assay of sialic acids : /. Biol. Chem. 234 1971-1975
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 91, Number 6, November 1982, pp. 587-597.
© Printed in India.
Cellular sites of steroid synthesis in the oviparous teleost fish
(Cyprinus carpio L.) : A histochemlcal study
SARDUL S GURAYA and SURINDERPAL KAUR
Department of Zoology, College of Basic Sciences and Humanities,
Punjab Agricultural University, Ludhiana 141 004, Punjab, India
MS received 30 December 1981 ; revised 26 July 1982
Abstract. Hi sto chemical techniques for lipids were employed to study the steroid
synthesizing cellular sites in the ovary of teleost fish (Cyprinus carpio L.). The
cellular sites of steroid biosynthesis appear to be the ovulating corpora lutea, inter-
stitial cells, and special thecal cells of developing follicle. They possess the cyto-
logical and histochemical features of well-established steroid gland cells. The
functional significance of histochemical changes in the granulosa cells of post-
ovulatory follicles in the teleost ovary has been discussed in the light of recent
researches on corresponding cells in the ovaries of other vertebrates. The
corpora atretica are merely the large yolky eggs in the process of their degeneration
and resorption.
Keywords. Cellular sites ; steroid synthesis ; histochemistry ; lipids; ovary ; teleost.
]. Introduction
Histochemical techniques mainly for A5-3/J~hydroxysteroid dehydrogenase
(3^-HSDH) and electron microscopy have been employed for the study of cellular
sites of steroid synthesis in the ovaries of some teleosts (see Bara 1965; Guraya 1976,
1978a, 1979 ; Nagahama et al 1978 ; Kagawa et al 1981). However, the precise roles
of atretic yolky eggs (corpora atretica or corpora lutea of atresia), postovulatory
follicles (corpora lutea of ovulation) and the scarcely developed stroma, together
with its interstitial (or thecal gland) cells, in relation to steroid biosynthesis, are
still not known. In most of the previous studies carried out with the routine
histological techniques the corpora atretica have been claimed to be the main site
of steroid biosynthesis in the teleost ovary (Ball 1960 ; Chan et al 1967). This
opinion was not shared by others (Dodd 1960 ; Polder 1964 ; Guraya et al 1975,
1977). No any attempt has been made so far to study the nature of lipid changes
during the involution of postovulatory follicles or corpora lutea in teleosts (see
Guraya 1979 ; Kagawa et al 1981). This study using histochemical techniques for
587
588 Sardul S Guraya and Surinderpal Kaur
lipids describes the lipid changes of follicles, postovulatory follicles, corpora lutea
of ovulation, corpora atretica or preovulatory corpora lutea and interstitial
(thecal gland) cells in the ovary of the scale carp, Cyprinus carpio L.
2. Materials and methods
The ovaries of oviparous teleost fish (scale carp Cyprinus carpio Linn.) were used.
The fish were obtained from the fishery pond of the Punjab Agricultural Univer-
sity, Ludhiana. This fish usually breeds in the months of February and March in
the Punjab waters. During the breeding season, two mature males and a female
were put in the hapa. The spawning usually occurred in the early hours of the
morning. Weeds were also added in the hapa for the attachment of eggs. The
recovery of eggs from hapa was the criterion for ovulation. After spawning, the
ovary was removed and transferred into physiological saline solution. The ovarian
material was collected at 12 hr intervals from different females. It was also collected
after an interval of one day. Thereafter, the material was collected on alternate
days. These time intervals were taken into account from the time of spawning.
After washing off the blood in physiological saline the ovary was immediately fixed
in freshly prepared fixing fluids. The details of histochemical techniques used were
the same as those reported previously (Guraya 1968).
3. Observations aad discussion
The vascularized thecal layer of follicle during the preovulatory period consists
of fibroblasts and does not show any appreciable development of sudanophilic
lipids-containing cells which could easily be demonstrated with the light micro-
scope. The distribution and histochemical nature of lipids in the follicular epi-
thelium are the same as those reported for the teleost Channa (Guraya 1976, 1978a).
The cytoplasm of follicle (or granulosa) cells shows some sparsely scattered, deeply
sudanophilic lipid droplets which give positive reactions for phospholipids, no
Schultz-positive substances (cholesterol and/or its esters) are observed.
The follicular epithelium of the developing follicle in the ovaries of some teleosts
may show a positive 3j3-HSDH activity which is also accompanied by the presence
of enzymes of the citric acid and the pentose-phosphate cycles (Guraya 1976, 1978a,
1979). The two enzymes 3j8- and 17^-HSDH are not only present in the granulosa
cells but also in the cortical cytoplasm during the last phases of follicle growth
in the fish ovary. It is still not known whether the presence of these enzyme
systems indicates synthesis and secretion of steroid hormones by the granulosa
cells of developing follicle in vivo, or simply indicates their potentialities for
steroidogenesis.
3.K Postovulatory follicles
The granulosa cells after ovulation develop sudanophilic lipid droplets of variable
size during different stages of evolution and involution of postovulatory follicles
Histochemistry of teleost ovary and steroidogenesis
589
Figures 1-6. 1. Portion of postovulatory follicle in stage I showing sudanaphilic
lipids in the follicular epithelium (FE) and some thecal gland cells (TC) of thecal
layer. Lumen (L) is seen, x 400. 2. Portion of postovulatory follicle in
stage 2 showing sudanophilic lipids in granulosa luteal cells and thecal gland cells
(TGC). Thecal layer (TL) shows some vacuolated cells (vc). Lumen (L) is
reduced. X 400. 3. Portion of posto,vulatory follicle in stage 5, showing" heavy
accumulation of sudanophilic lipid droplets in the granulosa luteal cell" mass
(GLM). The celts have separated from each other, x 400. 4. Postovulatory
follicles in stage 6, showing heavy accumulation of sudanophilic lipids in
degenerated granulosa luteal cells (DGLC). The amount of sudanophilic lipids
has also increased in the thecal layer (TL). x 100. 5. Higher magnification
of portion of postovulatory follicle shown in figure 4 showing accumulation of
sudanophilic lipids in degenerated granulosa luteal cells (DGLC). The thecal layer
also shows cells (TGC) filled with sudanophilic lipids. x 400. 6. A portion of
ovary showing sudanophilic lipids in residual granulosa luteal cells (RGC) and
interstitial cells (ic) distributed in the stroma (s). The latter does not show such
lipids. X 100.
Sardul S Guraya and Surinderpal Kaur
1C
Figure 7. A portion of ovary showing sudanophilic lipids in residual granulosa
luteal cells (RGC) and interstitial cells (ic). Stroma proper (s) does not show such
lipids. X 400.
Histochemistry of teleost ovary and steroidogenesis 591
(figures 1-7), which consist of first phospholipids, then phospholipids and trigly-
cerides, and finally triglycerides, cholesterol and/or its esters and some phospho-
lipids (table 1). Besides the lipid droplets, they also develop diffusely distributed
sudanophilic lipids (lipoproteins) in their cytoplasm (figures 1, 2). Similar lipid
droplets and diffuse lipoproteins are also developed during the transformation of
granulosa cells into the luteal cells in other vertebrates (see Guraya 1976).
Diffusely distributed lipoproteins apparently derive from the membranes of
smooth reticulum described for the granulosa cells of postovulatory follicles in
teleosts and other vertebrates (Hoar andNagahama 1978 ; Nagahama et al 1976,
1978 ; Guraya 1976, 1979 ; Kagawa et al 1981).
The thecal layer of postovulatory follicle in the present fish shows some hyper-
trophied cells with sudanophilic lipid droplets arid diffuse lipoproteins (figures 1, 2)
which correspond to the special thecal cells in the spent follicles of other teleosts
(Nagahama etal 1978.; Kagawa etal 1981). These special thecal cells give a positive
reaction for 3j8-HSDH and show ultrastructural features of steroid gland cells such
as greatly developed agranular endoplasmic reticulum and numerous large mito-
chondria with tubular cristae (see Nagahama et al 1978 ; Kagawa et a! 1981).
Generally when lipid droplets are abundantly present in the steroid gland cells,
storage is taking place and when the amount is less, the steroid hormone is being
released (Guraya 1976, 1978a, b, 1979). According to this concept, the granulosa
cells in stages 1, 2, 3, which contain a few lipid droplets of the postovulatory
follicles (table 1), may be functioning in the secretion of some steroid hormone
(see also Guraya 1976, 1979). This suggestion is also supported by the fact that
the granulosa cells during these stages show organelles and enzyme activities
related to steroid biosynthesis (see Guraya 1976, 1979 ; Hoar and Nagahama
1978 ; Nagahama et al 1976, 1978 ; Kagawa et al 1981).
The degenerating granulosa cells in stages 4, 5 and 6 of postovulatory follicle
(table 1) apparently function in the storage of hormone precursor, as supported
by the accumulation of highly sudanophilic, cholesterol-positive lipid droplets
(figures 3-7) which constitute the precursor material stored within the steroid
gland cells (Guraya 1976, 1978b, 1979). The regression of steroidogenesis during
the later life of postovulatory follicle in teleost ovary is also supported by the
gradual disappearance of its enzyme activities and alterations in organelles related
to steroidogenesis (Lambert and van Oordt 1974 ; Guraya 1976, 1979 ; Nagahama
et al 1976, 1978). The minimum enzyme 3^-HSDH activity is reached between the
third and fourth day after spawning in the zebra fish (Lambert and van Oordt
1974), indicating a short functional life of postovulatory follicle in the ovi-
parous teleosts (Guraya 1976, 1979) as also supported by the results of the present
study,
3.2. Corpora atretica (or preovulatory corpora luted)
The first change, which occurs during the atresia of ripe yolky eggs, is the develop-
ment of large sudanophobic vacuoles in the region of cortical vacuoles (figures 8, 9).
As the atresia advances, highly sudanophilic fatty yolk elements coalesce to forcq
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Histochemistry of teleost ovary and steroidogenesis
593
Figures 8-10. 8. Yolky eggs in its early stage of atresia, showing sudanophobic
vacuoles at the periphery (cv). Follicular epithelium and zona pellucida are
still intact. Germinal vesicle (GV) is also seen, x 400. 9. Corpus atreticum
in stage 3 showing accumulation, of sudanophilic lipids in granulosa cells. The yolk
has been ingested by the granulosa cells, x 400. 10. Showing degenerated
corpora (AF) in stage 4, filled with sudanophilic lipids. x 400.
Histo chemistry of teleost ovary and steroidogenests 595
highly sudanophilic masses (figures 8, 9, 10). The yolky contents of atretic follicles
are gradually digested and removed by the granulosa cells which, at the same time,
store lipids consisting of triglycerides and some phospholipids. Similar lipids
also accumulate in the granulosa cells of atretic follicles in other vertebrates
(Guraya 1976). In their later stages of resorption, atretic yolky eggs in the present
teleost also store cholesterol and/or its esters. The corpora atretica or c pre-ovulatory,
corpora lutea of lower vertebrates are usually believed to secrete steroid hormones
(see references in Browning 1973), But Guraya (1976) believes that they are
merely the large eggs in the process of their resorption as also observed in this
study. The various enzyme cytochemical investigations have also shown that
the corpora atretica of teleost ovary are merely the large yolky eggs in the
process of degeneration and resorption (Guraya 1976, 1979). The dense lipid
and cholesterol accumulations demonstrated in the atretic yolky eggs of the
present carp may be due to degenerative changes.
3,3. Interstitial cells
The interstitial cells in the ovaries of present teleost occur singly or in groups
in the stroma (figures 6, 7). Some of them appear to derive from the persisting
hypertrophied thecal gland cells of postovulatory follicles as well as from their
residual granulosa cells (figures 6, 7). They are filled with sudanophilic lipid drop-
lets consisting of triglycerides, cholesterol and/or its esters and some phospholipids.
The thecal gland cells, described by Nicholls and Maple (1972) in the wall of the
cichlid fish follicles, may be the interstitial (or thecal) gland cells having the
cytological features of steroid-secreting cells. These special thecal cells are believed
to be the only cells responsible for steroid production in the thecal layer of
salmonid fishes (Nagahama et al 1978 ; van den Hark and Peute 1979). Kagawa
et al (1981) have also identified special thecal cells in the wall of preovulatory
follicle of S. leucomaenis, which show mitochondria with tubular cristae and both
agranular and granular forms of endoplasmic reticulum, the latter being more
prominent. The results of these various studies have indicated that interstitial
cells form the important component of the teleost ovary. With the growth of
vitellogenic follicles, they get sparsely distributed in their walls and then are called
as special thecal cells or thecal gland cells. The presence of interstitial cells has
also been demonstrated in the ovaries of different vertebrates (see Guraya 1976).
The ovarian interstitial cells of these vertebrates possess the cytological and
histochemical features of well-established steroid gland cells (Guraya 1976). It
can also be presumed that the interstitial (or thecal gland) cells of present teleost
ovary are steroid secretors as they contain cholesterol-positive lipids in their
cytoplasm under certain physiological situations, and are associated with blood
vessels. This suggestion is further supported by the presence of 3j3-HSDH activity
indicative of steroidogenesis in the ovarian interstitial (or thecal gland) cells of
teleosts (Lambert and van Oordt 1974 ; Guraya 1976, 1978a, 1979) as well as
by the various electron microscope studies (see Nagahama et al 1978 ; Kagawa
et al 1981). In the ovaries of teleosts investigated by Lambert and van Oordt
596 Sardul S Guraya and Surinderpal Kaur
(1974), the 3/?-HSDH positive interstitial cells are mainly distributed in the stroma,
as well as against the follicle wall. They show conspicuous fluctuations in their
distribution and enzyme contents with the ovarian cycle. The physiological
significance of the interstitial cells remains doubtful as in the Swordtail where
they show clear cytochemical indications of steroid metabolism, but in the zebra fish
they lack any glucose-6-phosphate dehydrogenase activity (Lambert and van
Oordt 1974). Kagawa et al (1981) have attributed the secretion of high
progesterone levels to the special thecal cells of postovulatory follicle in the
white-spotted char rather than to its granulosa luteal cells.
References
Ball J N 1960 Reproduction in female bony fishes ; Symp. Zool. Sac. London 1 105-135
Bara G 1965 Histochemical localization of A5-3^-hydroxysteroid dehydrogenase in the ovaries
of a teleivst fish Scomber scomber L.; Gen. Comp. EndocrinoL 5 284-296
Browning H C 1973 The evolutionary history of the corpus luteum ; Biol. Reprod. 8 128-157
Chan S T H, Wright A and Phillips J G 1967 The atretic structures in the gonads of the
rioe-field eel (Monopterus albus) during natural sex reversal ; /. Zool. (Lond.) 153 527-539
Dodd J M 1960 Gonadal and gonadotrophic hormones. In Marshall's physiology of Reproduc-
tion (ed.) A S Parkes (London : Longmans, Green and Cc.) Vol. I Part II pp. 417-558
Guraya S S 1968 Histochemical study of graaulosa (follicular cells) in the preovulatory and
postovulatory follicles of amphibian ovary ; Gen. Comp. EndocrinoL 10 138-146
Guraya S S 1976 Recent advances in the morphology, histochemistry and biochemistry of
steroid-synthesizing cellular sites in the non-mammalian vertebrate ovary ; Inter. Rev. Cytot.
44 365-409
Guraya S S 1978a Maturation of the follicular wall of non-mammalian vertebrates. In The
vertebrate ovary (ed.) R E Jones (New York : Plenum Press) pp. 261-329
Guraya S S 1978b Recent advances in the morphology, histochemistry, biochemistry and
physiology of interstitial gland cells of mammalian ovary ; Int. Rev. Cytol. 55 171-245
Guraya S S 1979 Recent advances in the morphology and histochemistry of steroid-synthesizing
cellular sites in the gonads of fish ; Proc. Indian natn. Sci. Acad. B45 432-461
Guraya S S, Kaur R and S-.xena P K 1975 Morphology of ovarian changes during the
reproductive cycle of fish, Mystus tengara (Ham.) ; Acta Anat. 91 222-260
Guraya S S, Toor H S and Kumar S 1977 Morphology of ovarian changes during the
reproductive cycle of the fish Cyprinus carpio communis (Linn.) ; Zool. Baitrage 23 405-407
Hark R van den and Peute J 1979 Cyclic changes in the ovary of the rainbow trout, Salmo
gairdneri, with special reference to sites of steroidogenesis ; Cell Tissue Res. 199 289-306
Hoar W S and Nagahama Y 1978 The cellular sources of sex steroids in teleost gonads ; Ann-
Biol. Anim. Biochim. Biophys. 18 893-898
Kagawa H, Takano K and Nagahama y 1981 Correlation of plasma estradiol-17^ and
progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-
spotted char, Salvelinus leucomaenis ; Cell Tiss. Res. 218 251-277
Lambert J G X> and van Oordt P G W 1974 Ovarian hormones in teleosts ; Fortschr Zool
22, 340
Nagahama Y, Clarke W C and Hoar W S 1976 Ultrastructure of putative steroid producing
cells in the gonads of coho (Oncorhynchus kisutch) and pink salmon (Oncorhynchus gorbuscha •
Can. L Zool. 54 1128-J139 ?
Uistochemistry of teleost ovary and steroidogenesis 597
Nagaharaa Y, Chan K and Hoar W S 197S infrastructure of putative steroid producing cells in
the gonads of coho (Oncorhynchus kisutch) and pink salmon (Oncorhynchus gorbuscha) ;
Can. J. Zool 56 2508-2519
«
Nicholls T J and Maple G 1972 Ultrastructural observations on possible sites of steroid
biosynthesis in the ovarian follicular epithelium of two species of cichlid fish, Cichlasoma
nigrofasciatiim and Haplochromis multicolour ; Z. Zellforsch 128 317-335
Pearse AGE 1968 Histochemistry (London : Churchill Ltd.)
