(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

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

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-S 3 (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, ) 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 (C 6 H 5 OH), 2,4-diratrophenol ((NO 2 ) 2 C 6 H a OH) 
and pentachlorophenol (sodium salt; C 6 Cl 5 ONa) 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 LC 50 ) of these chemicals and combinations kept in triplicate for 30 days 
(96 hr LC 50 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 -25 M 
sucrose solution, with a Potter Elvehjem homogenizer. Homogenates were 
centrifuged at 900g under cold conditions (5-0 1-0C) 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 







I 
I 






q 

*C5 



03 

I 

o 





g 

I 



W ^ 



T) 



TTTT 






g ? 



11 



H 



ft c 
1 O S ^ CA 



1 + 



+] 



+ 



O O CO O M3 O 



O O 

I-H rf 

M 



H-H-fl-fm-H-W-H 
0000*0000000*000 



o\ oo *sO 'i ci r~* i/^ ci 

T 7 7 7 7 T 7 " 



I I I I i 



7 + 7 T T i T 



tc^T- 

T77T77T 



<N 

-H -H -H 41 -H 



Ills -O l 
fi a M 6 fi a a e 



o 
V 



p . 



I? 



S. 
<5" 
fi 

e 

8 43 *-* 

J .5 V 

a IS * 



a 



2 CM '^ 
O c 



OH "71 *- 

.2 S 2 "- 

.|? o o ^ 
.^j c3 ^S 



o 



c 



!" 



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 km 2 , 1-52 km 2 and 0-01 7 km 2 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 km 2 and 0-009 km 2 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 O f monkeys were also taken. 

3. Study areas 

3.1. Asarori forest 

The study site (32 km 2 ) 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 


32km 3 


Jan. 1974 to Dec. 75 


400 


13 


Chakia Forest 


24km 3 


Aug. 1977 to July 78 


70 


3 


Urban area 


40km 3 


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 (km 2 ) over which the group was seen moving and 

foraging during one year period. 

Area (km 2 ) 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 km 3 ) 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 km a , 
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 km 2 . 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 



7C-41C 

1088mm 

A few trees in Sankat 

Mochan temple 

High 



Human influence 
Other wild mammals Jackal (in Sanfcat 
Mochan temple) 



7C-41C 
1088mm 
Scrub forest 

Medium 

Jackal, pig, leopard ( ?) 



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) 


(km 2 ) (km 2 ) 


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 km 2 . 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 km 2 
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 














km 2 


(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 








km 2 


km 2 


(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 








* 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 km 2 . 
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 km 2 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 km 2 . 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 km 2 to 1-81 km 2 with 
a mean of 0-48 km 2 . 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 km 2 . 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 km 2 ) than those 
of temple groups msan 0-0 17 km 2 ). 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, th e 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 km 2 . 

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. v al 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 (LC 100 ) 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 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 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.3 Ppm 

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>js0ssion 

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 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 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 K m value is 4-5 x 10~ 2 M. 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 4C 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 3 ml, 3 M sucrose 2 ml and enzyme extract 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 s to ; 



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 10-80C 
(figure 2). Sucrase showed optimum activity at 50 C 9 although remaining quite 
active from 20-6C. The activity increased slowly from 1Q-40C but steeply 
ftom 40-50C; 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 D 9 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 (K m 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 (K m value) of hepatopancreatic sucrase as calculated from the collected 
data is 4-5 x 10~ 2 M (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 1963 5 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 (30C 
for gut, 35C for salivary glands; Agarwal 1976), Acy rtho siphon piston (35C; 
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 invrtasc 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. (A n im. 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 fc 3 ) 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 
x 3 =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 ; x 2 stall width and ^ 3 ~Slill weight. 

Percentage contributions 
to total variability of 
Piincipal components shell peramttcrs 

I Component 1*000000 *i 95-800 

+0-999043 * 3 
+0-992294 x 9 

II Component 1-000000 x 1 0'013 

+ -0-50000C * 2 
+0-500000 x 3 

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 ; XiI 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 -jc x ) x s = 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 -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 wa s 
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 CC1 4 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 0035 


0-0030 




0-0041 


Feiithiou y = 1 7825* + 1-6888 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* + 9507 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 10 4 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 LD 90 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/cm 2 x 10~ 3 compared with 416//g/ 
cm 2 x 10~ 3 by 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/cm 3 


X 10- Ji 


Chemical assay 


fig/cm 2 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 


LD 50 

(jag/cm 8 ) 


Fiducial 
limits 
( /ig/cm 2 ) 


LD 90 
(jig/cm 2 ) 


Fiducial 
limits 
(^g/cm s ) 


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) 


: 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/cm 2 , 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 LD 50 values. The deposits of fenitrothion were far in excess 
(92 times) of their intrinsic toxicity (LD 50 ) 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/cm 2 ) 
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 (LD 50 ) 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 LD 90 of LD 50 . 

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/ 
cm 2 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/cm 2 ) 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 





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 












AT^,.~ ... -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/cm 2 ) 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 





83 


0*30 




Chem. assay 


I 


7-39 


2' 


97 


1-83 


1 


04 





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 





85 


0-28 




Chem. assay 


II 


7-83 


3- 


02 


1-91 


1 


05 





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 





85 


o-io 




Chem. assay 


I 


7-24 


2 


79 


2-15 


1 


26 





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 





70 


0-10 




Chem. assay 


II 


7-44 


2' 


79 


1-39 


1 


04 





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 





42 





15 


BDL 




Chem. assay 


I 


7-10 


1- 


56 


0-85 





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 





19 


BDL 




Chem. assay 


II 


7-1.3 


1- 


44 


0-85 





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 





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 





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) 





12) 


(0 


67) 


(0-.12) 


Trichlorphon 


Bioassay 




7*22 


2' 


81 


1-97 


1 


24 





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 





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 





fl 






3? 




"o 


^ 




& 






1? 


^3?^^ P: S s JQ g ??SSS;^ ^ e ^SvS 









^ 




d 




5 




<-H 


o 




1 


1 




1 




1 


0? 

la 


*o o o o s ^*ooS ooo^^ ooooo 
^.. .-... /inoo s ^ *OOOON 


d 
o 




S 


r 


o OO^ Y - (O ,66vb^6 66w?T-40 


t< 










d 




O 







o 




1 > 


1 




<J 




i : 


3 
Er 

a 


r9g!S2 ' : : : : : jq g p 3 <8 

w 6 6 ffl 6 6 




i 









>. oo oo 

















i : 




^"" fOOs^tfi^ ^ OOO*/)/*iv*> ^ OO f^ f3 ^ 


d 




m d 


o 




^ 










>r- 








S S 1 & & 


ON 




^ 


fc, 


^ 1 CN 


Q 


1 


tt' 



>> 


Ci- 7^^<^^i--< "^ ro.^ri-'t^ c* <** V> ^. <* *sj ^^ ^ 






<*-< 








& 


3 s 


^ 


g r^cs ^ coc^^r^^ | ror.^^e, g rV.^^^^ 


It 




it 




ctf O w?- S 


"3 


c 






o w (^4 


S 


o 






*-> ^o 










4Ji ^ Ci *^ 




to 








o 


8 






E o 8 





"o 

-C 

i 




1 


IiS^^ gg^^^j ^ prtjvo^OO OooVD^^t' 


1 

S 


<* 


'^ 




^ ^ V-. ^ ^ ^ ^^^^^ 


*X3 




O 


' 






e 


i 


* 






I 


;_< 


5" 









.3 


*e 


ST* 




pjj 


1 




4 


sl^ iilBl 1<32;> ^i^sag 


v? 


tM 




. :j. 