Polder J J W 1964 On the occurrence and significance of atretic follicles (preovulatory corpora
lutea) in ovaries of Bitterling Rhodeus amarus (Bloch) ; Prod. Kon. Ned. Akad. Net. 67
218-222
*' K°vembe*
Bevelopment of the incretory organs in the eyestalk of freshwater
prawn, Macrobmchium kistnensis
M S MIRAJKAR, R SAROJINI and R NAGABHUSHANAM
Department of Zoology, Marathwada University, Aurangabad 431 004, India
MS received 9 February 1982 ; revised 20 September 1982
Abstract. The development of the neurosecretory cells in eyestalk is studied in
zoea, juvenile and adult stages of the Macrobmchium kistnensis. In zoea, the
ganglionic mass of the eyestalk compreses of three parts viz. medulla externa, medulla
interna and medulla terminalis. The future sensory pore x-organ is characterized: by
onion bodies near medulla terminalis and a vacuole closely applied to the outer
surface of the eyestalk. The monopolar giant neuron is observed in zoea. In juvenile
prawn, in addition to zoeal features the sinus gland makes its appearance. At this
stage, neurosecretory cells are discernible in the medulla externa and terminalis. In
adults, the eyestalk presents well developed lamina ganglionaris, medulla externa
ganglionic x-organ (m.e.g.x.), medulla interna ganglionic x-organ (nu.g.x.) and
medulla terminalis ganglionic x-organ (m.t.g.x.).
Keywords. Macrobrachiwn ; incretory organs ; lamina ganglionaris ; medulla
externa ganglionio x-organ (m.e.g.x.) ; medulla terminalis ganglianic x-organ
(nU.g.x.) ; giant neuron.
1. Introduction
The neurosecretory system of the eyestalk plays an important role in hormonal
regulation in crustaceans (Adiyodi and Adiyodi 1970). However, only a few works
deal with the development of these neurosecretory centres. Pyle (1943) was the first
to describe the histogenesis and cyclic phenomena of the sinus gland and x-organ
in Homarus americanus and Pinnotherus maculatus. Later, Dahl (1957) in Crangon
allamani and Matsumoto (1958) in Potamon dehani studied the embryology of the
eyestalk. Hubschman (1963) and Elofsson (1969) gave a detailed account of the
development of neurosecretory sites in the eyestalk of Palaemonate's and Penaeus
duorarum, respectively. Recently, Bellon-Humbert et al (1978) reported the
development and location of neurosecretory and sensory sites in larva and post-
larva ofPalaemon serratus. Most of the studies, reported so far, are mainly concerned
with marine crustaceans. Freshwater species have received only a meagre attention
in this regard. Therefore, the present study was undertaken to trace the develop-
ment of the incretory organs in the freshwater prawn, Macrobrachium kistnensis
from zoea to adult.
599
600 M S Mirajkar, R Sarojini and R Nagabhushanant
2. Material and methods
Macrobrachium kistnensis were collected from Kham river, near Aurangabad. The
berried females from the collection were maintained separately in glass bowl.
After 29-30 days of incubation, the zoea hatched out. The zoea entered the
juvenile phase after 10-11 days (Kulkarni 1972). The eyestalks of zoea, juveniles
and adults were removed and fixed in the Bouin's fixative for 24 hrs. Paraffin
sections of 8 //m thickness were prepared and stained with Gomori's Aldehyde
fuchsin (Ewen 1962) and Mallory's triple stain.
3. Results
3.1.
Ontogenesis of the neurosecretory cells of the eyestalk
3. la. Zoea : After hatching (within 24 hrs) the zoea measured 4-5 mm. The
eyes were sessile and cuticle of the cephalothorax was continuous over it. Omma-
tidia were quadrangular with black pigment. Internally the three medullae viz.
externa, interna and terminalis were clearly visible (figure 3a). The monopolar
neuron was present near the medulla terminalis (figure 3b). The future sensory
pore x-organ (in the form of vacuole) was observed near dorsal surface of the eye-
stalk (figure 3c). It did not show any connection with the onion bodies of the organ
of Bellonci which is situated in the ganglionic mass of the medulla terminalis.
3.1b. Juvenile: The juveniles ranged between 11 to 14mm length. In this
stage, the incretory structures were more clearly visible in the lamina ganglionaris
and three medullae. The sinus gland, which measured 8'5-/i diameter, was present
$)»M<?dulla extern*
sinus gland
l^dulla interna
granlnuron
Organ of Bellonoi
Figure 1. Diagrammatic representation of the eyestalk structures, A — dorsal
view and B — ventral view.
Development of incretory organs in M. kistnensis
601
Zoea
Metamorphosis
Juvenile
Figure 2. Diagramatic orientation of the incretory organs of the eyestalk of
M. kistenensis.
Abbreviations : ME— Medulla externa ; MI— Medulla intcrna ; MT— Medulla
terminalis ; SG— Sinus gland ; GN — Giant neuron.
in between medulla externa and medulla interna. It showed distinct affinity for
aldehyde fuchsin (figure 4a). The giant neuron measured 12 // in diameter and
had 2 to 3 nucleoli (figure 4b). The presumptive neurosecretory sites revealed the
neurosecretory cells particularly in the medulla terminalis and medulla interna
region (figure 4c, d).
3.1c. Adult eyestalk structure (figures 1, 5a) : The optic ganglion consists
of four parts : (i) lamina ganglionaris, (ii) medulla externa, (iii) medulla interna
and (iv) medulla terminalis. The neurosecretory cells are absent in lamina gang-
lionaris, but are present in the remaining three parts and are termed as medulla
externa ganglionic x-organ (m.e.g.x.), medulla interna ganglionic x-organ (m.i.g.x.)
and medulla terminalis ganglionic x-organ (m.t.g.x.). The giant neuron is present
in between medulla interna and terminalis (figure 5b). The sinus gland (2 1-26 p)
is located in between medulla interna and externa. The organ of Bellonci or sensory
pore x-organ (SPX) is situated in the medulla terminalis region and is embedded in
the neurosecretory cells along with onion bodies (figure 5c, d). The organ of
Bellonci opens to the outside through the sensory pore (figure 5e).
3. Id. Orientation of the eyestalk incretory structures'. During metamorphosis
the internal structures of the eyestalk turn through 180°. However, the internal
structures retained their original connections to each other. As a result, the dorsal
structures are shifted to the ventral side and the ventral structures to the dorsal
side in the juvenile prawn. The giant neuron which is present at the dorsal side
in zoea turned to the ventral side in the juvenile and the onion bodies also change
position, opposite to the giant neuron. The sinus gland in juveniles is located above
the giant neuron at medulla externa region. These hypothetical movements of
incretory structures of eyestalks are schematically represented in figure 2,
4. Discussion
Hanstrom in 1939 for the first time described the x-organ as c bunch of grapes *
in adult Homarus americanus. Pyle (1943) studied the histogenesis of x-organ
602 At S Mirajkar, R Sarojini and R Nagabhushandtii
in Homams americanus and Pinnothems maculatus and interpreted that the x-organ
is found in the eggs and it showed completely developed structure in adults. After
these basic studies, the Hanstrom's x-organ or sensory pore x-organ (SPX) or organ
of Bellonci was well studied by Hubschman (1963) in Palaemonetes, Elofsson (1969)
in Penaeus duorarum and Jacques (1969 a,b) in stomatopods. They described
the cavity under the cuticle in larval stages. In the present study of Macrobrachium
kistnensis zoea, the SPX was found like a vacuole below the exoskeleton of the eye-
stalk. The onion bodies, very few in number, were present in the ganglionic mass
of the medulla terminalis. However, Bellon-Humbert et al (1978) found the organ
of Bellonci with typical onion bodies in the zoeal stage of Palaemon serratus*
They did not find any vacuole below the cuticle, but described, the larval sensory
pore (LSP) in zoea, which has no correlation with future SPX. During metamor-
phosis, the main sensory pore (MSP) established connection with organ of
Bellonci.
In adult M. kistnensis the well developed SPX is located in medulla terminalis
region and opens out through sensory pore. The sensory pore and SPX are connected
to each other by a lumen. The sinus gland of juvenile M. kistnensis showed positive
and negative affinity with AF ; means upper part of sinus gland stained purple in
colour while lower part stained yellow. The sinus gland of Palaemon pauddens
(Hisano 1974) stained with Azan (blue, red, vermilon, orange and purple), CHP
(pink and gray), AF (AF + ve and AF — ve) and Mallory's triple (blue, orange
and greyish green). The giant neuron is a constant feature of the eyestalk during
development. Its presence from zoea to adult stage suggests that it may have some
role in development, especially after metamorphosis. Again, the occurrence of this
giant neuron before the appearance of the neurosecretory centres is significant.
Hubschman (1963) described in Palaemonetes that the giant neuron may be secretory
in nature and the secretion may accumulate in SPX.
The neurosecretory cells in the eyestalk of M. kistnensis are distributed in five
groups in adult prawns, but two groups (medulla externa and medulla terminalis)
show their existence in juveniles. In Potamon dehani the neurosecretory cells were
well developed before hatching (Matsumoto 1958). In Astaaus leptodactylus cell
types can be distinguished by their colouration at the time of hatching of the eggs
(Zielhorst and Van Harp 1976). The present results on M. kistnensis agree with
the earlier reports of Bellon-Humbert et al (1978). They have described the differen-
tiation of the neurosecretory cells of m.e.g.x. and m.tg.x. with metamorphosis.
The position of sinus gland, giant neuron and SPX in larvae change owing to
twisting of medullae along the longitudinal axis of the eyestalk (Hubschman 1963 ;
Elofsson 1969 ; Elofsson and Dahl 1970 ; Bellon-Humbert et al 1978). In
M. kistnensis the eyestalk incretory structures, after orientation, retain their constant
relations to each other though they are topographically changed. Thus, the
ventral side of the larva becomes dorsal in the adult and vice versa. The giant
neuron passes from distal to proximal position. Above the giant neuron the sinus
gland is situated and SPX shifts opposite to it.
Acknowledgement
One of the authors (MSM) expresses her thanks, to ICAR, New Delhi, for financial
assistance during the completion of research work.
Development of incretory organs in M. kistnensis
603
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604
M S Mirajkar, R Sarojinl and R Nagabhushanam
Figure 4. The ontogenesis of neurosecretoiy cells in the optic ganglion
of prawn, Macrobrachium kistnensis. a. The sinus gland of juvenile prawn
stained with Aldehyde fuchsin, showing AF + ve and ' AF — ve area, x 400.
b. The manopolar neuron in the juvenile prawn, stained with Aldehyde fuchsin'
X 400. c. The sensory pore x-organ of juvenile prawn, surrounded by neuro-
secretory cells, x 400. d. The differentiated neurosecretory cells of medulla externa
region in juvenile prawn, x 400.
Abbreviations : SG— Sinus gland; GN— Giant neuron; SPX— Sensory pore x-organ;
£{C— Neurosecretory cells.
Development of incretory organs in M. kistnensis
605
Figure 5,
606
M S Mirajkar, R Sarojini and R Nagabhushanim
Figure 5e
Figure 5. The eyestalk neurasecretory cells of M. kistn&isis. a. The eyestalk
showing lamina ganglionaris, medulla externa, medulla interna and medulla termi-
nalis with neurosecretory cells, x 150. b. The giant neuron of the eyestalk situated
in between medulla interna and medulla terminalis. c. The sensory pore x-organ
with onion bodies X 150. d. The onion bodies from the sensory pore x-crgan,
x 1000. e. The sensory pore of the sensory pore x-organ, x 150.
Abbreviations'. LM— Lamina ganglionaris ; ME— medulla externa ; MI — Medulla
interna ; MT — Medulla terminalis. GN — Giant neuron ; OB — Onion bodies ;
NO- Neurosecretory cells ; SP — Sensory pore.
Development of incretory organs in M. kistnensis 607
References
Adiyodi R G and Adiyodi K G 1970 Endocrine control of reproduction in decapod Crustacea ;
Biol. Rev. 45 121-165
Bellon-Humbert C H, Thijseen N J and Van Harp F 1978 Development location and reloca-
tion of sensory and neurosecretory sites in the eyestalks during the larval and post larval life
of Palaemon serratus; (Pennat) ; /. Mar. Biol. Assoc. U.K. 58 851-868
Dahl E 1957 Embryology of X-organ in Crangon allamani ; Nature London 179 482
Elofsson R 1969 The development of the compound eye of Penaeus duorarum (Crustacea,
Decapoda) with remarks on the nervous system ; Z. Zellforsch. Micros. K. Anant. 97 323-350
Elofsson R and Dahl E 1970 The optic neuropiles and chaismata of Crustacea ; Z. Zellforsch.
Microsc. K. Anant. 107 343-360
Ewen A B 1962 An improved aldehyde fuchsin staining techniques for neurosecretory product
irx insects ; Trans. Amer. Micro. Soc. 81 94-96
Hanstrom B 1939 Hormones in invertebrates, (Clarendon Press, Oxford) i-xii, 1-198
Hisano S 1974 The eyestalk neurosecretory cell type in the freshwater prawn, Palaemon paucidens
I A lights microscopic study ; /. Sci. Hokkaido Univ. Ser. VI. Zool. 193 503-514
Hubschman J H 1963 Development and function of neurosecretory sites in the eyestalk of
larval Palaemonetes (Decapoda ; Natantia) ; Biol. Bull 125 96-113
Jacques F 1969a Existence dun organe sensories dans le pendoncule ocularire des larves de
stomatopodes crustace's ; C.R. Acad. Sci. Paris (Ser. 17). 268 89-90
Jacques F 1969b Histogeneses des pendoncules ocutaires des larves de stomatopodes ; C.R.
Acad. Sci. Paris 20 565-596
Kulkarni M Y 1972 Studies on the biology of the freshwater prawn, Macrobrachium kistnensis ;
Ph. D. thesis, Marathwada University, Aurangabad, India
Matsumoto K L 1958 Morphological studies on the neurosecretion in crabs; Biol. J. Okayamo
Univ. 4 103-176
Pyle R W 1943 The histogenesis and cyclic phenomena of the sinus gland and X-organ
in Crustacea ; Biol. Bull. 85 87-102
Zielhorst A J and Van Harp F 1976 Development dus-systema neurosecreteur du pedoncule
oculaire des larves d Abacus leptodactylus Nordmann (Crustacea, Decapoda, Reptantia);
C.R, Acad. Sci. Paris series D 283 1755-1758
Proa Indian Acad. Sci. (Auim, ScL), Vol. 91, Number 6, November 1982, pp. 609-621,
© Printed in
Histological observations on tracheal growth during wing "
development in Oncopeltus fasciatus (Dallas) (Heteroptera; Lygaeidae)
MALLELA NIVEDITA
Department of Zoology and Applied Entomology, Imperial College, University of
London, London SW7 2 AZ, England
Present Address : Department of Zoology, C.K.M. Arts and Science College,
Warangal 506 006, India
MS received 25 February 19S2 ; revised 24 July 1982
Abstract. The development of the tracheal supply to the larval wing pad of
Oncopeltus fasciatus is described. The formation of lacunae is also described and
it is shown that their development precedes the growth of the associated tracheal
supply. Tracheae from anterior and posterior ends of the wing pad enter the
lacunae in second instar. The pattern of adult wing tracheation is well established
in the third instar. Where the Sub-costa, Radius and Medius arise from Costo-
radial trunk, the Cubitus, the first and the second anal tracheae arise from the
Cubito-anal trunk. Both groups are connected by a basal transverse connection.
Keywords. Oncopeltus fasciatus ; tracheae ; lacunae ; larval instar ; wing develop-
ment.
I. Introduction
Since the beginning of this century several papers have been published on the
histology of insect wing development. While much of the investigation has been
confined to Endopterygota (Powell 1904 , 1905 ; Marshall 1913 ; Kohler 1932 ;
Kuntze 1935 ; Behrends 1935 ; Hundertmark 1935; Waddington 1941). Only
a small body of literature exists on the wing development of Exopterygotes (Tower
1903 ; Sulc 1911 ; Beck 1920; Holdsworth 1940, 1942). Despite the number of
works on the histology of wing development, differences of opinion still exist
regarding the development of the lacunae, the tracheae and the relations between
the two.
In order to investigate the differentiation and the growth of wing epithelia and
to ascertain the relationship between the development of lacunae (blood space)
and tracheae, a detailed histological study of the large milk-weed bug, Oncopeltus
fasciatus (Dallas) has been undertaken,
609
Mallela Nivedita
Material and methods
A stock culture of Oncopeltus fasciatus at 24-28° C was maintained in plastic
containers, on a diet of decorticated sunflower seed and water.
Larval instars of various ages were removed from the cultures and part of thorax
and attached wing buds were extirpated and fixed in Bouin's fixative for 18 hrs
and dehydrated in ascending ethanol series up to 70%, followed by dioxan (2
changes), and subsequently embedded in paraffin wax. Serial sections 6 /an thick
were stained in Mallory's connective tissue stain (Hughesdon's modification) and
some in Phosphotungstic haematoxylin (Mallory's). Both of these techniques
were found to be good for obtaining full histological details of the developing
wings of O. fasdatus. The staining times in these solutions were subject to varia-
tion, depending on the age of the insect.
The study of earlier stages was made by reconstructions from the serial sections
as described by Holdsworth (1942).