^ir^vbi>vo vo r> i^* " " T 1 . y 7 H !> ro * i o\ T < 


cj 


8 






^*"^*C*-ONC> t^t^c^oot^- 


1? 


a 








eC 











*cS -"ti 


.2 








^o g 











^ 











l| 


4 








* "o 





*+ 


N 




<* sSrf 





Insecticides 
(at 0-05% cone. 




- i j .1 .8 

1 B il j B |i f f g 

t|| : |t f gjff la-.l&f | e lM| 

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/cm 2 ) for the 1st spray and 6-09/jg/cm 2 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/cm 2 (1st spray) 
and 8-94/^g/cm 2 , (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 t o 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 


Peare 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 1C. 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) = , Perc f * 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; 19S 1 *); 
spp. (Oliver et al 1970, 1971); H. vlrcswtis (Lukcfahr a nl 1966; Mctswr ri / 
1977) and Anthonimits grandis (Bailey ct al 1967; l>ougas I l >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 H t and H 2 (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 P t populations of interVArietai 
crosses have been presented in table 8. It was observed that Dttutat CJf-71 and 
non-pigmented F 2 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 mn l 

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 P1 gmented 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 


Y 2 Pupal period 








Xi Gossypol 


0-4473 


0-7992 




X% Tannins 


-0-2760 


0-4793 


0-0434 


y 3 Total developmental 








period 








Xi Gossypol 


1-9673 


2-1937* 




X 2 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 
T a 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 



b i t value 



YI Emergence 








Xi Go&ypol 


0-3281 


0-0446 




X Tannins 


- 17-1281 


3-1635* 


0-6104 


7 a Growth index 








X l Gossypol 


0-0027 


0-0052 




X 4 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 F 8 


80-00 




(b) Pigmorttcd F a 


46-67 


7. 


IXiulat x Lohit 






Non-pigmented F% 


80-00 




Pigmor^tod F a 


45-45 


8. 


Cernuum x G-27 






Non-pigmented F z 


62-50 




Pigmertted F 2 


32*43 




Average for F 2 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 





74 


d 


2. 


Lohit 


1- 


40 


c 





84 


d 


3. 


* Pigmented F 3 


1- 


20 


b 





47 


c 


4. 


Cernuum 


1- 


24 


b 





41 


be 


5. 


CJ 73 


1- 


02 


a 





31 


b 


6. 


* Nonpigmeitted F 3 


0- 


99 


a 





17 


a 




CD at 5% (0 


o- 


10 







12 





* Samples from the F a 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-H a . 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 

' O or> " q *o -. 




% / 

'%8SS^ 



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 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 





tf 

e 
cs 



/ R Dhanze and K C Jayaram 



^ 



0^^^^^vo^^^^^ 

^^oSHot/S-ioT l 7*'T'7 l S-^-'7 l 7*'f7'7 
T-I \ / O ^ "^ ^ \/ \/ \/ \/ \; \/ \; \/ \/ \/ \/ \/ 



<-? " O -< O w *-* 

v 8 v v v 

V 



vvvvvvvvvvv 



> V 8 



g- 



leg 

p*^ 




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 



Dhanze and K C Jayaram 



cS 

5 

<3 

! 

^ 

's 



8 

H 






late (Thunbe 



of A 



37 spec 



-H 
X 










? 




& 
8 



| 

'"eS 



1 

4 



Biometric studies of the genus Anns 



85 



I 



fc 




o 
S 



g 

41 





i 







8 

4) 



?o 

I 




CO OO 

co co 
<3- vo 



N rt VO 00 '- ' f- 

> . . i" O co *n ON f-~ 

VO ON "^ i- ONOOCO tf) 

^ONOOO in co oo ^ 



o o o 04 o 



*^ vo i-* co 



O 
co 



vo 

*-( 
jo 



;_J 04 



i- n 
o^ b 



^ 55 

i-r <n O 



-H -H -H -H 



1-1 ON 

4i 41 : 



41 

00 



oo 

4^-H 






S'-H 



3 



CO 

oq <x> 



^ 2 ^ 



<NI 
<N[ 
oo 



i - 



41 



41 



vo n 

41 4^' 

oo oo 

fc 2 

oo -^ 



14] 

00 



r- vo 



SON 


oo -t^ 1 

vi \0 



r? c^ 

J, 

o t^ 




. 

33 



41 -H 



00 -^ 



ON ^* 

t> 2: 

t-f ^t 



'IS 
II 



O JU 
V^ vp 



iS 2 



r^ 0\ 

g; oo o 



r-< OO 



l> 

b 



22 S 



gsa^g g 

<N"8 ? <^ ^ 3 



oq 

41 



86 



/ R Dhanze and K C Jayaram 



c 
% 



F 
"5 

I 



s 



| 

i 



&s 



S 2* 

Is 



A 

Q 



& 



I 



1 



5 

1 



i 




SSsSjSsISSgg 

<>r18 | o-$? N( p t r < ? )< ^ ( ? 5 vo 



11 






vo QO *-< co O 

sgsss 

r- * r- " -* 



-H -H -H -H -H -H -H -H 41 5 -H 



-H 



S " JN w$ 



'S?^? 

^S^^'J j!3SeB"V^? ^ 

III IP 



?M 




3 



.s 

1 

* 



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, 



/ R Dhanze and K C Jayaram 



pecimen 






8 



I 




o ^ P; ? P S T 1 

io-7- rt 



r- 

00 

-H 



co ^ 



8' 

O ' 

O ' 





7 



41 



OO 
00 



' 1 t 



OO , 
O o 
co co 



oo 
co co 



i> vo r- 

1-4 r> c>l 

oo o rf 




o 



CNJ V> 

o r> 



r-. vo O O 
00 CO 00 J CS 

pp ^ 



co 



r-i ^ 00 



oo co C; 

r .^ 9 



00 ^ 






^~H-H -H4i 



* ^ ^, S ?! 



N 



co 
en - 



P c<i r-- 

O\ c^ oo 



VO 



1 



Isi 



II 
II 






.2 

"cS 





e 



Biometric studies of the genus Arius 




90 



J & Dhanze and K C Jayafam 



i 



o 

s 



s 

"53 

** 

O 

g 



o 
tf 



* 

1! 



J -a 



1 

5 



8 







vvvv 



v v 



Sv^ 



vv 



00600000006.6 

vvvvvvvvvv vv 






s* 

,fc 






Siometric studies of the genus Anus 



I I ll 



l 17 fg 19 20 21 22 23 24 25 26 



-i 1 r- 



8 I C 



23 26 7,7 20 29 30 3( 



III 



IS ' ' '7 18 19 20 . 21 22 23 24 25 26 27 



A L 



IV 



W " 

Graphs I-IV 



IS 17 It 19 tO 



92 



Dhanze and K C Jayararri 



A I C 



V SL / 



'8 39 40 41 42 43 



VI 



6364 63 66 67 / " 69 M 



70 71 72 73 74 75 



VII 



20 21 22 23 24 25 / 26 27 28 29 30 31 32 33 



A L 



VII) 



-* 76- 



32 34 56 38 60 



Graphs V-VU1 



Mometric studies of the genus Arius 



93 




IX 



62 4 



68 TO 71 74 76 70 90 62 84 86 90 




J c 



JH 36 3f CO 62 , 64 



' I mi 

70 



I" " ' ' \ 
A L- 


_j_ , 




i i _. I 


ft i 1 ffiffi It 




^ U^-^uJr, 




XI 

J" "L '"i _ i 'i ... .1 i __. i _,. , r ,.. i._..i i j 



27 28 29 30 31 M 



34 35 36 37 39 39 40 



XII 



1 - -L-. - -L 



14 15 16 17 16 / 19 20 21 22 23 



Graphs IX-XII 



)4 J R Dhanze and K C Jayaram 



t> il mm il 



XII) 



40 44 48 52 56 60 64 66 72 76 80 



l.i mkm I 



XIV 



20 24 2ft 32 36 40 44 48 52 56 60 



li da il 



fe rtr 



48 53 56 60 64 



I I I I 



XVI 



^ 



ir ao ai "ti tV 







Graphs XIH-XVI 



Biometric studies of the genus Arms 



95 




xvn 



40 42 44 46 46 / 50 52 54 56 39 40 62 64 



XVil! 



M 16 l 20 22 



26 2 30 32 34 36 



XIX 



36 40 42 44 46 /4 / SO 52 54 56 5 00 62 

HCW>/LCPD> 



XX 



50/32, 54 56 58 6O 62 64 66 6g 



Graphs XVII-XX 
Graphs I- XX (See for Captions in p. 98) 



96 



/ R Dhanze and K C Jayaram 





| 




ON 


3 


I 


1 

"o3 

3 


I 


a 
-3 





5 1 


CO 


I 


? 


3 


CO 


c3 

O 





O 


? 


I 


04 




ON 




oi 




ON 




O 




O 




to 

ON 




ON 




0\ 


.1 



2 






8 



ON 
cm 



O 



ts 

zfi "^ 



"S 
WJ 






. o 







ON 

i 

CO 

ON 



I 
1 

cS 



Qj 



i 



g s 

8 2 



l11 



g 



i 10 

O ON 



8 



fjf? 



ot- 






O ON 

oo oo 
vo vo 



1 ^ 

i VO 



58 



00% 



88 

O O 



S 88 



5 

St.5 



It 



' 



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 t of 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 u k 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 4C. 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 O vojd 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 ^* 



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 KMnO 4 /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 B r swal 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, 
r ilapia 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 



n 
















+2 




4_> O 












S 




VT- c3 


co VO O 


CO 


CO 


CONOCO Of^f^^O 


00 


| 




2 


T) in 


oi 


rt 


ojooo'Ac* 1 * OC^CN 

Ol 01 T-( T-H 


CO 


s 


1 


*o 












o 


.S 


"S 


o r- eo 





o 


leoOO O O t** t- 


o 


*0 


s 


O 


r> 00 rf 


vo 


00 


oo co t^ w^ vo *O) t*~^ co 


vb 




8 


1 












f? 


o 5 


tJ 












B 


Z 


53 


CO CO CO 


CO 


CO 


Ot> OeOOC^Oco 


00 


S 


M 





,,-j ^.j. ^ 


O 


ON 


rHCOV - iOCO'O'nvO 


ON 


1 


^ 





co (N ^ 


CO 


r-( 


CO CM Ol 01 t-H T-, 




W 

CS 




13 1 
















ijl 


l>- O OO 
* CO -H 


^ 


ON 


CO CO CM CM ^ 01 ' 


*5 


1 


1 


cS O 


"^f" CO Vl 
"CJ* ^f- fSj 


5 


CO 


^-cooocMo^oono 

Tt CO CO CO t O -< 


00 


13 
















55 




s 












i 




ell 


ON oo eo, 


C^ 





t*- NO V) r^ O " i ^" ON 
OlOlOlc^T-i'^-^T-,^ ' 


01 


* 


t- 


60 ^ 

r 


OO CM 00 

*-< 01 


s 


CM 


lOOQ^coVOr-^OlO 

<M ^-i 01 <^ Ol -< 


OJ 






-^ 










CO 
















01 


1 




1^ 










_ 







S > 


oo cT <n 


s 


^ 


^SS^ >2'>L- 


5? 


xTJ, 




1.1 


vo ^T r- 


oT 


01 


Z2' s^ O sZl 1 
01 V) CO Os CO t> CO I 


;r 


a 

cS 














'"^ 


| 




3 




ri 





O. t*~ t* 1 " CO C3 OO to I 


01 


'~ 


W 

| 






vo 

01 


ON 


oo ON 01 en vo oi in 1 


CM 


CO 


t 












Ol 


IT 


1 

c3 


i 












j! 




1 


I 1 


01 ^ O 

^ ^, r-< 


vo 


- 


O\ t> 00 VO | <Ti CO 1 


C? 


eS 

JS 


1 


w 










<-^ 


II 


l-< 
t 1 


g 




01 


CO 


*< 01 T-M I en i | | 


5 


Is 
















O -M 

^ S? 