3. Results
3.1. Mesothoracic wing
3 . la. First instar : Histological study of the thoracic segments of newly
hatched first instars (figure 1) reveals the presence of wing pads, that appear like
minute flanges, measuring 90-100 /*m in length on either side of the thoracic
segments. Each rudiment appears as a hollow, flattened outgrowth of the body
wall and consists of an outer cuticle, and an inner layer of epidermal cells bounded
by a thin, non-cellular basement membrane.
The first instar wing pads are supplied by a single trachea arising from the longi-
tudinal tracjieal trunk towards the anterior side of each thoracic segment (figure 9A).
Renters the base of the wing pad anteriorly and runs posteriorly terminating bet-
ween the thorax and the base of the wing disc, measuring 40-50 jum in length and
l-l'5jum in diameter.
2-4 days after eclosion, the wing pads show a constant gradual growth in their
size. As the, larva grows, the epidermis detaches itself from the cuticle, the cells
elongate, and their ends meet those of. the opposite surface (figure 3a, b). A
small intercellular . space appears in .the anterior region of the wing pad, just in
front of the trachea, that, enters 'the base of the wing pad and runs across (figure
3b). This trachea is \ probably the future Costo-radial trunk.
Prior to the first ecdysis, the new cuticle is thrown into folds. The epidermal
cells become unilaminar in arrangement again, but the fully formed pharate
second instar wing pad is still enclosed in the old cuticle. Though there is single
trachea throughout the first instar, in the late pharate second instar, another
branch (35-40 /nn long and 1-1 • 5 /m in diameter) has been observed to grow
out from the longitudinal tracheal trunk near the posterior margin of the meso-
thoracic segment (figure 9B) and enters the wing rudiment posteriorly. The two
tracheal branches, now lying in the wing pad, grow towards each other.
3.1b. Second instar: In the newly moulted second instar, the wing pads
qn each thoracic segment double in size, 120-130 /on long. In the fully grown
Growth during wing development in 0. fasciatus (Dallas)
611
Figures 1. la, 1, Section through the mesothoracic wing pad of the newly formed
ittstarx450. la. Wing pad at higher magnification x 1SOO.
612
Mallela Nivedita
Figure 2. Section through the mesothoracic wing pad of II instar x 450.
Figure 3. (see captions in p. 614)
Growth during wing development in O. fasciaius (batlas)
(3b)
25//m
mit
bm
oen
(4)
(6)
CP
614
Mallela Nivedita
-cut
(7)
mac-
(8)
Figures 3-8. 3a. Section through the developing wing pad of I instar showing the
detachment of the cuticle, elongation of epidermal cells, meeting those of opposite
surface x 900. Camera Lucida drawings of : 3b. Section through 'the developing
wing pad of I instar, showing the detachment of cuticle. 4. Section through
the II instar wing pad showing the formation of middle membrane. 5. Section
through the II instar wing pad after 40 hrs showing mitotic division in epidermal
cells. 6. Section through the newly moulted in instar wing pad showing the
presence of lacunae. 7. Section through the III instar at 80 hrs after moulting
showing five lacunae with their individual tracheae. 8. Section through the IV
instar wing pad showing prominent lacunae enclosing tracheae.
Abbreviations: bin: basement membrane; cp : cytoplasmic process; cut: cuticle ;ep:
epidermis ; exsp : exuvial space ; gl : gland cell;icsp: intercellur space; lac: lacuna;
mac: macrotrichea ; mfl : moulting fluid ; mit : mitotic division ; mm : middle
membrane ; n : nucleus ; o :oenocyte ; t .: trachea ; tl :tracheole ; wr: wing pad.
second instars, these wing rudiments look like swollen dark areas on the dorso-
lateral regions of the thoracic segments (figure 2). The epidermis consists of a
single layer of cells ostensibly syncitial in nature. The basement membranes of
apposed layers have come close together and a middle membrane has formed
but lacunae have not yet developed near the membrane nor are tracheae asso-
ciated with it. A small space is observed interiorly at the base of the wing pad,
just in front of the middle membrane, indicating the beginning of the first formed
Growth during wing development in 6. fasdatus (Dallas)
615
Figure 9. Schematic diagram to illustrate Course of tracheae in developing meso-
thoracic wings from first to third larval instars.
Abbreviations', A. First instar ; B. Late pharate second instar ; C. Newly moulted
second instar; D. 40 hour ; second instar. E. 72 hour second instar ; F. Pharate
third instar; G. Newly moulted >third instar : atr: anterior trachea ; c-r : Costo-
radial ; Cu : Cubital ; Cu-a : Cubito-anal ; M : Medial ; ptr : posterior trachea ;
R : Radial. R -f M ; radio-medial ; Sc : Sub-costa ; tbt r transverse basal ; th :
mesothorax ; tl ; tracheoles , wr : wing rudiment.
P.(B>-12
61 6 Mallela Nivedita
lacuna (figure 4, icsrj). The two tracheae which appeared at the end of the first
instar enter from opposite directions of the wing pad, and run across the base.
The anterior trachea, a large branch, now measuring 60 /on in length and 3-4/xm
in diameter is the Costo-radial trunk. The posterior branch, regarded as the
Cubito-anal trunk, now measures 40 /im in length and 1 • 5-2 /zm in diameter.
They have grown very close together and are almost in contact, but a trans-
verse basal connection has not yet developed between them. From the Costo-
radial trunk, a small branch is seen growing towards the wing pad for the first
time (figure 9C). It is only 6-10 /nn in length at this stage.
The wing pads grow in size (180 /mi long) and 40 hrs after the first ecdysis,
the cells increase in number and the unilaminar arrangement is lost (figure 5).
The position of the tracheae is the same as that of the early second instar, and
the newly grown branch of the Costo-radial trunk shows a little further extension
(figure 9D). At a distance of 18 /mi from its origin from the Costo-radial trunk,
this branch penetrates the wing pad for a further 12-1 8 /nn. It is the first branch
to enter the wing pad epithelium and grows into the space at the base of the wing
pad which is formed earlier. 72 hrs after ecdysis, this branch penetrates the wing
pad epithelium to a depth of about 42 /nn and then bifurcates. Each branch is
24 /nn in length (figure 9E). Just in front of this main branch, a further fine
branch has grown from the Costo-radial trunk to form the most anterior tracheal
branch of the wing pad, the Sub-costal trachea. The anterior bifurcation of the
next branch (i.e., the first to enter the wing pad) is the Radius, and the posterior
bifurcation is the Media. The Costo-radial and Cubito-anal trunks have now
grown very near to each other (figure 9E).
Prior to the second ecdysis, the Sub-costal, Radial and Medial tracheae show
increased growth inside the wing pad. By the coalescence of the trunks, the
transverse basal trachea has also been established completely and a separate
Cubital trachea has started to grow from the Cubito-anal trunk (figure 9F).
The growth of the metathoracic expansions is rather slow during the second
instar and the inner surfaces of the two layers of the wing pad have not fused.
3.1.c Third-instar : The wing pads of newly moulted third instar larvae show
that the wing membranes of both the surfaces have come to lie very close together
and a fused middle membrane is formed by the apposed basement membranes
(figure 6). The cuticle covering the wing pad is at first very delicate. The
epidermal cells are tall, conical, with oval nuclei, usually towards the base of each
cell. The pointed apices of some epidermal cells meet at the middle membrane,
while those of others are curved to associate with the ends of neighbouring cells,
and leave an intercellular space. Some of these intercellular spaces merge to
form lacunae, for the haemolymph. In the newly moulted third instar such spaces
even extend up to the tip of wing pad (figure 6). Five prominent lacunae were
found in the middle of the wing pad, prior to the appearance of trachea in them.
The tracheae at this stage are still very short and confined to a small region at
the base of the wing pad.
At this stage the framework of adult tracheation ;has also been .established.
The Sub-costa enters the wing pad, just beneath the humeral 'angle. It is about
120 /mi long and bundles of tracheoles arise from it to supply the tip of the wing
pad. These tracheoles are not visible in some sections, but in others they are
Growth during wing development in O. fasciatus {Dallas) 617
very clear. The Radius and the Media run inside the second and third lacunae
respectively for a distance of 120-1 30 jam and give rise to bundles of tracheoles
to supply the tip of the wing pad. The anteriormost branch of the Cubito-anal
trunk the Cubital trachea, is 6-10 /mi long and enters the fourth lacuna of the
wing pad (figure 9G). It also gives off bundles of tracheoles. Towards the
tip of Cubito-anal trunk, two separate bundles of tracheoles grow out and enter
the remaining lacuna to indicate the future growth of the first and second anal
tracheae.
By 70 hrs after the second ecdysis the lacunae are very prominent. The anterior-
most four tracheae, the Sub-costa, Radius, Media and Cubitus have now exten-
ded towards the tip of wing pad, only 40-50 jum away from the apex. The fifth
trachea (the first anal trachea) runs parallel to the base.
By 80 hrs after the second ecdysis, the epidermis has reached its maxium thick-
ness (20 /rai). Proliferative cell division is still in progress, though it is now
about to decline in intensity (figure 7). Though they are very much crowded
together to a thickness of several layers, each nucleus is associated with a tail-
like process that runs towards the basement membrane. All the five lacunae
(Sub-costa, Radius, Medius, Cubitus and 1st Anal) contain their individual
tracheae which run up to the tip of the wing pad (figure 9G).
3. Id. Fourth instar : In the fourth instar, the cuticle covering the wing pad
is very thin and delicate. The epidermis of the wing pad is thin, consisting of a
single layer of cells. The basement membranes of the apposed layers remain
close together as a middle membrane, except around the lacunae (figure 8).
The five prominent tracheae running throughout the length of the wing pad grow
to about 150/nn from the tip. Now there is also a well established sixth lacuna
which runs parallel to the base of the wing pad and is occupied by a second anal
trachea. It is fully grown ia the late fourth-instar. All six lacunae correspond
in arrangement with the veins of the adult wings, which will be formed later
by differential sclerotisation of the integument adjacent to the lacuna. By this
time, the basic pattern of adult venation and tracheation have been established
completely. Groups of tracheoles arise from each nodal point in order to supply
air to every part of the wing pad.
Except for their diameter and length, the pattern of tracheation is same in the
fifth instar wing pads and in newly moulted adult wings (figures 11, 13).
3.2. Metathoracic wing
The essential features of the tracheal growth in a developing metathoracic wing
are identical to those of a mesothoracic wing (figure 10). The rudiments of
both anterior and posterior trachea arise at the end of 1st instar, which penet-
rate into the anterior Costo-radial trunk and a posterior Cubito-anal trunk.
During the second instar a transverse basal connection is established between
them, and a branch each from the Costo-radial and Cubito-anal tracheae penet-
rate the wing pad epithelium (figures 10D, E), A lacuna develops prior to the
second ecdysis. During the third instar stage, the wing pad development records
a rapid growth. The tracheal branching is fully established. Six lacunae develop
the tracheal branching penetrates these lacunae. In the fourth instar stage,
618
Mallela Nivedita
atr
atr
wr-
Cu
B
cu-a
D
cu-a
c-r
F,
Figure 10. Schematic diagram to illustrate course of tracheae in developing meta-
thoracic wings from first to fourth larval instars.
Abbreviations : A : first instar ; B : Late pharate second instar. C. : Newly moulted
second instar ; D : Late Pharate third instar ; E : Newly moulted third instar ; F^ :
Pharate fourth instar; G: Newly moulted fourth instar. ; 1A, 2A : anal tracheae; atr :
anterior trachea ; C-r : Costo-radial ; Cu : cubital ; Cu A : Cubito-anal ; M :
Medial ; ptr : posterior tracheae ; R : Radial ; R + M : Radio medial ; Sc : Sub*
postal ; tbt ; transverse basal ; th3 : metathorax ; wr : wing rudiment,
Growth during wing development in 0. fasciatus (Dallas)
619
the two tracheal trunks are joined by the transverse basal connection. ^ • v
tracheal ramifications are complete and the entire tracheal system assumes
framework of an adult wing. In the fifth instar, the basic pattern is the same
as that of the fourth instar, except an increase in the length of trachea and m
the number of tracheoles (figures 12, 14).
4. Discussion
It is established that the lacunar system corresponds to the venational system. But
opinions differ regarding the developmental relationship of the lacuna and the
trachea. Marshall (1913) while studying Platyphylax designatus (Tricheoptera)
erg
V - -Sc
rv-R+M
(ID
(12)
Figures 11-12. 11. Tracheation of right fare wing pad of fourth instar larva of
Oncopeltus (including basal connection). 12. Tracheation of right hind wing pad
of fourth-instar larva.
620
Mallela Nivedita
cuag~-
(14)
O-Smm.
Figures 13-14. 13. Tracheation of fifth instar fore wing pad (including basal
connections). 14. Tracheation of hind wing pad of fifth instar larva of Oncopeltus
including basal connection. The sub-costa is a very small trachea.
Abbreviations: 1A, 2A : anal ; erg : Costo-radial ; Cu: Cubital ; Cuag: cu bito-anal;
R : Medial; R -h M : Radio-medial ; Sc : Sub-costal ;t 1 : tracheoles ; tht : transverse
basal.
stated that the lacunae develop in the wing pad of the last larval stage and that
the tracheae do not enter until the wing is averted at pupation. Hundertmark
(1935), while dealing with the histology of Tenebrio molitor wings, reported that
the tracheae grow into the newly everted wing disc and later the lacunae are
formed about them. Kuntze (1935) in his studies of Philosamia cynthia (Lep.)
observed the formation of the lacuna earlier than the entrance of trachea. All
these are Endopterygotes. Holdsworth (1940, 1942) in his histological studies
of the wing pads of Pteronarcys proteus (Plecoptera) found that the precursors
of the veins in the nymphal wing pad are the lacunae, the free spaces surrounded
by spongy columnar epidermal cells. The trachea and nerves grow into these
channels only after their patterns have been established.
In the early first instar wing pads of O. fasdatus the epidermal cells increase
in size and undergo mitotic division. The divided cells elongate and their inner
ends meet those of the opposite surface. During this stage a small inter-cellular
space is seen towards the anterior region of the wing pad. The subsequent forma-
tion of lacunae in the wings of O. fasdatus is quite similar to that of Pteronarcys
proteus (Holdsworth 1940, 1942). The tracheae grow into these lacunae indi-
9ating that even in O. fasdatus lacunar envelopment precedes the entrance of
Growth during wing development in 6. fasciatus (Dallas)
tracheae. Holdsworth's conclusions are, therefore, extended to another quite
unrelated order of Exopterygata. Whether the condition found by Hundert-
mark (1935) is a secondary specialization found in Coleoptera or whether there
exist considerable variations in the sequence of lacunar and tracheal develop-
ment, require more elaborate study.
The growth of metathoracic wing pads is slow during the second instar, but in
both meso and metathoracic wing pads complete pattern of adult tracheation is
well established in the fourth instar.
Acknowledgement
The material in this paper was included in my thesis, submitted for the Doctor
of Philosophy at London University. I am deeply indebted to my learned super-
visor Dr R G Davies, Reader in Entomology, Imperial College, for his able
guidance.
References
Beck H 1920 Die Entwicklung Flugelgeaders bel Phyllodromia (Blatta) Germanica ; Zool.
Jahrb. Anat. 41 377-410
Behrends J 1935 Ueber die Entwick-lung des Lakunen. Ader-und Tracheen systems Wahrend
Puppenruhe im Fiugel der Mehlmotte Ephesita kuhniella Zeller ; Z. Morph. Okol. Tiere.
30 573-596
Holdsworth R P 1940 The histology of the wing-pads of the early instars of Pteronarcys proteus
Newport (Plecoptera) ; Psyche. 47 112-119, 714-715
Holdsworth R P 1942 The wing development of Pteronarcys proteus (Pteronarcidae : Pleco-
ptera) ; /. Morphol. 70 431-462
Hundertmark A 1935 Die Entwicklung der Fiugel des Mehlkafers Tenebrio molitor, met beson-
derer Beruk schtingung der Hautungsvorgnge ; Z. Morphol Okol. Tiere. 30 506-543
Kohler W 1932 Die Entwicklung der Fiugel beider Mehlmotte Ephestia kuhniella Zeller mitt
besonderer Berucksichtingung des Zeichnungmusteis;Z.Morp/z0/. Okol. Tiere. 24 282-6&1
Kuntze H 1935 Die Flugelentwicklung bei Philosamia cynthia Drury, mit besoderer Beruck-
sichtigung des Geaders der Lekunen und derTracheensysteme; Z. Morphol. Okol. Tiere 30
544-572
Mallela N 1979 Histology and ultrastructure of wing development in Oncopeltus fadatus
(Dallas) (Hemiptera : Heteroptera) ; unpublished thesis, London University, London.
Marshall W S 1913 The development of a caddis fly, Platyphylax designates Walker ; Z. f.
Wiss. Zool. 105 574-597
Powell P B 1904, 1905 The development of wings of certain beetles and some studies on origin
of wings of insects ; /. New York Entomol. Soc. 12, 13 237-243, 5-23
Sulc K 1911 Uber Respiration, Tracheen system, Und Schaumproduction der Schaurncikaden
Larven ; Z. f. Wiss. Zool. 99 147-188
Tower W L 1903 The origin and development of the wings of Coleoptera ; Zool. Jahrb. Anat.
17 517-572
Waddington C K 1941 The genetic Control of wing development in Drosophila, /. Genet. 41
71-139
Proc. Indian Acad. Sci. (Ariim. Sci.), Vol. 01, lumber 6, November 1$S2, pp. 623-623.
© Printed in India.
The functional demography of adrenal glands in Rattus mdtada
pallidior in Indian desert
B D RANA
All India Coordinated Research Project on Rodent Control
Central Arid Zone Research Institute, Jodhpur 342003, India
MS received 10 March 1982 ; revised 12 August 1982
Abstract. In this paper, the seasonal fluctuations in adrenal glands and its relation-
ship with their body weights, reproduction activity and population density of soft-
furred field rat, Rattus meltada pallidior in the Indian desert has been discussed.
Results of the present study revealed that the left adrenal gland in both the sexes of
rodents was found to be significantly heavier (P < 0-01) than the right one. The
paired adrenal of female rats was significantly heavier (f < O'OOl) than those of
males. The seasonal variations in adrenal weights of pregnant females were found
to be significantly heavier (P < 0-01) than those of nonparous females. The
adrenal weights of male, pregnant and non-pregnant female rats were found to be
significantly correlated with their body weights. Results of this study further
revealed that changes in adrenal weights in Rattus m. pallidior are functions of body
weights which are regulated by the availability of food and its nutritional level*
Keywords. Rattus meltada pallidior ; adrenal glands ; body weight ; pregnant
females ; reproduction activity ; population density*
1. Introduction
It has been postulated that adrenal weight is the function of population density
in Albino rats and in wild house mice of confined density (Christian 1955 ; 1956;
Christian and Davis 1955). Clarke (1953) , Christian (1962) and Southwick
(1963) stated that fighting and social-interactions enhance adrenal weights*
Contrarily, Southwick (1958) and Rudd and Muthen (1963) did not observe
adrenal enlargements due to fighting in house mice (Mus musculus) and Pocket
gophers (Thomomys umbrinus) respectively.
In view of the confirmity, the present study on relationship of adrenal gland
with their body weights, reproduction activity and population density was under-
taken in the free living population of soft-furred field rat, Rattus meltada pallidior
in Thar desert.
623
P. (B)— 13
624 S D Rana
2. Materials and methods
The Rattus meltada pallidior (45 c?c? and 43 ??) were captured during January 1978
to December 1978 from Bisalpur (25° T N-73° 10' E) in Western Rajasthan.
Later on, the rats were weighed, sexed, dissected and both right and left adrenal
glands were preserved in 10% formaldehyde. The preserved adrenal glands were
weighed on semimicro balance to the nearest 0-001 g.
3. Results
3.1. Difference between right .and left adrenal gland
The left adrenal gland was found to be significantly heavier than the right one
in both the sexes of rats (table 1).
3 . 2. Difference between sexes
The right and left adrenal gland of female rats were found to be heavier than
those of the males, but the significant (P < 0-001) difference was noticed in case
of left ones (table 1). Similarly, on an average the paired adrenal weights of
female rats were significantly (p < 0 • 01) heavier than those of male rodents. The
mean monthly paired adrenal weights of females were found to be significantly
heavier (P < 0-05, P < Q-01) than those of male rats, almost throughout the
year. However, the male adrenals were recorded significantly heavier (P < 0*01)
during July and October (table 2). The relative as well as absolute adrenal weights
of pregnant females were observed significantly heavier (P < 0 • 01) than those
of nonparous females (table 3).
3.3. Seasonal trend through the year
The fluctuations in adrenal weights of both male and female exhibit a peak during
February, July and October in former sex and February-March and September-
Table 1. Absolute adrenal weights (mean ± S.E.) of Rattus meltada pallidior.
Average adrenal weights (mg)
Sex No,
Right Left Paired *t' between
Male
45 6'98-hO'63
8-52+0-71
13-46+1-05
1 and 3 =2-65
(D
(3)
(5)
(P< 0-01)
Female
43 7-79+0-42
9-55+0-47
16-48+2-41
2 and 4= 3- 74
(2)
(4)
(6)
(P < 0-001)
3 and 4= 2'03
(P< 0-05)
1 and 2 = 1*44
Functional demography in Rattus meltada pallidlor 625
Table 2. Seasonal fluctuations in adrenal weight of Rattus meltada pallidior.
Paired adrenal weights
Mean ± S.E.
IVJLUillLll^
Males Of = 45)
Females (n = 43)
L UVLWV^IJ
males and females
January
10-12 ± 5-95
9-22 ±5- 30
0-39
February
18- 65 ±0'46
17-53 ±8-04
0-44
March
10-00 ±0-00
IS- 55 ±0-75
5-09**
April
9-44 ±2-05
13-10±2-06
4-93**
May
9-00 ±0-00
13-00 ±1-00
2-85*
June
12- 36 ±2- 58
14- 96 ±3- 58
2-27*
July
23-18 ±2-58
14- 96 ±3- 58
2-27*'
August
10-90 ±1*35
16-55 ±2-08
2-09*
September
11-00 ±0-00
19- 90 ±0-07
5-83**
October
18-55 ±1-50
16-50 ± 1-50
2-13*
November
15-83 ±0-58
22- 25 ±2- 20
3-02*
December
12-51 ±3-51
18-05 ±1-88
3-40*
* =/>< 0-05 ; ** =
0-01
November in latter sex (table 2). Thereafter, in females, they remained almost
constant throughout the year. Whereas, in case of males remarkable decrease
from March to June was observed. The lowest adrenal weights were found
during August to September in case of males (table 2).
Table 3. Adrenal weight in relation to prevalence of pregnancy.
Adrenal weight
f <o-OJ
Adult females
— , , « t ' between
Pregnant (n = 28) Nonparous (n =15)
Absolute
IS- 35 ±1'32
16* 52 ± 0-60
1 and
2 = 3.21**
(1)
(2)
Relative
42- 14 ±0-93
3S-48± 1-38
3 and
4 = 5-94**
(3)
(4)
626 B D Rana
3.4. Adrenal weight in relation to body weight
The adrenal weights of male, pregnant and not pregnant female metads were found
to be significantly correlated (r = 4- 0-592, P < 0*01, r = + 0*609, P < O'Ol
and r = -f 0'890, P < O'Ol) respectively with their body weights. The fluctua-
tions in the changes of male and female adrenal weights were found to be almost
in similar pattern throughout the year (figures 1 and 2), which suggests that
weight of adrenal gland in Rattus meltada pallidior is influenced by body weight.
^ 0-— O MALE ADRENAL WEIGHT
tv
E • t> MALE BODY WEIGHT
A M J J
MONTHS
Figure 1. Paired adrenal weights of male Rattus m. pallidior in relation to their
body weight.
O o FEMALE ADRENAL WEIGHT
• • FEMALE BODY WEIGHT
62-
6O-
58-
54-
| 52
!< a
- 23
I 21
5-
SIT-
^ 15
ui
o 13
o n
JFMAMJJASONQ
MONTHS
Figure 2, Paired adrenal weights of female Rattus m. pallidior in relation to female
weight.
Functional demography in Rattus meltada pallidior
627
3.5. Adrenal weight in relation to reproduction activity
The females JR. m. pallidior litter throughout the year with two peaks one in
March to April and another in July to November (Rana and Prakash 1981). The
adrenal weights of females R. m. pallidior show a parallel fluctuation trend with
the prevalence of pregnancy (figure 3), suggesting an increase in adrenal weights
with the enhanced female fertility. The adrenal weights are influenced by preg-
nancy stress is further confirmed by the data presented in table 3, where both the
absolute as well as relative adrenal weights of pregnant female rodents are signi-
ficantly heavier (P < 0-01) than those of nonparous females (table 3).
0—0 f EM ALE ADRENAL WEIGHT
• • PERCENT PREONANT FEMALE
100
90
| 80
HI 70
S 50
20
g25-
22°'
I19
z 10
5 5
JFMAMJ JASON D
MONTHS
Figure 3. Paired adrenal weights of Rattus m. pallidior in relation to female fertility.
10-
9-
8-
6-
a 4-
8 3
«T 2
* 1
*20-
die-
ui
SI6.
S l4"
I 12-
5 10
« --- • ADRENAL WEIGHT
° - ° TRAP INDEX
JFMAMO JASON 0
MONTHS
Figure 4. Variation in paired adrenal weights of Rattus m. pallidior in relation, to
tfyeir population density.
628 B D Rana
3.6. Adrenal weight in relation to population density
The pooled adrenal weights of both male and female rats showed three peaks in
July, November and February, whereas, the population density was found to be
low during these peak levels, indicating reverse pattern (figure 4). This may be
explained by the facts that their numbers do not influence the seasonal variations
among adrenal gland. The trap indices exhibited two peaks, one in December
and the second in April-June and August. These peaks do not have any relation-
ship with their adrenal weights.
4. Discussion
A striking similarity in the fluctuations of adrenal weight and body weight in adult
male and female indicate that changes in variations among them are influenced
by body weights which are regulated by the availability of food and its nutritional
level. Similar observations were made among other species of Indian mammals
(Prakash et al 1969 ; Jain 1971 ; Rana et al 1975 : Rana 1981).
Selye (1936) argued that an increase in adrenal weight is due to the pregnancy
stress, on the other hand, Christian (1962), Christian and Davis (1964) suggested
that this enhancement is a reflection of the social-interactions, the frequency of
which usually exhibited an increase during breeding season. Similarly, in the
present study the significant differences in the adrenal weights of pregnant and non-
par ous R. meltada points out that physiological processes of reproduction might
have an impact on the adrenal weights. Similar observations were made in
jR. c. cutchicus (Rana et al 1975), T. indica (Jain 1971) and Jack-Rabbit, Lepus
californ!cu5 melanotis (Herrick 1965). Whereas no relationship was found
between male fecundity and adrenal weights decreased (figure 5), however, percent
O O MALE ADRENAL WEIGHT
« 9 PERCENT MALE FECUND
K30!
90
o 80
z
o 70
so
ki 25
e:
tu
s
at 10
iu
2£ S
-T-
~T 1 1~
A M J J A 5
MONTHS
Figure 5. Paire4 adrenal weights of Rattus m. pallitfior in relation to male fecundity.
Functional demography in Rattus meltada paltidior 629
male fecuad rats showed a decline trend during November. The adrenal glands
did not reflect much change in their seasonal trend. During summer season
when breeding activity in male metad had ceased, a second minor peak in adrenal
weights was exhibited.
The average number and the adrenal weights of Woodchucks, Marmota monax,
are closely associated and fluctuations found among them follow a parallel
trend throughout the year which tends to indicate that weight of adrenal glands
is influenced by population density (Christian 1962). Whereas, the results of
present study on free living population of R. meltada suggest that the increase
in population density decreased the weight of adrenal gland.
Acknowledgement
Author expresses his deep gratitude to Dr H S Mann, Director of the Institute,
and Dr Ishwar Prakash for encouragement and for providing necessary facilities.
References
Christian j j 1955 Effect of population size on the adrenal glands and reproductive organs of
male mice in population of fixed size ; Am. J. Physiol. 182 292-300
Christian j j 1956 Adrenal and reproductive responses to population size in mice from freely
growing populations ; Ecology 37 258-273
Christian j j 1962 Seasonal changes in the adrenal glands of Woodchucks (Marmota monax) J
Endocrinol. 7 431-447
Christian j J and Davis D E 1955 The reproduction of adrenal weight in rodents by reducing
population size ; Trans. 20r/z North Am. Wildlife Conf. pp. 177-184
Christian J J and Davis D E 1964 Adrenal glands in female voles (Microtus pennsylvanicus)
as related to reproduction and population size ; J. Mammal. 47 1-18
Clarke J R 1953 The effect of righting on the adrenals, thymus, and spleen of the vole (Microtus
agrestis) ; J. Endocrinol. 9 114-126
Herrick E H 1965 The black-tailed Jack rabbit Kansas N. Endocrine Studies; Kansas Univ>
Sti. Butt. 140 73-75
Jain A P 197! Adrenal weight in the Indian gerbil, Tatera i. indica Hardwicke, as related to
bodyweight and reproduction activity ; 4 279-288
Prakash Ishwar, Dass B and Taneja G C 1969 Adrenal weight in relation to body weight and
reproduction activity in the Indian desert hare, Lepus nigricollis day anus ; /. Anat. Soc.
India 2 42-47
Rana B D 1981 Variations in the weight of the adrenal glands of Suncus murinus sindensis ;
Annals Zool 18 115-124
Rana B D and Prakash Ishwar 1981 Reproduction biology of the soft-furred field rat, Rattus
meltada pallidior (Ryley 1914) in the Rajasthan desert ; /. Bombay nat-Hist. Soc. (In press).
Rana B D, Prakash Ishwar and Jain A P 1975 Variation in the weight of adrenal glands of
Cutch-Rock rat, Rattus cutchicus (Wroughton) ; Mammalia 39 479-486
Rudd R L and Muthen D A 1963 Adrenal gland responses to experimental manipulations of
captive pocket gophers (Thomomys unibrinus) ; /. Mammal. 44 451-466
Selye H 1936 Thymus and adrenals in response of the organism to injuries and intoxications ;
Br. J. Expt. Pathol. 17 234-248
Southwick C H 1958 Population characteristics of house mice living in English corn ricks :
density relationships ; Proc. Zool. Soc. London 131 163-175
Southwick C H 1963 Adrenal weights and eosinophil levels of Peromyscus leucopm in relation
to social climate ; Bull. Ecol. Soc. Am. 44 130 (Abstr.)
Proc. Indian Acad. Sci. (Anini. Sci.), Vol. 91, Number 6, November 1982, pip. 631-631
© Printed in India.
Description of three new species of Drosophila (Scaptodrosophila)
from Orissa, India
J P GUPTA and K K PANIGRAHY
Genetics Laboratory, Department of Zoology, Banaras Hindu University,
Varanasi 221 005, India
MS received 29 March 1982 ; revised 8 October 1982
Abstract. Drosophila koraputae, D. neomedleri and D. puriensis all belonging to
the subgenus Scaptodrosophila are described as new species. Their taxonomic
relationships, based on the morphology and male genital structures, are established.
Keywords. Drosophilidae ; Drosophila koraputae ; D. neomedleri ; D. puriensis.
1. Introductions
Until recently very little has been known concerning the Drosophilid fauna of
Orissa (Gupta 1972 ; Dasmohapatra et al 1981). This paper deals with the
descriptions of three more new species collected recently from a wild area in
Koraput district of Orissa*
2. Taxonomic descriptions
2.1. Genus Drosophila Fallen
Drosophila Fallen, 1823, Diptera Sueciae Geomyzides, 2 : 4 Type species :
Muscafunebris Fabricius ; Sweden*
2.2* Subgenus Scaptodrosophila Duda
Scaptodrosophila Duda, 1923, Mus. Nat. Hungarici, Ann. 20 t 37. Type species :
Scaptodrosophila.
Scaptomyzoidea Duda ; New Guiena
Paradrosophila Duda, 1923, Mus. Nat. Hungarici, Ann. 20 : 43. Type species 3
Drosophila pictipennis Kerte'SZ ; New Guiena.
Pugiodrosophila Duda, 1924, Arch. Naturg. 90A (3) : 203. Type species :
Drosophila pugionota de Meijere ; Simalur.
631
P.(B>-14
632 J P Gupta and K K Panigrahy
Xiphidiochaeta Duda, 1925, Mus. Nat. Hungarici, Ann. 22 : 200 (improper
replacement name for Pugiodrosophila ; type : D. pugionota de Meijere).
Pholadoris Sturtevant, 1942, Univ. Texas publ. 4213 : 28. Type species :
Drosophila victoria Sturtevant ; United States.
2.3. Drosophila (Scaptodrosophild) koraputae, sp. nov.
2. 3 a. Head, $ and $ : Arista with 4 dorsal and 3 ventral branches in addition
to terminal fork. Antennae with second segment reddish brown ; third seg-
ment brown. Frons including ocellar triangle pale brown. Orbitals in ratio
of 7 : 4 : 11, anterior reclinate orbital closer to proclinate than posterior reclinate.
Vibrissa strong, second oral not differentiated. Palpi pale, slender, with 3-4
marginal setae. Carina brown, broad and high. Face and cheek dark brown,
greatest width of cheek 0'16 the greatest diameter of eye. Clypeus dark browne.
Eyes dark red.
2.3b. Thorax, $ and $ : Acrostichal hairs somewhat irregular, in 8-40 rows.
Prescutellars well developed. Anterior scutellars convergent ; posterior scutellars
crossing each other. Anterior dorsocentral two-fifth length of posterior dorso"
central ; distance from anterior dorsocentral to posterior dorsocentral about half
the distance between two anterior dorsocentrals. Mesonotum brown ; with a
rectangular dark brown dorsal median patch on posterior half. Scutellum pale
brown dorsal median patch on posterior half. Scutellum pale brown, with lateral
sides black. Humerals two, outer thicker and long. Thoracic pleura dark brown,
with a faint pale stripe. Sterno-index about 0-7. Legs yellowish brown, pre-
apicals on all three tibiae ; apicals on first and second tibiae.
2.3c. Abdomen, $ and $ : Abdominal tergites yellow, 2T-3T with dark brown
medially interrupted uniformly broad black bands, Sternites brown.
2.3d. Wigs, c? and $ (figure ID) : Clear, d bristle one ; C3 bristles on basal
about three-fourth of third costal section. Halteres white.
2.3e. Periphallic organs (figure I A) : Epandrium yellowish brown, pubescent,
broadened below, with 5 bristles on upper half and 15 closely placed bristles on
lower half. Surstylus small, with 8-9 black, stout teeth arranged in a row on
outer margin and a few fine setae ventrally. Cerci elongate, pubescent, with 22
small bristles.
Table 1. Average wings indices calculated from 1Q<$<$ and 4
C-index 4K-index 4C-index 5X-index
;^Male
Female
2'3$
2-52
2-0
2-1.2
1-0
1-16
2*0
1-92
Average length of wing 2* 7S mm (c?) ; 2* 9 mm (?)
Average length of body 2- 49 mm ($) ; 2*7 mm ($).