1 




G 


C 


SSS^sr^oTT 


"^ 


|2 




& 


2 Os <* 


t^ 


-* 


oc -^ t- M ^ ^ w , 


^T 

















vo 


<o i 




^ 












* "| 




eS 


^ C^ ^ 

1-4 W <> 


0* 


S 


oj ^c^v^^oT'T'T*' 


^ 


a i 




2 

PQ 


eo co vo 


- 


^ 


VO SO ^ ON \0 | | 


OO 

I 


il 


















1 

*J 

g 






fi fc 

*5 *c? jjj 


01 


^ 

OI 


ilj.lll 

^frSoN^^OiO 


S 



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 Jif 2 =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 X 2 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 X 2 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 3T 2 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 JP 2 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 som p > 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 worm 8 
(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 



<g 






s 



sl 



ll 



S 

13 fa bb 

fS-tSg 

S?*!* 

"i*i i 

5 s * o'S 

i|I;s 

r^i 

Hill 

2 "g ^ ^ 

in 11 



J5 

O 










^* f? 

s| S 

o " 2 -S 
8 | g | 



S^~ 

O V5 

2 'If 
S'-S e 



s, 

"35 







^ 
O 



to 

eS 

O 
P 



i 



o 
T 



I 

OJ 



II 

3 



e 

"i 



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. (Aim. 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% ) 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% ), even though 
during the first month, growth and health was apparently better in the lower 
salinity grades (4-11, 10-21 and 16-31% ). 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% ) 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% at 26 2C 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% ). At 31-56% (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% ) 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 A f6 A fm . 

Growth efficiency 10 d y ,= /d ? 10 



Where -I/, = amount of food eaten, A fm = 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% . 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% ), even though during the first month growth and health 
were apparently better in lower salinities (4-11, 10-21 and 16-31% ). 

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 % 




Y=0-1128X + 9.3739 



10-21% 




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 (% ) 














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% and 
33*60% (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% ) and the gradual decline in the rate of growth for 
fish maintained at the two lower salinity grades (4-17% ). 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 Xt-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% ) and 6l-3%(28-51% ) whereas it was as high as 
97-61 %(4-ll% ); 94-66% (10-21% ) and 87-99% (16-3 1% ) 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% 10- 


21% 1<5-31% 


22-41% 


28-51% 


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% 10-21% 16-31% 


22-41% 


28-51% 


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% 


10-21% 


*x 


D 22-41% 


28-51% 


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 hierarchy 9 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 2C. 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% ) 
strengthen the above conclusion. 

Using the regression formula for growth in length in the 28- 51% (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 


meanSE 


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 

R t 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 










TRICHOCRAMMA 


1 8 




<2Ha2 


. sp.) 


1 








^o 








a 








20 











_ , 






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 38-0C). 



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 4393i50-3 a 

1 pair of parasites 
released from day 5 
to 15 199-3 25-7 b 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 








2nd instar nymph 


80 


8 








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 



9 




* 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 31-8C) 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 
r ely 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 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 


3-40-49 


9 1-55 


23-2iO-9* 


35-6l- 


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 


20 


3-8 : 0-4 


6-42-58 


12-22 


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 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 89-922-4 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 ^beyond 1 the resorption of corpus 
luteum or clutching is multiple. Yet, it has often been observed that the counts 
of corpora lutea in the k 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), an d 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 and 1 
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 . 1966 V 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, 1948 5 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 'RuffGymnocephalus 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 NH 3 -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 CO a produced/volume of O 2 consumed; Ammonia 
tient, AQ = the volume or mole : mole relation of NH 3 -^N excreted to O a 
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 O a con- 
ption, CO 2 production and NH 3 -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 0-5C 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,, KH 3 ^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 

0.07 
I. 2 
KO 



ON 



:R 



x 

UJ 



2 

2 
I- 
O 
ID 
O 
O 

<r 
a. 



T 



a: 

x 








0.8 

0.6 
I 

5 
I 00 



50 
40 

30 
100 



50 
40 





1. 




J_ 



10 20 30 40 

RANDOM ACTIVITY (COUNTS/HOUR) 

Figure 1. Oxygen consumption;, carbon dioxide production, NH a -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 (O 2 consimption, CO a production and NH 3 -*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 




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) o f o a consumption, CO 2 produc- 
tion, NH 8 -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 



O 3 log Y = 1, 69888 -4- 00677 X 

CO a log F== 1/60628 -f 0/00852 X 

NH 3 -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 + * 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 O 2 consumption, CO 2 production, NH 3 -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 


O a consumption 


64-4 2-1 


50-0 


81-8 3-7 


62-6 


CO a production 


55-6 2-4 


40-4 


70-6 4-3 


48-7 


NH 3 -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 NH 3 -^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 and 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 


1259ll-3 


86 


6-10 


1541M 


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 


18l-2 6 


16-20 


101'2 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 


121'5 


1-9 


16-20 


80-1 


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 75710-7 87 

6-10 61ll-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 O a 
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), O 2 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- 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 O 2 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 O 3 consumption, ammonia/O s 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; MoanS.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 ratio t 

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 = * o64 
P <0-01 



OD O 



10 20 30 

2, Frequency of 2LA inversion 





r= -Q-322 


r = + 0-193 


40 


40 


P,NS 




oo 


oo 6 


o 30 


o o o 30 


L ^o 


I 2 ' 

V 

- 10 





*- 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- 

inC 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 (r m ) and 
finite (X) rates of increase, net reproductive rate (R Q ) 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 (m f ). 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 (r m ) and finite rate GO were calculated. 
The values of % (pivotal age in days), l x (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 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- * 0188ool * 2 T 



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 d x 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 iJL 9 = 2ft 2 3, where 2ft 2 " is a constant, then by integration we get 

/, = /o<r* v - (1) 

Patting IJk = ?> a nd fi, = 1 - P 



and, by differentiation we have, 
dQ x = 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 = 2ft 2 f" &er**dX 9 



from which, by integration we get, 



. 
2h 

The observed and the smoothed values of 1 9 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 ; 



l m = i-ooe- ' 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 = R = Z k = 20- 411 ; x k x = 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 (Dl tt /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 m g 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 (R ) 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 (r m ) 

__ In R 9 __ log e R o __ 3-0161 
r<w = "rT "" ~ 86-464 

3. Fir.ite rate of increase in numbers 

(A) = Natural antilog of r w 
= Natural antilog of 0-0349 

4. Corrected generation time (T) 

^ In It* log e^o 3-0161 

Tsss , = s= . 

r m r m 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. 

(r w .= 0-0349) 



Pivotal age 

in days, 

1 


L. 

2 


3 


4 


Percentage age-d&tributioo 
100 0L, <r r m<+D 

5 





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 


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 


-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 (r m ) 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 


27-10l-85 


17-600-87*** 




Blank slide 


27-16l-87 


12-41 0-64 


Female 


Male sebum 


31-13l-93 


16-700'38*** 




Blank slide 


3a-90l-91 


13-72Q-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 


& 


.2 




cn v5 en 


1 


8 


fe 


^f 01 00 C* 

^H b b o 


O 



1 




-H 41 


. 


C5 




* 


O 


.2 




^j en *>O ON 


o 

1 


1 


^ 


Tf <S OO O 


T-< O O 




P 




-H -H 


1-1 






.s 






<n 








E, t-^ en oo 


tf 




Ci 


S p * 


1 


? 




^ *?, i t 7, 
m -H ^ -H 


o 








en 


o 















I 


Q 


* 


cn 
?L ON C5 ^O 
cl <* 


i 






41 ^41 


g 




* 


1 


W) 


^ 


i ? ?? 


x 


1 




vtj O ^ O 

JJ 4- 

~n TI 


VI 


8 




* 








* 


c3 


1 

c 


^ 


4t 

TX ^H Vfi G 

s ? *f 


1 


1 




"* 41 4 


.2 








*> 






cs 






* 









1 ^ ss 


< 


W) 

fl 




^ 41 ^4 





*y| 















-H 


a 




# 


^ fm ^ 






Jri en Tf r- 


rrt 




j^ 


vo en oo C N 


*5 






<L b bo 


o 






-H 4 


3 4* 







* 

o *i <X ^ 


^1 


to 

s 




'-* -H 4 


1| 


S 

rt 




* 

* 




?/5 


. 


* en ^ 


s* 




^ 


C*4 ^ <2 


1 








c? 1-t 






S 






H 










V-t o 


s s 


^ 


o *^ 


5 


. 


H ^ 


** s 


2S 


^ 'tt 


o 




S ^ 


21 




1 s 




o 






CO 


S 



8 

6 

V 



o 
6 
V 



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-680-37 
1-800-31 


3-250-38 
3-750-42 


3-58iO-45 
3-90:tO-36 


2'46iO-37 
3-280*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 < 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 grgaria$ 
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 





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 



-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 



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 



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 



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 



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 



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 
C pairs mating tion per per pod 

male female 



20 Male 5 


8 682-23 1-6 1 


48 


Female 


99i2*82 




25 Male 5 


14 720-55 2-8 2 


59 


Female 


104l-57 




30 Male 5 


21 78.1'39 4-2 4 


76 


Female 


113l-93 




35 Male 5 


26 91l-56 5-2 5 


81 


Female 


121 2-82 




37 Male 5 


28 87 1-59 5-6 7 


87 


Female 


1162-95 





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*2Q0-86 





80 



90 



Male 87; 00 1-56 28 

Female 116'002-95 

Male 75 -40 1-99 23 

Female 105- 20 2- 23 

Male 67-4Ql-02 14 

Femele 95-40l-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 1 7-0 27 x 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, 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; T 1? anterior testis ; T 2 , 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- 8-19 7 7 
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 




in 

(M 




m 
evi 

O 



m 
CM 



JffS 

.ai?i 

g 'tf '3j o 



8 'T tf 
? 5? a "5 



<N 

C 



00 




ID 
(M 



290 



A Jayaprakash and Y S Rao 






<M 

o 



> 6 

fr , r r-j Q 







in 
U. 



QJ ;* -j 
^ i C TO 

a-bh e 



,-! s 



o <o 2T 

Ed . S - O tJ 

" fl 

TJ O .. S 



H 



5 

-4 > O 





to ^ 



E 

a. 



UJ 



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 1C (Average of 4 



Time in hr 



Number of second stage juveniles' penetrated 

Epidermis Cortex Endodf;rmis Total 

and 
Pericycle 



2 


2 






2 


4 


10 




. . 


10 


6 


20 


4 


. 


24 


S 


28 


8 




36 


10 


29 


5 




34 


12 


10 


18 


t 


28 


14 


7 


25 




32 


16 


2 


31 


.. 


33 


18 


. . 


35 


2 


37 


20 


1 


20 


10 


31 


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 



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 


38 27 


112 98, 


,115 195 


120 198 


Average No. of 
egg masses/day 


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 cm 2 . 

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 







39nN303< 



Sediment-ostracode relationship 



301 



X U3UVM 3IMV9HO 



* * # 

5 = i 

.j ~ 
u f> w 

I Bv&l 






D-;-=:.v.- :-;-; : -.-.-.-.- 
Cfer.v ;--. i 




OEESZZSS 
QES5SSI 

DESESSS 


I 

(n 2 

I 


lEHSSTTTrrr. 
E77ST7-. -,:?." 


-R- & 

- = 5 >, a 

S* O 


FTTmTTTxrrrr- 


S cU 

2 ^3 > 


ftv.'.?..;;...v^. 

flETT^V^TTv- : 

E-. . . . j , . . : -- 


< *Cf w 

s "I 
** "^Z3 


:.v:. ..:.::::::... i 

IK^^T.-,:. .,-.- 

Ifcv.>:-.-.;-.v..--.^ e 


:l Jf 

3 * !l 


OSS55MZI2 = 


S3 


f-.-.-.^V,l.-.,.g. 
H;:.-V:^:=.:vr: -v.::: . 


l 

- -1-a 


w.-.-.V.:,-----.:-. 1 ".: 

HESSiSS- 
nrTT^nrTTT-, 


* S> j 

o T f5 
N - O 3 

S S - ^ 


nssisnzs* 
ri... ^ < 


w i 


mJT^r^r^ 
[ET^^rrS. 


.s "K 

*2 


CEZIHZSSi- 

EmsznEino 


>> 

1 ^ * 
'S -22 

i S s 


(ESv-v::-^, 
OESSEEESo 

i^^M 

E222ZS3IS] < 


* s 
is 

I.S 


SHTTT^-r:. 

(ESSHSSEI- 

QLv. v. ::....; ;-.-.. x 

[BBIBII ' " -r 


! 

? H 






K5S.y:',.'.!...:,,u 




IJL-. ;.;:'. '.-.' ;.;;> .:] ^ , 





f* o 01 j 



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 *v t s*i> ,u 
0-01", DDT for 7, $.. 24 in <>. 41 ! Ill, ! ii4 
I or ahbrcviitii^ 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 

S310NIW 01 / SlNflOO -W39 








o <i o 



o o o o o 

oT / siNnoo * 



- 

O 

I 
I 






L F$&&&^ 

f^.K.;g 




O o O O O O O 



OL / SINOOD H39 



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 


102431* 
43352 


1457 


Female and female 


Female one 
Female two 


73349* 
50449 


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 


19023 
281 26** 
21328 
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 LC 50 
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 
O a 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 (LC 50 ) 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 LC 50 values including the upper and lower limits (ULC 50 
and LLC 50 ) 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 LC 50 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) 


LC 50 values 
(mg/1) 


Regression equation 


DDT 


24 
48 


0- 

o- 


14890 
12280 


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- 


15GOa- 
9750 


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 C 50 values 
(mg/i) 


P,egrossion equation 




Oirs) 




















DDT 


24 


2 


813Q- 


453 


log 


y 


= 8- 


6338- 


bg^ + 


M219 




48 


2' 