Drosophila species from India
633
Figure 1. (A-D). Drosophila koraputae sp. nov. : A. Penphalhc _ organs :,
L nov. B. Phallic organs ; C. egg-guide ; D. male wing. (E-H). DrosopMa
Lwtien. E. Periphallic organs; F. Phallic organs; G. Decastemum ,
H. Male wing.
2 3f Phallic organs (figure IB) : Aedeagus pale, bifid, broadened below basal
apodeme of aedeV straight, about one and half times as long as aedeagu.
Anterior gonapophyses pale, club shaped with 2 subapical sensrtla. HyPa°™
mSy project; with two pairs of long stout submedian spmes, ventral fragma
hemispherical.
?.3g. Egg-guides (figure 1C) : Lobe yellow, elongate with 13 margmal teeth,
apL'al tooth with broad tip, and with 11 discal teeth, upper five bns le ^
Holotype A India : Narayanpur, Koraput D.stnct, Onssa April 1981, Colls.
Gupta and Panigrahy.
Paratypes:? c? <?, 2??, collection data same as holotype.
All type specimens are at present deposited in the Museum Departmen to
Zoology, Banaras Hindu University, Varanast, Indm. 2 $ $ and 1 $ fr
634 j p Gupta and K K Panigrahy
the paratype series are also deposited in the " Drosophila collection" of Depart.
meat of Biology, Tokyo Metropolitan University, Tokyo, Japan.
2.3h. Relationships : This species in the brunnea group closely resembles
D. scutellimargo Duda, but it distinctly differs from it, having a rectangular, dark
brown dorsal median patch on posterior half of mesonotum (no median patch
in Scutellimargo), 2T-3T with dark brown medially interrupted uniformly broad
apical bands (2T-3T brownish yellow, with white fluorescence in Scutellimargo),
anterior gonapophyses club shaped, with 2 subapical sensilla (large, dorsally
curved with many sensilla in Scutellimargo), hypandrium with 2 pairs of submedian
spines (1 pair in Scutellimargo), ventral fragma hemispherical (almost squarish
in Scutellimargo).
Distribution : India.
2.4. Drosophila (Scaptodrosophild) nemeodleri : sp. nov.
2 . 4a. Head, <J : Arista with 4 dorsal and 2 ventral branches in addition to
terminal fork. Antennae with second segment reddish brown ; third segment
pale brown. Frons pale brown, ocellar triangle dark brown. Orbitals in ratio
of 8 : 2 : 10. Second oral thin, about half the length of vibrissa. Palpi pale
brown, slender, with one prominent apical and 2-3 fine ventral setae. Carina
dark brown, moderately ridged. Face and cheek dark brown, greatest width of
cheek 0*14 the greatest diameter of eye. Clypeus black. Eyes dark red.
2.4b. Thorax, <$ : Acrostichal hairs somewhat irregular, in 6-8 rows. Anterior
scutellars nearly convergent ; posterior scutellars crossing each other. Anterior
dorso central half the length of posterior dorsocentral ; distance from anterior
dorsocentral to posterior dorsocentral about two-fifth the distance between two
anterior dorsocentrals. Mesonotum and scutellum unicolorous, blackish brown,
tip of scutellum white. Thoracic-pleura blackish brown. Sterno-index about
0-6. Legs brown, coxae, femora and tibia of fore legs blackish brown ; coxae
and femora of midandhindlegs dark brown ; tarsal segments of all legs yellowish
brown, joints lighter. Preapicals on all three tibiae ; apicals on first and second
tibiae.
2.4c. Abdomen $ : 1 Tergite pale yellow, 2T with narrow medially interrupted
brown apical band, 3T with medially interrupted broad band, the remainder ter-
gites completely dark brown. Last two sternites light brown.
2.4d. Wings, $ (figure IH) : Hyaline. Ci bristle one ; C3 bristles on basal
about two-fifth of third costal section. Halteres white.
Table 2. Average wings indices calculated from 11 $<$.
C-index 4F-index 4C-index 5^T-index
Male 1-8 2-21 1'31 2-0
Average length of wing 2 -47 mm ($) ; Average length of body 2 '74 nun (<J).
Drosophila species from India 635
2.4e. Periphallic organs (figure IE) : Epandrium brown, pubescent broadened
below and narrowly projected at heel, with 12 bristles running from the middle
of posterior margin downwards. Surstylus small, with 9 small dissimilar teeth
arranged in a straight row on outer margin and 6 short dorso-medial and a few
fine setae ventrally. Cerci brown, pubescent, narrow and elongate, with 7 upper
long and 5-6 smaller bristles. Decasternum (figure 1G) brown, with lateral
pieces inwardly projected.
2.4f. Phallic organs (figure IF) : Aedeagus brown, short and stout, apically
pointed and hairy. Basal apodeme of aedeagus straight and thick, about twice
as long as aedeagus. Anterior gonapophyses pale, narrow, finger like having
4 basal sensilla and 4 equidistantly placed upper sensilla. Hypandrium with 2pairs
of strong submedian spines, inner pair little longer. Ventral fragma rounded
distally.
2.4g. Holotype cf, India : Narayanpur, "Koraput District, Orissa, April, 1981
Colls. Gupta and Panigraphy.
2.4h. Paratypes ; 8 <J <£, collection data sams as holotype.
All type specimens are at present deposited in the Museum, Department of
Zoology, Banaras Hindu University, Varanasi, India. 2 <$ $ from the paratype
series are also deposited in the " Drosophila Collection " of the Department of
Biology, Tokyo, Metropolitan University, Tokyo, Japan.
2.4i. Relationships : This species resembles D. medleri Tsacas and Chassagnard
in having somewhat similar male genital structures, but distinctly differs in
having mesonotum with no silvery stripes (mesonotum with four brown broad
stripes having silvery fluorescence, in medleri), surstylus with a row of 9 small
dissimilar teeth arranged in a straight row (with a row of 11 similar strong
teeth arranged in a concave row in medleri)^ ventral fragma rounded distally
(almost rectangular in medleri).
Distribution : India.
2.5. Drosophila (Scaptodrosophila) puriensis, sp. nov.
2.5a. Head, <J and $ : Arista with 4 dorsal and 3 ventral branches in addition
to long terminal fork. Antennae with second segment reddish-brown ; third
segment pale brown. Frons including ocellar triangle dark brown. Orbital s
in ratio of 8 : 5 : 12. Vibrissa strong, second oral not differentiated. Palpi pale
brown, slender with 3-4 marginal setae. Carina brown, narrow, high and some-
what broadened below. Face and cheek brown, greatest width of cheek 0-12
the greatest diameter of eye. Clypeus dark brown. Eyes dark red.
2.5b. Thorax, $ and $ : Acrostichal hairs very small, in 8-10 rows between
dorsocentrals. Anterior scutellars nearly parallel ; posterior scutellars crossing
each other. Anterior dorsocentral half the length of posterior dorsocentral ;
distance from anterior dorsocentral to posterior dorsocentral about one-third
the distance between two anterior dorsocentrals. Mesonotum shiny dark
brown, anteriorly lighter, with narrow and faint longitudinal streaks in the line
9f dorsocentrals. ScuteHum blackish brown with yellowish tip. Humerals two?
636
/ P Gupta and K K Panfgrahy
Subequal? outer strong. Thoracic pleura blackish-brown. Sterno-index about
0*6, Legs : Dark brown, tarsal segments slightly lighter. Preapicals on all
three tibiae ; apicals on first and second tibiae.
2.5c. Abdomen, £ and $ : 1 Tergite pale yellow. 2T-4T with shiny broad black
bands, the remainder tergites uniformly black. Sternites black.
2.5d. Wings, $ and $ (figure 2Z>): Clear Ci bristle one ; C3 bristleson basals
three-fourth of third costal section. Halteres white.
Table 3. Average wings indices calculated from 12 <$<$ and 14 $$.
C-index 4F-index 4C-index S^-index
Male '
Female
2-36
2-48
2-0
2-12
1*0
1-18
1-87
1-72
Average length of wing 2-52 mm ($) 2*66 mm ($) ; Average length of body 2 '81 mm
B
Figure 2 (A-D). DrosopMla puriensis sp. nov. A. Periphallic organs ; B. P^allig
organs ; C. c^g-guide ; D. Male wing.
Drosophita species from India 63?
2.5e. Periphallic organs (figure 2A) : Epandrium yellowish brown, pubescent,
broadened below with 3 long bristles on upper half and 12 similar bristles on lower
half. Surstylus small, with 6 small teeth arranged in a straight row and 4 dorso-
medial and 2 ventral setae. Cerci pale yellow, large elongate, pubescent, with
17 long and 4-5 stout setae ventrally.
2.5f. Phallic organs (figure 2B) : Aedeagus pale, bifid, crescentric in lateral
aspect ; narrowing distally ; basal apodeme of aedeagus about one and half times
as long as aedeagus. Anterior gonapophyses pale, blade like, narrowing apically,
contagious with aedeagus, with 4 equidistantly placed marginal sensilla. Posterior
gonapophyses fused together forming a triangular process. Hypandrium with a
pair of submedian spines of moderate length. Ventral fragma hemispherical.
2.5g. Egg-guides (figure 2C) : Lobe yellow, elongate with 23 small marginal
teeth and 5 discal bristles. Basal isthmus thick and short.
2.5h. Holotype <J, India : Narayanpur, Koraput District, Orissa, April 1981,
Colls. Gupta and Panigrahy.
2.5L Paratypes : 8 <J <J, 11 $$, collection data same as holotype.
All type specimens are at present deposited in the Museum, Department of
Zoology, Banaras Hindu University, Varanasi, India. 2 # <J and 1 <j> from the
paratype series are also deposited in the " Drosophila Collection " of the Depart
ment of Biology, Tokyo Metropolitan University, Tokyo, Japan.
2.5j. Relationships : This species somewhat resembles D. parabrunnea Tsacas
and Chassagnand, but it distinctly differs from it in having surstylus with 6 sparsely
placed stout teeth (with a group of tightly placed 12 strong teeth in Parabrunnea),
anterior gonapophyses with 4 equidistantly placed sensilla (with numerous scattered
sensilla in parabrunnea), egg-guide with 28 teeth, apical teeth placed apart (with
40 teeth, five apical teeth tightly placed in parabrunnea), submedian spines of
moderate length (usually long in parabrunnea).
Distribution : India.
Acknowledgements
The authors are thankful to Dr T Okada, Emeritus Professor, Department of
Biology, Tokyo Metropolitan University, Tokyo, Japan, for his help in confirming
the identifications and to Prof. M S Kanugo, Head of the Zoology Department, for
facilities. One of us (KKP) is thankful to UGC for awarding Teacher Fellow-
ship under the Faculty Improvement Programme.
References
Dasmohapatra D P, Tripathy N K and Das C C 1981 Distribution of different species of
Drosophila in Khallikote Ghats, Ganjam District, Orissa, India ; D.I.S. 56 45
Gupta J P 1972 D. orissamto, A new species of Drosophila from Orissa, India, Oriental Ins.
6 561-563
PROCEEDINGS OF THE
INDIAN ACADEMY OF SCIENCES
(Animal Sciences)
VOLUME 91. JANUARY-DECEMBER 1982
INDEX
THE INDIAN ACADEMY OF SCIENCES
BANGALORE 56O O8O
PROCEEDINGS (Animal Sciences)
Volume 91, 1982
SUBJECT INDEX (Animal Sciences)
\bdominal ganglion
Microanatomy of the 7th abdominal ganglion
and its peripheral nerves in the scorpion
Heterometms fulvipes 225
Accessory cells
The annual reproductive cycle of Achaetobo-
nellia maculata (Echiura : Bonellidae) 569
Acid phosphatases
Acid phosphatase activity in tissues of Notop-
terns notopterus chronically exposed to
phenolic compounds 7
Adaptive modification
Differences in home ranges of rhesus monkey
(Macaco, mulatto) groups living in three
ecological habitats 13
Adrenal glands
Histology and hi sto chemistry of adrenal
glands of Indian mongoose Herpestes edwardsii
edwardsii (Geoffroy) 113
The functional demography of adrenal glands
in Rattus meltada pallidior in Indian desert
623
Albumen gland
Histological and histochemical studies on the
albumen gland and capsular gland of Thais
bufo (Lamarck) (Mollusca : Gastropoda)
407
Aldrin effects
Effects of aldrin on serum and liver consti-
tuents of freshwater catfish Clarias batrachus
L. 27
Ammonia quotient
Metabolic rates and quotients in the cichlid
fish, Tilapia mossambica (PeUrs) in relation
to random activity 217
Anagrus spp Gonatopus
Studies on egg and nymphal parasites of rice
planthoppeis, Nilaparvata lugens (Stal) and
Sogatella furcifera (Horvath) 165
Anti-andro&enicity
Synthesis of 4-methyl (6,7-£-telrahydrGbenzo-
furano)-coumatin and its contraception like
properties in male rabbits (Oryctolagus
cuniculus) 577
Antibiotic
Effect of some antibiotic compounds iri
cotton on post-embryonic development of
spotted bollworm (Earias vittella F.) and the
mechanism of resistance in Gossypiunt
arboreum 67
A/O ratio
Branchial protein metabolism of freshwater
fish Tilapia mossambica (Peters) during acute
exposure and acclimation to sublethal alka-
line water 235
Aplocheilus lineatus
Toxicity of certain pesticides found in the
habitat to the larvivorous fishes Aplocheilus
lineiitus (Cuv. and Val.) and Macropodus
cupanus (Cuv. and Val.) 323
Argulus mangalorensis
A new species of Argulus Muller (Crustacea :
Branchiura) with a note on the distribution
of different species of Argulus in India 375
Ariidae
Some biometric studies of certain closely
related species of the genus Arius (Pisces :
Siluriformes : Ariidae) 79
Arius species
Some biometric studies of certain closely
related species of the genus Arius (Pisces i
Siluriformes : Ariidae) 79
Artocarpus chaplasha Roxb.
Life and fecundity tables for the longicorn
beetle borer* Olenecampius bilobus (Fabricius)
(Coleoptera : Cerambycidae) 249
Assimilation efficiency
Effect of salinity on the survival and growth
of Chanda (=*Ambassis) gymnocephalus (Lac.)