2770- 


310 


log 


y 


= 9- 


87^- 


log x + 


1-4720 


Ekalux 


24 


3- 


6590- 


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- 


9620- 


479 


log 


y 


= 13-2989-log x- 


-4-2607 




48 


4- 


5940-557 


log 


y 


= 10 


5503 


log x~ 


-1-9859 


Sevin 


24 


14' 


7300- 


590 


log 


y 


= 35 


2288-logx- 


- 36-1552 




48 


13' 


9100- 


380 


log 


y 


= 44 


0285 


log x - 


-45-3320 



The higher LC 50 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) 


LC 50 

(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 LC 50 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 LC 50 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 LC 50 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 (4C) 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~ 4 M) 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 
periodApril 



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 ^ US f 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. r ty 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. m a - - 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 





14 


2 


25 


6 


19 


III 





20 


2 


25 


2 


23 


II 





26 


2 


25 


2 


23 


II 





38 


2 


25 





21 


I 


4 


2 


5 


25 


11 


14 


V 





14 


5 


25 


2 


23 


IV 





20 


5 


25 


4 


21 


III 





26 


5 


25 


2 


23 


II 





38 


5 


25 





22 


I 


3 


2 


10 


25 


8 


17 


VI 





14 


10 


25 


4 


21 


IV 





20 


10 


25 


4 


21 


III 





26 


10 


25 


2 


23 


II 





38 


10 


25 


2 


20 


I 


3 


2 


15 


25 


6 


19 


VI 





14 


15 


25 


7 


18 


IV 





20 


15 


25 


6 


19 


IV 





26 


15 


25 


3 


22 


II 





3$ 


15 


25 


4 


18 


I 


3 


2 


25 


25 


7 


18 


VI 





14 


25 


25 


3 


22 


IV 





20 


25 


25 


3 


22 


IV 





26 


25 


25 


3 


22 


II 





38 


25 


25 


3 


22 


I 






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 muSleS;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 (PM300- 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) 


7-000-63(<5) 


II 


LD 6 : 30 


36 


5 


8-00-00(7) 


285 -71 21 -03 (7) 


ni 


LDLD 6 :6 :1 : 11 


24 


6 


7-50'50(6) 


216-0022-49(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 - 20C. 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 3 a -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 A 4 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 C 3 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 NSC S 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 V on 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 
o 



1 

O 

4 



o 

I 

I 

! 
i 



g 



in 



3 fa O 
CU o 




"8 !- 



Z^o^o 



~H 



O 



-HoO 



i 



41 






^t- 

wi 




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 



100 


5 D Josh 


i 










g 


o* o' 


cs oo" 


^ 


c^ 


Si 


o 


vn c? oo 


o <"^ 


4r 


r^ 


'o 


"? 


O 2 VD xj-i ^ ! 


I L 


rH A 


Tt ON 


d 












s 





r-i T-^, eo cA ** eo 


-* S ^ c^ 


CO Ol 


^ 0, 


^ 












:5 . 


ex 










ua 


' ^ S 


>jr -^ ^HS ^j-^ 


^^^ -^ " 


.^ 


^^. 






vo ol oo 


C4 "^ 


O4 


iO 


1 


1 
tj 


ON C5 ON 

*~* CS. CO 
(S, C5 VD OO .O 


V ^ 

i I 
i {} 


T 

ON (0 


1 

iO 


3 


" S 


<i> in ^ ^ JJJ O 








< 


to 










o 


*5* .-- 


X-s , . 


to 


o 


^ 


*s 


* 'O 


v T^ 


oo . 




o" 


1 


I | 


oo >o 

r-4 CH 

1 I O , 

tn 10 i/i o - 


i s 


co 


{0? 


<*- 


*< ^ 


'OO 

ct i i cO O co 


^ i. <= s '. 


c4 r4 


<b S- 


o 




N^' N^ / 








JD 












-a 


g 


X"- /- ~* >K 


^^ 






s 


ba """ '-""' 


o 9 o 


to 'r 5 


Cn 


co 


* 


"g t^ 


* C4 00 ^ 


CNL ^ 


CO 


C4 




Si * ~j 


In ^- ^ 00 ^ 




in L 


I 


<s 


^ SX 




t~^ CA ^4* T^ 


^* ^[* 


t*" ^^ 


a 


t-H 


^o 4j. ^i- r- S r- 


^ ^ ^ O, 


<si 53, 


rU ^ 


I 


!* 


^ ^ ^ 3 








5? 


1 1 


o > ? 


co "* 





00 


*>, 


^ *""> 


^ co ^ 


" ^ * 


TJf' 




*2 


* *^ 




1 t 


I 


I 


I 


*" i 


? ? t 


co vp 

* ^5 


^ 


t 
O 





*?* 


t i 








^3 


tJ^r 


CO ? 
** CO 


00 





V5 


*? 


S 


r-< ^ In 

cxi fj i 


co Y 


V 


T 


"""^ 


r* ^/ *""' 


ci <^ T 


r 1 Y* 


. 


r-" 







rf ^ S 


^ C5 


n-4 


o 


j 


,^ C j-^, 

as ^ <3\ 













!! *^ '"i k 


Tn vb ^ ^ "^ ^ 


^ rt oo 


^ 


10 o 





. # *g 


^ t>> 2 ?{ 2 S 


f\3[ CM t < i < 


** r^ 


v-t cNl 


H 


^c2 


T. tit It 


'j OO CO ^^5 


r A 


1 I 







O 00 CM O 


^ 00 


^ T^ 


r-< ,-K 





^ j< rt 


^ ^ ^ C4 










as *^ ^J 













1 ll^ 


in 








fj 


Jg 'o S S 


^4" i/j CO CO OO CO 

Tii i i 

ON OO -* 0^*0 


S^ X 

ON ^ vx 


CO CO 

o v^ 


IO 


11 


1 8^-* 


CO 




C4 c4 


r-i r-t 


11 

w 


li 


III oo co 


2 X 

T 


? 


O 
1 


A C3 


* "v 


^ co co 


^ X 


^c 


f- 


v< c3 


^ 15 









o 


! *5 


2 o; 


* 












r O\ co 


IO v 










,-t d co 








. O 


o 


1 I 1 


CO 

f 


CO 


o 


T " 1 :J3 


r* 


ds c^ ~j 

C"4 *^* 


10 ^ 


c$ 


. 


ll 






^1 




J-l 




*o ^ 

I | 

i 


i 

*d T5 

o O 

**^ >* 
CM *-< T3 
O cj O O 
^ ^ JD 

o rt S O 
v4 W J H 


* i 
f - Is 

* 2 

^I B ^1 

^3 y O ^3t Q. 
2 0*2 2 EO 

J 


1 

8 

t/J 

ll 


1 

i s 
11 

w a 




1 


i-^ C4 CO 


^ V^ 


vo. 


^ 



New species of haematozoans 



401 



2 


sr 


CO ^ . ^ 


0^ 


00 


*3 


07^ 
<N 


? 


vO vo 


o <s 


' CO 


o 


Q* 


o, 


s- e 


s 


*"* 


"** 












*O 


o" 


, . 


^_^ 


.^ 


^^ 


Q 




o 










* 1 *~" H 




, o*\ 





T ( 


t 


^ s *" 


2 


7 7 

1-1 ON G\ vo 


VO Tf 


r ti 


J 


<<>, 


2< 


O, O 


S- 


*"* 2* 















"o 






T 


X-. 


o" 





o cr 


x 


x x ^ 


s 




CM 


* *-* 




T 7 


VO 7 


l/^ I 


*rt 


-25, 


x 


x x * ^9 


o o. 


T ~ l o 


g 












2 


CD" 




cT 


rT 


*-** 


O 


00, 


X 
X 


X * ^ 

x x ^ 


O CO 

e 


O CM 

o 


1| 


o 










*" 5 




X 


Xx ^ 


00 





O 

fl >> 


C3 


X 


X X 


TH 
oo 


! 


2 g 


00 




o 


o 


* 1 


! 












r "O 

tt 


VO 
o 


X 


XX ^ 


'OO 


<s 


O 

a| 


7 

CO 


X 


xx 2 


O 


s 


%| 












2 "o 












tt 












t-i O 












o ^cj 










00 


V V 


00 

1 


X 


x x x 


o 

I 




W 





X 


x x x 


6 


o 


|1 












H 'x 


X 


X 


X X 






s^ 








X 


X 


uJ w 


X 


X 


x o X 


X 


X 


11 












S| 


X 


X 


X t x 


X 


X 


S.9 






! 






^ 2 


X 


X 


vx \f\ 

A i- X 


X 


X 


>> ** 






o 






"S 


X 


X 


X r x 


X 


X 


S 


X 


X 


X ^ v 






OT JJ 


3 




X 


X 


X 


P< 













w w 


Distance of nua 
from a flagellar 
end 


Length of 
karyosome 


CM 

+* . *- 

sf 55 ^S 

So If J 1 
s & g | ? 
3 6l Ssi 


Width of 
kinetoplast 


J 

.P 

'l 


les aro quoted a 
phic forms. All 


CO 


Q\ 


o ~ r< 


to 


rt 


3 g 



402 



3 D Joshi 







**-! 

o 




,-N 0" X-^ 


s 




,*. O O '*"" % '*""* ^T"* ***** 






Vi9o/"i r * s ^CM^-* < '4"^ >1 ' ON. O. V"). i I 




3 a 


^^^^fMJsCM'T'^"[CM( 1 O^ V CM | 


. 


O vi 


/-5 *o CM . ** O O *^"S V"* OO 


C3 


o- 2 


vb * -rV ,-H rf ^ 


VJ 




x- . y-s O* 


x 


*5 S 


C5 O O O ->, O _. O 
vo (Nj m 2 en yj oo . v . ON. <-?. *^r . . 


1 


^ ^ *Si 


i"" ' ^in^vb C fkX t 7r-< C ? 1 ^' U p T ~ HC i i 1 ' 


r^ 


r" XO C 


^ o "^ O O ON 




^^ 


r-C^eJinc^^fnb 


e 






M 






o 


"7 


O <> CM x* 


^ 


|1 


CM r^ 1 2 <O .>v >/^ *-O O ""1 /> CM 
OOfNifMiOCMfrjONfrj 1 /^. Tf. ^* ON. . . ^ . 


i 


. w 


enlj^Tooi bT* ^5Pcn? ^ J* bT ^Y ^T 

'-H.'- H OCM.r-ioo O CO |/^ Tjf CS O 


a 1 


^ 8* 


SoOcMOO^^b b CM T-H 


2? 






^J *o 

C o 


ON" 


^ ^s 


*-* o 


r- 


9 o" T t^" 


o & 


^5 ON 

<O T-"< 

" ^5 


oS^c^^fn^b^ o 9 *T x X 
i/iJjbTooAooYr- VT> * "- 1 

<M . "r* O CM . o 


1 2 


o| 


CM 2x CM i, 


^ 


~- 




s 


53 ^ 


ON" 


o 2 


*-* CM 

^ 

53 flN 
&^ 
5 

o 


cT o ^ 9 

V X r-iS7 x x 2 ^ X X 


1 5 
s 1 


o ^ 


2^ S- i ^ 




^s 




i "! 






s 1 


"S ^ 


. 


, ^ 


S N 


irt ^ ON ON oo oo 
ON. . i uTi n n -r- X X 


o , 


"S t5 


S oo S . ^ ,1, 4- b 


"Mi ^> 


05 w 


CM 


55 


o^ 




5- 


^rs ^ 

lit 


Xx?5Txxxxx x 

I en 


1 8 

'1 


0^ 










G <;> 


9 t-, 




c 


|l 




a 1 


r*J 




-d ^ 


VI "^ 




tf M 




G H 


cO O 


Particulars 


P 2 4-> 
J3 *o v> * 

> * * *, S 

? 1 1 1 I ! i i 

11 S g o ^ 1 

C O riOe^QCQI-i 

gaa^al^i^jj 

"S'Sgl'Sls'S's'S'S 

i-f 5 .& * i s 'i s s 

CjS^'^ A IS t-J . C! *^"* 

w^ Jj CQ CQ kJ ^ u S^ .ij sH 

*^ * ^ "^ <^ ^* *^ ^ *^ HH P*^ 


All values are in rnicrotns 
tho or ganoid. 
Note ; All measurements \ 



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 NH 2 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 


NH 2 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 T f 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 K 2 ) 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 C r 
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 22936-2 25332'6 71090'4 91684'5 10l10*9 13414*2 

27 7'50-50 22739*6 24038*9 64982'2 85594*7 10312-1 142 9*7 

32 77-00-00 21632'S 23127'4 69188'1 74892'9 124ll-3 164 8'6 

37 5-0l-00 151 29- 7 167 24- 1 56279'3 6017T'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?ac of"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 



A 









uj 



30 



27 



32 



All 



i 



r 



Jj 


- 


^ 






L 


L n 




\\ 








r 


s 










y 




', 




/ 




1 


/ 




/ 




/ 
/ 




(MI 


/ 
/ 




/ 




/ 




M 1 










r 





22 



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 (AT 2 : %) 



Effect .of temperature on Eupterote moUifera 



421 



H 




Q MALE 


o 

2 50 




T 






. Q FEMALE 


o 




/* 








IT 




/ 




"1 




UJ 




X 






1 







X 




x 


1 1 







x 




X 




HI 


I 30 




x 


x 


x 


] 


^ 






x 




X 












x 






x 1 






x 




x 




. 








x 






/ 






x 




x 




. 