fry (Pisces : Centropomidae) 143
Bandicota bengalensis
Rhythmic oscillations in non-aggressive
social behaviour in Bandicota bengalensis 317
Barbital sodium
Durational effects of hemispayirg on ovarian
hypertrophy and estrous cycle in albino
rats 433
Index
Interruption of pregnancy by barbiturates
in albino rats 533
Base line susceptibility
Evaluation of warfarin against Taterc indica
and Meriones hurriance 463
Bat
Histochemical studies on non-specific esterases
in epididymis of the bat, Cynopterus sphinx
sphinx 329
Baya weaver bird
Steroid metabolism in target related to nuptial
plumage production in the Baya weaver
bird 361
Behaviour
Life history and behaviour of the cyst nema-
tode, Heterodera oryzicola Rao and Jaya-
prakash, 1978 in rice (Oryza saliva L.) 283
Bimodal
Rhythmic oscillations in non-aggressive social
behaviour in Bandicota bengalensis 317
Biological management
Seasonal fluctuations in the diet composition
of Rhinopoma hcfl'dwickei in the Rajasthan
desert 563
Biometric study
Some biometric studies of certain closely
related species of the genus Arius (Pisces :
Siluriformes : Ariidae) 79
Blackheaded bunting
Circadian basis for the photoperiodic response
in the male blackheaded bunting (Emberiza
melanocephald) 357
Blood
Three new species of haematozoans from fresh-
water teleosts (Pisces) 397
Blood flukes
. On some blood flukes (Spirorchiidae: Coeuri-
trematinae) from freshwater chelonians in
India 275
Body weight
The functional demography of adrenal glands
in Rattus meltada palUdior in Indian desert 623
Branchial metabolism
Branchial protein metabolism of freshwater
fish Tilapta mossanibica (Peters) during acute
exposure and acclimation to siiblethal alkaline
water 235
C. batrachus
Effects of aldrin on serum and liver consti-
tuents of freshwater catfish Glorias batrachus
L. 27
Seasonal variations in the phosphorus contents
of the muscle of catfish Clarias batrachus
L. . 423
Callosobruchus maculatus
Studies on preference of Callosobruchus
maculutus Fabricius to some high yielding
varieties of arhar (Cajanus cajan L.) 391
Capsular gland
Histological and histochemical studies on the
albumen gland and capsular gland of Thais
bufo (Lamarck) (Mollusca : Gastropoda) 407
Carbohydrates
Biochemical studies on the haemolymph and
heart muscle of normal and insecticide treated
cockroach Periplaneta americana L. 481
Caridea
Hepatopancreatic sucrase of Macrobrachium
lamarrei (Crustacea, Caridea, Palaemonidae)
33
Carnivores
Sediment polychaete relationship in the
Vasishta Godavari estuary 199
Caste differentiation
Observations on the natural history and
population ecology of the social wasp Ropa-
lidia marginata (Lep.) from Peninsular India
(Hymenoptera : Vespidae) 539
Cellular sites
Cellular sites of steroid synthesis in the
oviparous teleost fish (Cyprinus carpio L.) :
A histochemical study 587
Chanda commersonii
Effect of teleostean prey size and salinity on
satiation amount, satiation time and daily
ration in the glassy perchlet Chanda (= Ambas-
sis) thomassi (Day) (Pisces : Centropomidae)
507
Chanda gymnocephalus
Effect of salinity on the survival and growth
of Chanda (= Ambassis) gymnocephalus (Lac)
fry (Pisces : Centropomidae) 143
Chemical communication
Behavioural responses of the Indian gerbil,
Tatera indica to conspecific sebum odour of
the ventral scent marking gland 259
Chelonians
On some blood flukes (Spirorchiidae ;
Coeuritrematinae) from freshwater chelonians
in India 275
Chromium
The tannery industrial effluent effect on succi-
nate dehydrogenase activity pattern in a
freshwater snail, Pila gkbosa 427
Circadian
Circadian basis for the photoperiodic response
in the male blackheaded bunting (Emberiza
melanocephald} 357
Index
ill
Clibanarius longitarsus
Shell selection in the estuarine hermit crab
Clibanarius longitarsus (De Haan) 39
Coeuritrema
On some blood flukes (Spirorchiidae :
Coeuritrematinae) from freshwater chelonians
in India 275
Commensal species
A comparison of the electrophoretic haemo-
globin pattern of the commensal rodent
species 159
Copulation
Effect of temperature and humidity on the
development and fertility-fecundity of Acrida
exdtata Walk. 267
Corpora lutea
Interruption of pregnancy by barbiturates in
albino rats 533
Corpus luteum
Transabdominal migration of o,va in some
freshwater turtles 189
Corvospongilla lapidosa
Ecobiology of Corvospongilla lapidosa
(Annandale 190&) (Porifera : Spongillidae) in
the Manjira Reservoir, Sangareddy, Andhra
Pradesh 553
Crab
Structure and seasonal changes in the testes
of a freshwater crab, Potamon koolooense
(Rathbun) 439
Crustacea
Hepatopancreatic sucrose of Macrobrachium
lamarrei (Crustacea, Caridea, Palaemonidae) 33
Cuticle
Structure and chemical composition of the
cuticle of Cirolana fluviatilis, Sphaeroma
walker i and Sphaeroma terebrans 57
Cynopterus sphinx sphinx
Histochemical studies on non-specific esterases
in epididymis of the bat, Cynopterrus sphinx,
sphinx 329
Dark ayclc
Orcadian basis for the photoperiodic response
in the male blackheaded bunting (Emberiza
melanocephala) 357
DDT
Effect of DDT on brain neurosecretory cells
of adult Poekilocerus pictus (Orthoptera :
Acrididae) 305
Detritus feeders
Sediment polychaete relationship in the
Yasishta Godavari estuary . 199
Diet composition
Seasonal fluctuations ID the diet composition
of Rhinopoma hardwickei in the Rajasthan
desert 563
Differential radio-sensitivity
Effect of x-rays on the somatic chromosomes
of the exotic fish, Tilapia mossambica 121
Dirosophila ananassae
Temperature-related chromosome polymor-
phism in Drosophila ananassae 243
Drosophila koraputae
Description of three new species of Drosophila
(Scaptadrosophila) from Orissa, India 631
Drosophila neomedleri
Description of three new species of Drosophila
(Scaptadrosophila) from Orissa, India 631
Drosophila puriensis
Description of three new species of Drosophila
(Scaptodrosophila) from Orissa, India 631
Drosophilidae
Description of three new species of Drosophila
(Scaptodrosophila) from Orissa, In$ia 631
Ductule cell
Electron microscopic study of the spermatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Earias
Effect of some antibiotic compounds in cotton
on post-embryonic development of spotted
bollworm (Earias vittella F.) and the mecha-
nism of resistance in Gossypium arboreum 67
Ecology
Ecobiology of Corvospongilla lapidosa
(Annandale 1908) (Porifera ; Spongillidae)
in the Manjira Reservoir, Sangareddy,
Andhra Pradesh 555
Electrophoresis
A comparison of the electrophoretic fcaemo*
globin pattern of the commensal rodent
species 159
Endoplasmic reticula
Electron microscopic study of t&Q sperma*
theca of Gesonula punctifrans (Aprididae :
Orthoptera) 99
Endosulfan
Toxic and sublethal effects of endosulfan on
Barbus stigma (Pisces : Cyprinidae) 523
Epididymis
Histochemical studies on non-specific esterases
in epididymis of the bat, Cynopterus sphinx
sphinx 329
IV
Index
Esteiases
Histochemical studies on non-specific esterases
in epididymis of the bat, Cynopterus sphinx
sphinx 329
Estrous cycle
Durational effects of hemispaying on ovarian
hypertrophy and estrous cycle in albino rats
433
Euchromatin
Electron microscopic study of the spermatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Eupterote mollifera
Effect of temperature OB food intake, growth
and conversion efficiency of Eupterote mollifera
(Insecta : Lepidoptera) 417
Euthynnus affinis
A comparative study on certain biochemical
aspects of red and white myotomal muscles
of the black skipjack tuna, Euthynnus affinis
Cantor 349
Evolutionary approach
The form-function relationship of veitebrates :
A selected review 207
External coincidence model
Circadian basis for the photoperiodic response
in the male blackheaded bunting (Emberiza
melanocephala) 357
Familiarisation
Behavioural responses of the Indian gerbil,
Tatera indica to conspecific sebum odour of
the ventral scent marking gland 259
Fatty acids
Biochemical studies on the haomolymph and
heart muscle of normal and insecticide treated
cockroach Periplaneta americana L. 48 1
Fecundity
Fecundity of a hillstream minor carp Puntius
chilinoides (McClelland) from Garhwal
Himalaya 487
Fertility-fecundity
Effect of temperature and humidity on the
development and fertility-fecundity of Acrida
exaltata Walk. 267
Fish
' Effect of x-rays on the somatic chromosomes
"of the exotic fish," Tilapia mossambka 121
Foetuses
Interruption of pregnancy by barbiturates in
albino rats 533
Food intake
Effect of temperature on food intake, growth
and conversion efficiency of Eupterote molli-
fera (Insecta : Lepidoptera) 417
Form-function relationship
The form-function relationship of vertebrates i
A selected review 207
Fuel reserves
A comparative study on certain biochemical
aspects of red and white myotomal muscles
of the black skipjack tuna, Euthynnus affinis
Cantor 349
Fulgoroidea
New natural enemy complex of some fulgoroids
(Insecta : Homoptera) with biological studies
of three hymenopterous parasites (Insecta :
Hymenoptera) 177
Callus domesticus
Effects of aqueous and Hpoidal extracts of the
wall of preovulatory follicles on the ovary
of growing chicks 473
Gerbil
Behavioural responses of the Indian gerbil,
Tatera indica to conspecific sebum odoui of
the ventral scent marking gland 259
Giant neuron
Development of the incretory organs in the
eycstalk of freshwater prawn, Macrobrachium
kistnensis 599
Glutamine
Branchial protein metabolism of freshwater
fish Tilapia mossambica (Peters) during acute
exposure and acclimation to sublethal alkaline
water 235
Glycogen
Biochemical studies on the haemolymph and
heart muscle of normal and insecticide treated
cockroach Periplaneta americana L. 481
Gonadotrophins
Interruption of pregnancy by barbiturates in
dbinorats 533
Gonoduct
The annual reproductive cycle of Achaeto-bo
nellia maculata (Echiura : Bonellidae) 569
Gossypium t
Effect of some antibiotic compounds in cotton
on post-embryonic development of spotted
bollworm (Earias vittella F.) and the mecha-
nism of resistance in Gossypium arboreum 67
Gossypol
Effect of some antibiotic compounds in cotton
on post-embryonic development of spotted
bollworm (Earias vittella F.) and the mecha-
nism of resistance in Gossypium arboreum 67
Growth efficiency
Effect of salinity on the survival and growth
of Chanda (= Ambassis) gymnocephalus (Lac)
fry (Pisces ; Centrapomidae) 143
Index
Gut contents
Bionomics of hiDstr earn cyprinids. III. Food
parasites and length -weight relationship of
Garhwal mahaseer, Tot- tor (Ham.) 493
Haematozoans
Three new species of haematozoans from
freshwater teleosts (Pisces) 397
Haemocyte
Electron microscopic study of the spermatheca
of Gesonula punctifrons (Acrididae : Or mop -
tera) 99
Haemoglobin pattern
A comparison of the electroplioretic haemo-
globin pattern of the commensal rodent
species 1 59
Haemolymph
Biochemical studies on the haemolymph and
heart muscle of normal and insecticide treated
cockroach Penplaneta americana L. 481
Handling
Effects of handling on oxygen consumption
and random activity in the freshwater mullet
Rhinomugil corsula (Hamilton) 469
Heart muscle
Biochemical studies on the haemolymph and
heart muscle of normal and insecticide treated
cockroach Penplaneta americana L. 481
Hemispaying
Durational effects of hemispaying on ovarian
hypertrophy and estrous cycle in albino
rats 433
Hepatopancreatic sucrase
Hepatopancreatic sucrase of Macrobrachium
lamarrei (Crustacea, Caridea, Palaemonidae)
33
Herpestes edwardsii edwardsii
Histology and histochemistry of adrenal
glands of Indian mongoose Herpestes edwardsii
edwardsii (Geoffrey) 113
Heterochromatin
Electron microscopic study of the spermatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Heterodera oryzicola
Life history and behaviour of the cyst nema-
tode, Heterodera oryzicola Rao and Jaya-
prakash, 1978 in rice (Oryza sativa L.) 2S3
Heterometrus fulvipes
Microanatorny of the 7th abdominal ganglion
and its peripheral nerves in the scorpion
ffeterometrus fulvipes 22,5
Himalayan riverine ecosystem
Bionomics of hillstream cyprinids. III. Food,
parasites and length-weight relationship of
Garhwal mahaseer, Tor tor (Ham.) 493
Hi sto chemical observations
Histochemical changes in Sataria cervi caused
by certain anthelmintics 135
Histochemistry
Structure and chemical composition of the
cuticle of Cirolana fluviatilis, Sphaeroma
walker i and Sphaeroma terebrans 57
Histology and histochemistry of adrenal
glands of Indian mongoose Herpestes edwardsii
edwardsii (Geoffrey) 113
Steroid metabolism in target related to nuptial
plumage production in the Baya weaver
bird 361
Histological and histochemical studies on the
albumen gland and capsular gland of Thais
bufo (Lamarck) (Mollusca : Gastropoda) 4j7
Cellular sites of steroid synthesis in the ovi-
varous teleost fish (Cyprinus carpio L.) ;
A histochemical study 537
Histological techniques
Microanatomy of the 7th abdominal ganglion
and its peripheral nerves in the scorpion
Heterometrus fulvipus 225
Histology-
Histology and histochemistiy of adienal
glands of Indian mongoose Herpestes edwardsii
edwardsii (Geoffroy) ^3
Histological and histochemical studies on the
albumen gland and capsular gland of Thais
bufo (Lamarck) (Mollusca : Gastropoda) 407
Structure and seasonal changes in the testes
of a freshwater crab, Potamon koolooense
(Rathbun) 439
Holistic approach
The foim-function relationship of vertebrates :
A selected review 207
Holocrine cells
Histochemical studies on non-specific esterases
in epididymis of the bat, Cynopterus sphinx
sphinx
329
Home range
Differences in home ranges of rhesus monkey
(Macaca mulatto) groups living in three
ecological habitats J3
vi
Index
Homing
Behavioural responses of the Indian
Tatera indica to conspecific sebum odour of
the ventral scent marking gland 259
Host tissues
A comparative study on the mineral compo-
sition of the poultry cestode Raillietina tetragona
Molin, 185& and certain tissues of its host 153
Hymenoptera
Obseivations en the natural history and
population ecology of the social wasp
Ropalidia marginata (Lep.) fiom Peninsular
India (Hymenoptera : Vespidae) 539
Incretory organs
Develop uent o the incretory orgars in the
eyestalk of freshwater prawn, Macrobrachium
kistneitsis 599
Inhibition of sper-matogenesis
Synthesis of 4-methyl (6, T-6-tetrahydrobenzo-
furano)-couraarin and its contraception like
properties in male rabbits (pryctolagus
cunlcidus) 577
Insecticide
Biochemical studies on the haemolymph and
heart muscle of normal and insecticide treated
cockroach Periplaneta americana L. 481
Insect pests
Seasonal fluctuations in the diet composition
of Rhinopoma hardwickei in the Rajasthan
desert 563
Inversion
Temperature-related chromosome polymor-
phism in Drosophila ananassae 243
Inversions
repatterning in Drosophila :
nasuta nasuta and D. kohkoa 1
IsoosmotJc
EUacfe of sublethal levels of DDT, malathion
and mercury on tissue proteins of Saro-
therodon mossambicus (Peters) 501
Juvenoid effect
A study of pupal -adult intermediates produced
with Juvenoid treatment of Spodoptem litwra
Fabr. pupae 337
.Kachuga tectum tectum
Transabdominal migration of ova in some
freshwater turtles 1 89
Kachuga smithi
Transabdominal migration of ova in some
freshwater turtles 189
2LA
Temperature-related chromosome polymor-
phism in Drosophila ananassae 243
3LA
Temperature-related chromosome polymor-
phism in Drosophila ananassae 243
Lamina ganglionaris
Development of the incretory organs in the
eyestalk of freshwater prawn, Macrobra*
Mum kistnensis 599
Larval instar
Histological observations on tracheal growth
during wing development in Oncopeltus
fasdatus (Dallas) (He teroptera: Lygaeidae) 609
Larvivorous fish
Toxicity of certain pesticides found in the
habitat to the larvivorous fishes Aplocheilus
lineatus (Cuv. and Val.) and Macropodus
cupanus (Cuv. and Val.) 323
Length-weight relationship
Bionomics of hillstream cyprinids, III.
Food, parasites and length-weight relation-
ship of Garhwal mahaseer, Tor tor (Ham.)
493
Lethal concentration
Toxic and sublethal effects of endosulfan on
Barbus stigma (Pisces : Cyprinidae) 523
Life and fecundity tables
Life and fecundity tables for the longicorn
beetle borer, Olenecamptus bilobus (Fabri-
cius) (Coleoptera : Cerambycidae) 249
Life history
Life history and behaviour of the cyst nema-
tode, Hetorodera oryzicola Rao and Jaya-
Prakash, 1978 in rice (Oryza sativa L.) 283
Light
Circadian basis for the photoperiodic res-
ponse in the male blackheaded bunting
Emberiza melanocephala 357
Lipids
Cellular sites of steroid synthesis in the ovi-
parous teleost fish (Cyprinus carpio L.) :
A histochemical study 587
Lissemys punctata punctata
Transabdominal migration of ova in some
freshwater tuitles 139
Longevity
Effect of temperature and humidity on the
development and fertility-fecundity of
Acrida exaltata Walk. 267
Liver constituents
Effects of aldrin on serum and liver consti-
tuents of freshwater catfish Clarias batra-
chus L. < 27
Index
vu
Luteotrophins
Interruption of pregnancy by barbiturates
in albino rats 533
Macrobrachium
Development of the incretory organs in the
eyestalk of freshwater prawn, Macrobra-
Mum kistnensis 599
Macrobrachium lamarrei
Hepatopancreatic sucrase of Macrobrachium
lamarrei (Crustacea, Caridea, Palaemonidae)
33
Macropodus cupanus
Toxicity of certain pesticides found in the
habitat to. the larvivorous fishes Aplocheilus
lineatus (Cuv. and Val.) and Macropodus
cupanus (Cuv. and Val.) 323
Male gamete
Electron microscopic study of the sperrnatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Manjira reservoir
Ecobiology of Corvospongilla lapidosa
(Annandale 1908) (Porifera : Spongillidae)
in the Manjira Reservoir, Sangareddy, Andhra
Pradesh 553
Marginal water bodies
Sediment-ostracode relationship in the Bimili
backwater and the Balacheruvu tidal stream
297
Medulla externa ganglionic x-organ
Development of the incretory organs in the
eyestalk of freshwater prawn, Macro-
brachium kistnensis 599
Medulla interna ganglionic x-organ
Development of the incretory organs in the
eyestalk of freshwater prawn, Macrobrachium
kistnensis 599
Metacercariae
Studies on some Tetracotyle Fillipi, 1859
metacercariae from fishes of Lucknow 515
Metaphire peguana
The effect of cephalic transection on the micro-
morphological changes in the ventral nerve
cord neurosecretory system of earthworm,
Metaphire peguana (Rosa, 1890) during
anterior regeneration 381
4-methyl coumarin
Synthesis of 4-methyl (6,7-6-tetrahydro-
benzofurano)* coumarin and its contracep-
tion like properties in male rabbits (Orycto-
lagus cuniculus) 577
M. hurrianae
Evaluation of warfarin against Tatera indica
and Merfynes hurrianae 463
Microanatomy
Microanatomy of the 7th abdominal ganglion
and its peripheral nerves in the scorpion
Heterometrus fulvipes 225
Microvilli
Electron microscopic study of the sperrna-
theca of Gesonula punctifrons (Acrididae :
Orthoptera) 99
Milieu interior
Effects of sub-lethal levels of DDT, malathion
and mercury on tissue proteins of Sarothero-
don mossambicus (Peters) 501
Mineral composition
A comparative study on the mineral compo-
sition of the poultry cestode Raillietina
tetragona Molin, 1858 and certain tissues
of its host 153
Multivariate analysis
Shell selection in the estuarinc hermit crab
Clibanarius longitarsus (De Haan) 39
Muscle cell
Electron microscopic study of the sperma-
theCa of Gesonula punctifrons (Acrididae :
Orthoptera) 99
Myoglobin
A comparative study on certain biochemical
aspects of red" and white myotomal muscles
of the black skipjack tuna, Euthynnus affinis
Cantor 349
Nasuta subgroup
Chromosomal repatterning in Drosophila ;
Drosophila nasuta ttasuta and D. kohkoa 1
Natural enemies
New natural enemy complex of some fulgo-
roids (Insecta : Homoptera) with biological
studies of three hymenopterous parasites
(Insecta : Hymenoptera) . 177
Natural sex attractants
Sex pheromone in a stomatopod crustacean
Squilla holoschista 367
Neurosecretory cells
Effect of DDT on brain neurosecretory cells
of adult Poekilocerus pktus (Orthoptera :
Acrididae) 305
The effect of cephalic transection on the
micromorphological changes in the ventral
nerve cord-neurosecretory system of earth-
worm, Metaphire peguana (Rosa, 1890)
during anterior regeneration 381
Neurosecretory material ......
The effect of cephalic transection on the
micromorphological changes in the ventral
nerve aord-neurosecietory system of earth-
Vlll
Index
worm, Metaphire peguana (Rosa, 1890) during
anterior regeneration 381
Nucleus
Electron microscopic study of the spermatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Notopterus notopterus
Acid phosphatase activity in tissues of
Notopterus notopterus chronically exposed to
phenolic compounds 7
Olenecamptus bilobus (Fabricius)
Life and fecundity tables for the longicorn
beetle borer, Olenecamptus bilobus (Fabri-
cius) (Coleoptera : Cerambycidae) 249
Oncopeltus fasciatus
Histological observations on tracheal growth
during wing development in Oncopeltus
fasciatus (Dallas) (Haeroptera : Lygaeidae)
609
Ontogenetic approach
The form-function relationship of vertebrates :
A selected review 207
Oocyte
Tue annual reproduc tive cycle of Achae to bone Ilia
mzcuiata (Echiura: BonellidaeJ 569
Oral toxicity
Evaluation of warfarin against Tatera indica
and Merioms hurrianae 463
Organic matter
Sediment polychaete relationship in the
Vasishta Gcdvaari estuary 199
Organic matter in sediment
Sediment-ostrocode relationship in the Binuli
backwater and the Balacheruvu tidal stream 297
Organophosphorus insecticides
Evaluation cf some c-rganophosphorus insecti-
cides against Dacus cucurbitae Coquillett on
peaon 45
Oryza sativa
Life history and behaviour of the cyst nema-
tode, Heterodera oryzicola Rao and Jaya-
prakash, 1973 in rice (Oryza sativa L.) 283
Ostracode assemblages
Sedhnent-ostracode relationship in the Bimili
backwater and the Balacheruvu tidal stream 297
Oxygen consumption
Effects of handling on oxygen consumption
and random activity in the freshwater mullet
Rhinomugil corsula (Hamilton) 469
Ova
Transabdominal migration of ova in some
freshwater turtles 189
Ovarian compensatory hypertrophy
Durational effects of hemi spaying on ovarian
hypertrophy and estrous cycle in albino
rats 433
Ovarian cycle
Seasonal changes in the ovary of a freshwater
crab, Potamon koolooense (Rathbun) 451
Ovarian histology
Seasonal changes in the ovary of a freshwater
crab, Potamon koolooense (Rathbun) 451
Ovaries
Transabdominal migration of ova in some
freshwater turtles 189
Ovary
Effects of aqueous and lipoidal extracts of
the wall of preovulatory follicles on the ovary
of growing chicks 473
Cellular sites of steroid synthesis in the ovi-
varous teleost fish (Cyprinus carpio L.) :
A histochemtcal study 587
Oviduct
Tiansabdominal migration of ova in some
freshwater turtles 189
Ovulation
Transabdominal migration cf ova in some
freshwater turtles 139
Palaemonidae
Hepatopancreatic sucrase of Macro brachium
lamarrei (Crustacea, Caridea, Palaemonidae) 33
Palatability
Evaluation of warfarin against Tatera indica
and Meriones hurrianae 463
Parasite
New natural enemy complex of some fulgoroids
(Insecta : Homoptera) with biological studies
of three hymenopterous . parasites (Insecta :
Hymenoptera) 177
Bionomics of hillstream cyprinids. III.
Food, parasites and length-weight relation-
ship of Garhwal mahaseer, Tor tor (Ham.) 493
Parasitocoenosis
Bionomics of hillstream cypiinids. III.
Food, parasites and length-weight relationship
of Garhwal mahaseer, Tor tor (Ham.) 493
Parasitoids
Studies on egg and nymphal parasites of rice
planthoppers, Nilaparvata lugens (Stal) and
Sogatella furcifera (Horvath) 165
Peripheral roots
Microanatomy of the 7th abdominal ganglion
and its peripheral nerves in the scorpion
Heterometrus fulvipes 225
Index
IX
Persistence
Evaluation of some organophosphorus insecti-
cides against Dacus cucurbitae Coquillett on
peach 45
Pesticide
Toxic and sublethal effects of endosulfan on
Barbus stigma (Pisces : Cyprinidae) 523
Toxicity of certain pesticides found in the
habitat to the larvivorous fishes Aplocheilus
lineatus (Cuv. and Val.) and Macropodus
cupanus (Cuv. and Val.) 323
Phagostimulant
Behavioural responses of the Indian gerbil,
Tc^tera indica to conspecific sebum odour of
the ventral scent marking gland 259
pH acclimation
Branchial protein metabolism of freshwater
fish Tilapia mossambica (Peters) during acute
exposure and acclimation to sublethal alkaline
water 235
Phenobarbital
Durational effects of hemi spaying on ovarian
hypertrophy and estrous cycle in albino
rats 433
Interruption of pregnancy by barbiturates in
albino rats 533
Phenolic compounds
Acid phosphatase activity in tissues of Notop-
terus notoptems chronically exposed to phenolic
compounds 7
Pheromone
Sex pheromone in a stomatopod crustacean
Squilla holoschista 367
Phosphorus contents
Seasonal variations in the phosphorus contents
of the muscle of catfish Glorias batrachus L. 423
Photo period
Orcadian ba?is for the photoperiodic response
in the male blackheaded bunting (Ember iza
melanocephala) 357
Phylogenetic adaptation
Differences in home ranges of rhesus monkey
(Macaca mulatto) gioups living in three
ecological habitats 13
pila globosa
The tannery industrial effluent effect on succi-
nate dehydrogenase activity pattern in a fresh-
water snail, Pila globosa 427
Pituitary
Interruption of pregnancy by barbiturates in
albino rats 533
Plasma membrane
Electron microscopic study of the sparmatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Poekilocems pictus
Effect of DDT on brain neurosecretory cells
of adult Poekilocems pictus (Orthoptera :
Acrididae) 3Q5
Polymorphism
Three new species of haematozoans from
freshwater teleosts (Pisces) 397
Population density
The functional demography of adrenal glands
in Rattus meltada pdlidior in Indian desert 623
Population ecology
Observations on the natural history and
population ecology of the social wasp Ropa-
lidia marginata (Lep.) from Peninsular India
(Hymenoptera : Vespidae) 539
Porifera
Ecobiology of Corvospongilla lapidosa (Annan-
dale 1908) (Porifera; Spongillidae) in the
Manjira Reservoir, Sangareddy, Andhra
Pradesh 553
Post-embryonic
Effect of some antibiotic compounds in
cotton on post-embryonic development of
spotted bollworm (Earias vittella F.) and the
mechanism of resistance in Gossypium
arboreum 57
Potamon
Seasonal changes in the ovary of a freshwater
crab, Potamon koolooense (Rathbun) 451
Potamon koolooense
Structure and seasonal changes in the testes
of a freshwater crab, Potamon koolooense
(Rathbun) 439
Poultry cestode
A comparative study on the mineral compo-
sition of the poultry cestode Raillietina tetra-
gona Molin, 1858 and certain tissues of its
host 153
Predator
New natural enemy complex of some fulgo-
roids (Insecta : Homoptera) with biological
studies of three hymenopterous parasites
(Insecta : Hymenoptera) 177
Effect of teleostean prey size and salinity on
satiation amount, satiation time and daily
ration in the glassy perchlet Chanda
(— Ambassis) thomassi (Day) (Pisces : Centro-
pomidae) 507
Pregnancy
Interruption of pregnancy by barbiturates in
albino rats 533
Pregnant females
The functional demography of adrenal glands
in Rattus meltada pallidior in Indian desert 623
Premating gestures
Sex pheromone in a stomatopod crustacean
Squilla holoschista 367
Preovulatory follicle
Effects of aqueous and lipoidal extracts of
the wall of preovulatory follicles on the
ovary of growing chicks 473
Principal cells
Histochemical studies on non-specific esteraces
in epididymis of the bat, Cynopterus sphinx
sphinx 329
Protein
A comparative study on certain biochemical
aspects of red and white myotomal muscles
of the black skipjack tuna, Euthynnus affinis
Cantor 349
Biochemical studies on the haemolymph and
heart muscle of normal and insecticide treated
cockroach Periplaneta americana L. 481
Proteolysis
Effects of sublethal levels of DDT, malathion
and mercury on tissue proteins of Sarotherodon
mossambicus (Peters) 50 1
Puntius chilionoides
Fecundity of a hillstream minor carp Puntius
chilinoides (McClelland) from Garhwal
Himalaya 4 £7
Pupal-adult intermediates
A study of pupal-adult intermediates produced
with juvenoid treatment of Spodoptera litura
Fabr. pupae 337
3RA
Temperature-related chromosome polymor-
phism in Drosophila ananassae 243
Raillietina tetragona
A comparative study on the mineral compo-
sition of the poultry cestode Raillietina tetra-
gona Molin, 1S58 and certain tissues of its
host 153
Random activity
Metabolic rates and quotients in the cichlid
fish, Tilapia mossambica (Peters) in relation
to random activity 217
Effects of handling on oxygen consumption
and random activity in the freshwater mullet
Rhinomugil corsula 469
Rattus meltada pallidior
The functional demography of adrenal glands
in Rattus meltada pallidior in Indian desert 623
Red and white muscles
A comparative study on certain biochemical
aspects of red and white myotomal muscles
of the black skipjack tuna, Euthynnus affinis
349
Regression coefficient
Bionomics of hillstream cyprinids. Ill
Food, parasites and length-weight relationship
of Garhwal mahaseer, Tor tor (Ham.) 493
Regeneration
The effect of cephalic transection on the
micromorphological changes in the ventral
nerve cord-neurosecretory system of earth-
worm, Metaphire peguana (Rosa 1890) during
anterior regeneration 381
Relative humidity
Effect of temperature and humidity on the
development and fertility-fecundity of Acrida
exaltata 26 7
Reproductive cycle
The annual reproductive cycle of Achaetobo-
nellia maculata Fisher (Echiura : Bonellidae) 569
Reproduction activity
The functional demography of adxenal glands
in Rattus meltada pallidior in Indian desert 623
Resistance
Effect of some antibiotic compounds in cotton
on post-embryonic development of spotted
bollworm (Earias vittella F.) and the mecha-
nism of resistance in Gossypium arboreum 67
Respiratory quotient
Metabolic rates and quotients in the cichlid
fish, Tilapia mossambica (Peters) in relation to
random activity 217
Respirometer
Effects of handling on oxygen consumption
and random activity in the freshwater mullet
Rhinomugil corsula (Hamilton) 469
Rhesus monkey
Differences in home ranges of rhesus monkey
(Macaca mulatto) groups living in. three
ecological habitats 13
Rhinomugil corsula
Effects of handling on oxygen consumption
and random activity in the freshwater mullet
Rhinomugil corsula (Hamilton) 469
Rhinopoma
Seasonal fluctuations in the diet composition
of Rhinopoma hardwickei in the Rajasthan
desert 563
Rhythm
Orcadian basis for the photoperiodic response
in the male blackheaded bunting (Emberiza
melanocephald) 357
Rhythmic oscillations in non-aggressive social
behaviour in Bandicota bengalensis 317
Rice planthoppers
Studies on egg and nymphal parasites of rice
planthoppers, Nilaparvata lugens (Stal) and
Sogatella furcifera (Horvath) 65
Index
xi
Ropalidia marginata
Observations on the natural history and
population ecology of the social wasp
Ropalidia marginata (Lep,) from Peninsular
India (Hymenoptera : Vespidae) 539
Routine metabolic rate
Metabolic rates and quotients in the cichlid
fish, Tilapia mossambica (Peters) in relation
to. random activity 217
Salinity
Effect of salinity on the survival and growth
of Chanda (= Ambassis) gymnocephalus (Lac.)
fry (Pisces :. Centropomidae) 143
Satiation
Effect of salinity on the survival and growth
of Chanda (= Ambassis) gymnocephalus (Lac.)
fry (Pisces : Centropomidae) 143
Effect of teleosteart prey size and salinity on
satiation amount, satiation time and daily
ration in the- glassy perchlet Chanda
(= Ambassis) thomassi (Day) (Pisces : Centro-
pomidae) 507
Scent marking
Behavioural responses of the Indian gerbil,
Tatera indica to. conspecific sebum odour of
the ventral scent marking gland 259
Seasonal changes
Structure and seasonal changes in the testes
of a freshwater crab, Potamon koolooense
(Rathbun) 439
Seasonal variations
Seasonal variations in the phosphorus contents
of the muscle of catfish Clarias batrachus
L. 423
Secretory dynamics
The effect of cephalic transection on the
micromorphological changes in the ventral
nerve cord-neurosecretory system of earth-
worm, Metaphire peguana (Rosa, 1890) during
anterior regeneration 381
Secretory granule
Electron microscopic study of the spermatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Sediment composition
Sediment polychaete relationship in the Vasishta
Godavari estuary 199
Sedimentological characteristics
Sediment-ostracode relationship in the Bimili
backwater and the Balacheruvu tidal stream 297
Serum
Effects of aldrin on serum and liver consti-*
tuents of freshwater catfish Clarias batrachus
^ 27
Setaria cervi
Histochemical changes in Setaria cervi caused
by certain anthelmintics 135
Shell selection
Shell selection in the estuarine hermit crab
Clibanarius longitarsus (De Haan) 39
Sialic acid
Synthesis of 4-methyl (6,7-&-tetrahydrobenzo-
furano)-coumarin and its contraception like
properties in male rabbits (flryctolagus
cuniculus) 577
Silica
Ecobiology of Corvospongilla lapidosa
(Annandale 1908) (Porifera : Spongillidae) in
the Manjira Reservoir, Sangareddys Andhra
Pradesh 553
Skin
Steroid metabolism in target related to nuptial
plumage production in the Baya weaver bird
361
Skipjack tuna
A comparative study on certain biochemical
aspects of red and white myotomal muscles
of the black skipjack tuna, Euthynnus affinis
Cantor 349
Social behaviour
Rhythmic oscillations in non-aggressive social
behaviour in Bandicota bengalensis 317
Social wasp
Observations on the natural history and '
population ecology of the social wasp
Ropalidia marginata (Lep.) from Peninsular
India (Hymenoptera ; Vespidae) 539
Soluble proteins
Branchial protein metabolism of freshwater
fish Tilapia mossambica (Peters) during acute
exposure and acclimation to sublethal alka-
line water 235
Spawning
Annual reproductive cycle of Achaetobonettia
maculata (Echiura: Bonellidae) 569
Sphaeroma terebrans
Structure and chemical composition of the
cuticle of Cirolana fluviatilis, Sphaeroma
walkeri and Spheroma terebrans 57
Speimathecal gland cell
Electron microscopic study of the spermatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Spicules
Ecobiology of Corvospongilla lapidosa
(Annandale 1908) (Porifera ; Spongilidae) in
the Manjira Reservoir, Sangareddy, Andhia
Pradesh 5??
xn
Index
Spirorchiidae
On some blood flukes (Spirorchiidae : Coeuri-
trematinae) from freshwater chelonians in
India 275
Spodoptera litura
A study of pupal-adult intermediates produced
with juvenoid treatment of Spodoptera litura
Fabr. pupae 337
Spotted bollworm
Effect of some antibiotic compounds in cotton
on post-embryonic development of spotted
bollworm (Earias vittella F.) and the
mechanism of resistance in Gossypium
arboreum 67
Squilla
Sex pheromone in a stomatopod crustacean
Squilla holoschista 367
Stable age-distribution
Life and fecundity tables for the longicorn
beetle borer, Olenecamptus bilobus (Fabricius)
(Coleoptera : Cerambycidae) 249
Standard metabolic rate
Metabolic rates and quotients in the cichlid
fish, Tilapia mossambica (Peters) in relation
to random activity 217
Steroid dehydrogenases
Steroid metabolism in target related to nuptial
plumage production in the Baya weaver
bird 361
Steroid synthesis
Cellular sites of steroid synthesis in the
ovivarous teleost fish (Cyprinus carpio L.) :
A histochemical study 587
Structural proteins
Branchial protein metabolism of freshwater fish
Tilapia mossambica (Peters) during acute
exposure and acclimation to sublethal alkaline
water 235
Structure and chemical composition
Structure and chemical composition of the
cuticle of Cirolana fluviatilis, Sphaeroma
walkeri and Sphaeroma terebrans 57
Sublethal concentrations
Toxic and sublethal. effects of endosulfan on
Barbus stigma (Pisces : Cyprinidae) 523
Succinate dehydrogenase
The tannery industrial effluent effect on succi-
nate dehydrogenase activity pattern in a
freshwater snail, Pila globosa 427
Sucrase
Hepatopancreatic sucrase of Macrobrachium
lamarrei (Crustacea, Caridea, Palaemo-
nidae) 33
Tannery effluent
The tannery industrial effluent effect on Succi-
nate dehydrogenase activity pattern in a
freshwater snail, Pila globosa 427
Tannin
The tannery industrial effluent effect on succi-
nate dehydrogenase activity pattern in a
freshwater snail, Pila globosa 427
Effect of some antibiotic compounds in
cotton on post-embryonic development of
spotted bollworm (Earias viiella F.) and the
mechanism of resistance in Gossypium
arboreum 67
Tatera indica
Behavioural responses of the Indian gerbil,
Tatera indica to conspecific sebum odour of
the ventral scent marking gland 259
Teleost
Cellular sites of steroid synthesis in the ovi-
varous teleost fish (Cyprinus carpio L.) :
A histochemical study 587
Teleostean prey
Effect of teleostean prey size and salinity on
satiation amount, satiation time and daily
ration in the glassy perchlet Chanda
(== Ambassis) thomassi (Day) (Pisces :
Centropomidae) 507
Temperature
Effect of temperature and humidity on the
development and fertility-fecundity of Acrida
exaltata Walk 267
Testes
Structure and seasonal changes in the testes
of a freshwater crab, Potamon koolooense
(Rathbun) 439
Tetracotyle pandei n.sp.
Studies on some Tetracotyle Fillipi 1859
metacercariae from fishes of Lucknow 515
Tetracotyle ramalingi n.sp.
Studies on some Tetracotyle Fillipi, 1859
metacercariae from fishes of Lucknow 515
Tetracotyle srivastavai n.sp.
Studies on some Tetracotyle Fillipi, -1859
metacercariae from fishes of Lucknow 515
Thais bufo
Histological and histochemical studies on the
albumen gland and capsular gland of Thais
bufo (Lamarck) (Mollusca : Gastropoda) 407
Tilapia mossambica
Effect of x-rays on the somatic chromosomes
of the exotic fish, Tilapia mossambica • 121
Metabolic rates and quotients in the cichlid
fish, Tilapia mossambica (Peters) in relation
to random activity 2J7
Index
Xlll
f. indica
Evaluation of warfarin against Tatera indica
and Meriones humanae 4.63
Toxicity
Evaluation of some organophosphorus insecti-
cides against Dacus cucurbitae Coquillett on
peach 45
Toxicity of certain pesticides found in the
habitat to the larvivorous fishes Aplocheilus
lineatus (Cuv. and Val.) and Macropodus
cupanus (Cuv. and Val.) 323
Toxic stress
Effects of sublethal levels of DDT, malathion
and mercury on tissue proteins of Sarotherodon
mossambicus (Peters) 501
Tracheae
Htstological observations on tracheal growth
during wing development in Oncopeltus
fasdotus (Dallas) (Heteroptera : Lygaeidae) 609
Tracheole
Electron microscopic study of the spermatheca
of Gesonida punctifrons (Acrididae : Orthop-
tera) 99
Transabdominal
Transabdominal migration of ova in some
freshwater turtles 189
Transmission electron microscope
Electron microscopic study of the spermatheca
of Gesonula punctifrons (Acrididae : Orthop-
tera) 99
Transpositions
Chr-ornosomal repatterning in drosophila :
Drosophila nasuta nasuta and D. kohkoa 1
Trypanoplasma
Three new species of haematozoans from
freshwater teleosts (Pisces) 397
Trypanosoma
Three new species of haematozoans from
freshwater teleosts (Pisces) 397
Turbidity
Ecobiology of Corvospongilla lapidosa
(Annandale 1908) (Porifera : Spongillidae) in
the Manjira Reservoir, Saugareddy, Andhra
Pradesh 553
Unimodal
Rhythmic oscillations in non-aggressive social
behaviour in Bandicota bengalensis 317
Variance
Bionomics of hillstream cyprinids. III. Food,
parasites and length-weight relationship of
Garwhal mahaseer, Tor tor (Ham.) 493
Varietal preference
Studies on preference of Callosobruchus
maculatus Fabricius to some high yielding
varieties of arhar (Cajanus cajan L.) 391
Vertebrates
The form-function relationship of vertebrates :
A selected review 207
Vitellogenesis
Seasonal changes in the ovary of a freshwater
crab, Poiamon koolooeme (Rathbun) 451
Warfarin
Evaluation of warfarin against Tatera indica
and Meriones humanae 463
White rats
Histochemical changes in Setaria cervi
caused by certain anthelmintics 135
Wing development
Histological observations on tracheal growth
during wing development in Oncopeltus
fascfatus (Dallas) (Heteroptera : Lygaeidae)
609
X-irradiated chromosome aberrations
Effect of x-rays on the somatic chromosomes
of the exotic fish, Tilapia mossambica 121
AUTHOR INDEX (Animal Sciences)
Advani Ranjan
Seasonal fluctuations in the diet composition
of Rhinopoma hardwickei in the Rajasthan
desert 563
Agarwal R A
see Sharma H C 67
349
Alexander K M
see Pillai N Gopinathan
AH Shamshad
Effect of temperature and humidity on the
development and fertility- fecundity of Acrida
exaltata Walk 267
XIV
Index
Ananthakrishnan T N
see Swaminathan S
177
Annapurna C
Sediment-ostracode relationship in the Bimili
backwater and the Balacheruvu tidal stream 297
Babu K Sasira
see Yellamma K 225
Balasubramanian N K
see Nair J Rajasekharan 143
see Jacob Sheila Susan 323
Baqui Abdul
Histochemical changes in Setaria cervi caused
by certain anthelmintics 135
Bentur J S
Studies on egg and nymphal parasites of rice
planthoppers, Nilaparvata lugens (Stal) and
Sogatella furcifera (Horvath) 165
Bhaskar M
Branchial protein metabolism of freshwater
fish Tilapia mossambica (Peters) during acute
exposure and acclimation to sublethal alka-
line water 235
Chaudhuri P S
see Nanda D K
381
Dalela R C
Acid phosphatase activity in tissues of
Notopterus notoptems chronically exposed to
phenolic compounds 7
Das C C
see Dasmohapatra D P 243
Dasmohapatra D P
Temperature-related chromosome polymor-
phism in Drosophila ananassae 243
Deecaraman M
Sex pheromone in a stomatopod crustacean
Squilla holo schist a 367
Dhanze J R
Some biometric studies of certain closely
related species of the genus Anus (Pisces :
Siluriformes : Ariidae) 79
Dixit V P
see Sinha Rakesh 577
Dobriyal A K
see Singh H R 487
Duda P L
Transabdominal migration of ova in some
freshwater turtles 1&9
Dutta Hiran M
The form-function relationship of vertebrates :
A selected review 207
Gadagkar Raghavendra
Observations on the natural history and
population ecology of the social wasp
Ropalidia marginata (Lep.) from Peninsular
India (Hymenoptera : Vespidae) 539
Gadgil Madhav
see Gadagkar Raghavendra 539
Ghosh D
see Pal S G 99
Govindappa S
see Bhaskar M 235
Grewal G
see Parshad R K 473
Gupta J P
Description of thrde new species of droso-
phila (Scaptodrosophila) from Orissa, India
631
Gupta NK
see Tandon V 275
Gupta V K
see Duda P L 189
Guraya S S
see Parshad R K 473
Cellular sites of steroid synthesis in the
ovivarous teleost fish (Cyprinus carpio L.):
A Mstochemical study 587
Hameed S F
see Kashyap N P 45
Humaira Khatoon
see Baqui Abdul 135
Jayaprakash A
Life history and behaviour of the cyst
nematod*3, Heterodera oryzicola Rao and
Jayaprakash, 1978 in rice (pryza sativa L.) 283
Jayaram K C
see Dhanze J R 79
Jashi B D
Three new species of Haematozoans from
freshwater teleosts (Pisces) 397
Joshi N V
see Gadagkar Raghavendra 539
Joshi P C
Structure and seasonal changes in the testes
of a freshwater crab, Potamon koolooense
. (Rathbun) 439
Seasonal changes in the ovary of a freshwater
crab Potamon koolooense (Rathbun) 451
Kalode M B
see Bentur J S 165
Kashyap N P
Evaluation of some organophosphorus insecti-
cides against Dacus cucurbitae Coquillett on
peach 45
Indek
itaur Surinderpal
see Guraya Sardul S 587
Khan M A
see Rao I Seshagiri 553
Khan S Ajmal
Shell selection in the estuarine hermit crab
Clibanarius longitarsus (De Haan) 39
Khan T N
Life and fecundity tables for the longicorn
beetle borer, Olenecamptus bilobus (Fabricius)
(Cerambycidae) 249
Khanna S S
see Joshi P C 439
see Joshi P C 451
Kotak V C
Steroid metabolism in target related to
nuptial plumage production in the Baya weaver
bird 361
Krishnamoorthy R V
see Shakunthala Sridhara 317
Kumar N V Nanda
see Rao M Guruprasada 427
Kumar Vinod
Orcadian basis for the photopericdic response
in the male blackheaded bunting (Emberiza
melanocephala) 357
Leela Vallabhan D
Structure and chemical composition of the
cuticle of Cirolana fluviatilis, Sphaeroma
walkeri and Sphaeroma terebrans 57
539
249
Afahabal A S
see Gadagkar Raghavendra
Haiti P K
see Khan T N
Malhotra Sandeep K
Bionomics of hillstream cyprinids III Food,
parasites and length-weight relationship of
Garhwal mahaseer, Tor tor (Ham.) 493
Manna G K
Effect of x-rays on the somatic chromosomes
of the exotic fish, Tilapia mossambica 121
Manoharan T
Toxic and sublethal effects of endosulfan on
Barbus stigma (Pisces : Cyprinidae) 523
Mathur R P
Evaluation of warfarin against Tatera indica
and Meriones hurriame 463
Meera Agrawal
see Sinha Rakesh 5//
Menon G K
see Kotak V C 361
Mewa Singh
see Pirta Raghubir Singh
13
Mirajkar M S
Development of the incretory organs in the
eyestalk of freshwater prawn, Macrobrachium
kistnensis 599
Mohan P Murali
see Yellamma K 225
Mohd. Idris
Behavioural responses of the Indian gerbil,
Tatera indica to conspecific sebum odour of
the ventral scent marking gland 259
Mote L T
Histochemical studies on non-specific esterases
in epididymis of the bat, Cynopterus sphinx
sphinx 329
Murthy Ramesh Chandra
see Padma Saxena 33
Murthy V Krishna
see Bhaskar M 235
Nadakal A M
A comparative study on the mineral compo-
sition of the poultry cestode Raillietina tetra-
gona Molin, 1858 and certain tissues of its
host 153
Nagabhushanam R
see Mirajkar M S 599
Nair J Rajasekharan
Effect of salinity on the survival and growth
of Chanda (= Ambassis) gymnocephalus (Lac.)
fry (Pisces : Centropomidae) 143
Effect of teleostean prey size and salinity
on satiation amount, satiation time and
daily ration in the glassy perchlet Chanda
(= Ambassis) thomassi (Day) (Pisces : Centro-
pomidae) 507
Nair K Vijayakumaran
see Nadakal AM 153
Nair N Balakrishnan
see Nair J Rajasekharan 143
see Sheila Susan Jacob 323
see Nair J Rajasekharan 507
Nalavade M N
see Mote L T 329
Nanda D K
The effect of cephalic transection on the micro-
morphological changes in the ventral nerve
cord-neurosecretory system of earthworm,
Metaphire peguana (Rosa, 1890) during
anterior regeneration 381
Natarajan P
A new species of Argulus Muller (Crustacea :
Branchiura) with a note on the distribution
of different species of Argulus in India"
375
XVI
Index
Natarajan R
see Khan S Ajmal 39
Nauriyal B P
see Singh H R 487
Nirupama Agrawal
Studies on some Tetracotyle Fillipi, 1859
metacercariae from fishes of Lucknow 515
Nivedita Mallela
Histological observations on tracheal growth
during wing development in Oncopeltus
fasciatus (Dallas) (Heterptera : Lygaeidae) 609
Om Prasad
Effect of DDT on brain neurosecretory cells
of adult Poekilocerus pictus (Orthoptera :
Acrididae) 305
Padma Saxena
Hepatopancreatic sucrase of Macrobrachium
lamarrei (Crustacea, Caridea, Palaemonidae) 33
Pal S G
Electron microscopic study of the sperma-
theca of Gesonula punctifrons (Acrididae :
Orthoptera) 99
Palanichamy S
Effect of temperature on food intake, growth
and conversion efficiency of Eupterote mollifera
(Insecta ; Lepidoptera) 417
Panigrahy K K
see Gupta J P 631
Parshad R K
Effects of aqueous and lipoidal extracts of
the wall of preovulatory follicles on the
ovary of growing chicks 473
Peer Mohamed M
Metabolic rates and quotients in the cichlid
fish, Tilapia mossambica (Peters) in relation
to random activity 217
Effects cf handling on oxygen consumption
and random activity in the freshwater mullet
JRhinomugil corsula (Hamilton) 469
Pillai N Gopinathan
A comparative study on certain biochemical
aspects of red and white myotomal muscles
of the black skipjack tuna, Euthynnus affinis
Cantor 349
Pirta Raghubir Singh
Differences in home ranges of rhesus monkey
(Macaca mulatto) groups living in three
ecological habitats 13
Ponnuchamy R
see Palanichamy S 417
Pradhan M S
A comparison of the electrophoretic haemo-
globin pattern of the commensal rodent
species 159
Prakash Ishwar
see Mohd. Idris 259
see Mathur R P 463
Prasad S S
see Srivastava US 337
Rajalakshmi Bhanu R C
Histological and histochemical studies on the
albumen gland and capsular gland of Thais
bufo (Lamarck) (Mollusca : Gastropoda) 407
Rajasekarasetty M R
see Ramesh S R l
Raju P Varada
Histology and histochemistry of adrenal
glands of Indian mongoose Herpestes edwardsti
edwardsii (GeofTroy) 113
Ramalingam K
Effects of sub-lethal levels of DDT, malathion
and mercury on tissue proteins of Sarotherodon
mossambicus (Peters) 501
Ramalingam K
see Ramalingam K 501
Rama Sarma D V
see Annapurna C 297
Ramesh S R
Chromosomal repatterning in drosophila :
Drosophila nasuta nasuta and D. kohkoa 1
Rana B D
The functional demography of adrenal glands
in Rattus meltada pallidior in Indian desert 623
Rao A Purushotham
see Reddy G Surender 481
Rao D Srinivasa
Sediment polychaete relationship in the
Vasishta Godavari estuary 199
Rao I Seshagiri
Ecobiology of Corvospongilla lapidosa
(Annandale 1908) (Porifera : Spongillidae) in
the Manjira reservoir, Sangareddy, Andhra
Pradesh 553
Rao K Hanumantha
see Rajalakshmi Bhanu R C 407
see Raju P Varada 113
Rao M Appaswamy
see Saras wati B Patil 433
see Saraswati B Patil 533
Rao M Guruprasada
The tannery industrial effluent effect on succi-
nate dehydrogenase activity pattern in a
freshwater snail, Ptla globosa 427
Rao YS
see Jayaprakash A 283
Index
xvn
235
235
sddanna I?
?ee Bhaskar M
ddy G Surender
Biochemical studies on the haerdolymph and
aeart muscle of normal and insecticide treated
cockroach Periplaneta americana L. 481
ddy G Vemananda
see Bhaskar M
in Mangal
we Bentur J S 165
raswati B Patil
Durational effects of hemispaying on ovarian
rypertrophy and estrous cycle in albino,
•ats 433
"nterruption of pregnancy by barbiturates in
ilbino rats 533
:ma D V Rama
we Rao D Srinivasa 199
:oj Rani
tee Dalela R C 7
rojini R
we Mirajkar M S 599
ikila Khan
'ee Nirupania Agrawal 515
ikunthala Sridhara
Rhythmic oscillations in non-aggressive sodal
jehaviour in Bandicota bengalensis 317
axma H C
Effect of some antibiotic compounds in cotton
>n post-embryonic development of spotted
>ollworm (Earias vittella F.) and the mecha-
iism of resistance in Gossypium arbor eum 67
sila Susan Jacob
Coxtcity of certain pesticides found in the
labitat to the larvivorous fishes Aplocheilus
ineatus (Cuv. and Val.) and Macropodus
wpanus (Cuv. and Val.) 323
yamasundari K
we Rajalakshmi Bhanu R C 407
tgh H R
Fecundity of a hillstream minor carp Puntius
Minoides (McClelland) from Garhwal,
Himalaya ; 487
tgh Munshi
?ee Sharma H C 67
ighal R N
rhe annual reproductive cycle of Achaeto-
jonellia maculate* Fisher (Echiura : Bonelb'dae)
569
iha Rakesh
synthesis of 4-methyl (6,7-Metrahydrobenzo-
'urano)-couraarin and its contraception like
properties in inale rabbits (Oryctolagus
Som R C
see Manna G K j2l
Srivastava U S
A study of pupal-adult intermediates produced
with juvenoid treatment of Spodoptera litura
Fabr. pupae 337
Srivastava V K
see Om Prasad 305
Subbiah G N
see Manoharan T 523
Subhashini K
see Yellarnma K 225
Subramoniam T
see Deecaraman M 357
Swaminathan S
New natural enemy complex of some fulgoroids
(Insecta : Homoptera) with biological studies
of three hymenopterous parasites (Insecta :
H>-menoptera) 177
Tandon V
On some blood flukes (Spirorchiidae :
Coeuritrematinae) from freshwater chelooians
in India 275
Tewary P D
see Kumar Vinod 357
Thangaraj T
see Palanichamy S 417
Tripathy N K
see Dasmohapatra D P 243
Verma S R
see Dalela R C 7
Vir Satya
Studies on preference of Callosobruchus
maculatus Fabricius to some high yielding
varieties of arhar (Cajanus cajan L.) 391
Yagana Bano
Effects of aldrin serum and liver constituents
of freshwater catfish Clarias batrachus L. 27
Seasonal variations in the phosphorus contents
of the muscle of catfish Clarias batrachus
L. 423
Yellamma K
Microanatomy of the 7th abdominal ganglion
and its jieripheral nerves in the scorpion
Heterometrus fulvipes 225
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