20 






x 




' 


x 




22 27 32 37 


20 






16 




--] 


s 5 


T 


\\ x 




12 

UJ 




fl, 


F 

r x 


1 ^ 
X 

x 






O 




X. 




x 






u. 






M X 






/ 


u. 




x 


i 








uj 8 






1 X 




f 


X 1 


z 


22 27 32 37 


2 




oc 




45 


. 






CM 






ft ' 


* 35 


- 


P" 




rti 




-' 


x 




1 

9 


25 


I 


X 


x 








n 




x 








r 1 


x 






X 






1 


x 








r J 


x 






X 




\y\ 




x 






15 




1 ^x 




, 


X 


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 ad 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 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 


-!- 9'863'45 








N.S. 


do. 


10% 


24 hr 


+ 17-69l-52 








P< 0-05 


do 


10% 


10 days 


16-43l-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 


+ 27'892-2 








P< 0-001 


do. 


100 ppm 


3 hr 


+28'672-4 








P< 0-05 


do. 


25 ppm 


24 hr 


15- 98 4- 3 








N.S. 


do. 


2'5 ppni 


10 days 


+20'72M5 








P< 0-05 


Tannin (Wattle extract spray dried) 


100 ppm 


3 hr 


+ 30-763'0 








P< 0*05 


Tannin 


100 ppm 


24 hr 


5'300-48 








P< 0*05 


do. 


20 ppm 


10 days 


19'762-34 








P< 0-05 


Sodium chloride 


5000 ppm 


3 hr 


+ 9-42+4-2 








N.S. 


do. 


5000 ppm 


24 hr 


+ 8-76M7 








P< 0-05 


do. 


1 000 ppm 


10 days 


31-163-67 








P< 0-05 


Calcium chloride 


1000 ppm 


3 hr 


+ 2-55l-5 








N.S. 


do. 


1000 ppm 


24 hr 


+ 1-281-1 








N.S. 


do. 


500 ppm 


10 days 


+ 13'991-61 








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 O 3 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 io n 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 O a 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<0001) 
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 17-180"95 


21-571'91* 


25-56 


15 18-461'85 


27-770-98** 


50-43 


20 15-400'30 


30'24l-94*'** 


96*40 


25 16- 92 0- 94 


28-06l-99** 


65-54 


30 17-780-26 


29-31l'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) 


3'00'0 


3-l0-2 


I'OiO'O 


l-20-2 


15(5) 


2'90-5 


2'00-3 


2-80-2 


2'80-2 


20(5) 


2'8Q'3 


2-50-l 


3'00'2 


4-0 0-0 


25(5) 


3'00'2 


3'l0-3 


4'40-2 


4'6G'l 


30(5) 


2'70'2 


2*90'2 


6'00-3 


5-80-2 



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 
Labhsetwa r 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 4 H '-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 Si e 
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. Reproductiv e 

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 




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 (Zrn.) 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 c r ab, 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 LD SO 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 LD SO . 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 LD 50 / 8> lethal feeding periods (LFP SO and LFP 9S ) 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 LD 50 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. 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 (LFP SO and LFP 98 ), their 95% confidence 
limits and slopes of the probit regression lines. The slope of the probit regression 
line and LFP SO 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 MeanS.E. 
(percent) 


- Mortality 


Days to 


death 


Died 


Survived 


Mean 


Range 






Tatera indica 








2 


0-0125 


14-3C0 -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 


85-4514'60 


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 


114-10l4-95 


103'572345 


S/12 


8-4 


5-12 


10 


114-41 21-59 


148-8316'18 


S/10 


8-1 


5-14 


14 


173-1616'98 


... 


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. 


LFP 60 

(days) 


L*P 98 

(days) 


T. indica 


0-0125 
0-025 


1-920-10 
M30*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 
1-890-08 


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-950-35 4- 21 0-41 O'OOl 6/12 

0*0125 14(2) 2-480-45 4'200-59 0'05-0'02 5/12 

Meriones hurrianae 

0-025 14(2) 5-460-98 7'161*07 030-0 '20 11/12 

0-0125 14(2) 4-070-76 6'940'81 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 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 LFP 98 (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 



ll 


t s * " ! - 


v> rf oo co 

rH 


J^ fl> 


OO S^^ _ 






^ rt 




t5 


1- 




3 3,1 


J ^1 ~ 


00 VO T> CO 








1 


*^ S ^S 




?i 


^,t> 




1> 


j^v CO 3 4) 




o c 

H ;=! 

Pu </> 


S | 9 " ^1 2 

00 o "^t- 


T^" OO *"< CO 


4- 


CD *^ & 


<* 


u 


w n ^ "ft Z 




l 


15 ^ ^ 




2 


S si 




S 2 * 


CJ <^v ^ ^ 


OO CD ^t" T* 


8 3 o 


11 15 




2.1 
3 


v. 1 f 1 
>w- 


O I*- 00 VO 

M *-< 


15 

IS 


r- 

o 

I - " ; ; 


so Tf co ; 


d 


s 




"3 t3 t? 





^ t- ** co 


1 3g 


s* 6 = 




T-H 


VD 


f 







g co : 


1 


o oo 6 : : 




- g 


^ ^ 


S 




^ 


CO 




1 x-s 


.5 




'So 


CO 




"8 o * 


^p 




o ^ > 


.2 


1 


^o S 7 
^ -3 J 8 -S 3 

t.f fill 1 


1 It 1 1 

^ o g o 

| S S S 8 


< 


f 1 5 | g 1 1 


g t- c* co m 




f * -f 1 1 3 S 


' o s & 




093 


S 



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 (C 26 H 28 O 14 , 
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 (LC 50 ) 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 
06 


0-2 
0-4 


0-25 
0-75 


0-15 
0-25 



Total 

carbohydrates N 107039 102050 78038 70038 84032 91021 9QO23 

A 91047 35C70 62062 65062 57078 72040 61050 

N 92032 87025 68029 63036 74016 76029 73026 

A 77065 75040 51049 44018 43019 70025 53724 

Proteins N 100039 90050 60026 36019 72026 740?4 76017 

A S4053 80060 50060 32077 56077 64011? 60077 

N 120033 110030 70029 52027 82030 88016 90029 

A 102098 98060 60050 44020 70077 780101 72076 

Fatty acids N 30019 28025 19017 160d=26 23027 24026 20026 

A 24034 21030 16020 14022 18518 19519 18021 

N 40023 38015 28024 23028 32021 36018 34020 

A 33032 30025 24019 20021 26020 28528 27544 

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 2580l04 2*0095 172089 163092 184093 2lOO78 205096 

A 191880 190080 147092 1350101 152098 170098 1658101 

N 2270128 2220100 164082 156086 172089 191789 18889 

A 152087 1470100 106078 98072 114051 130549 125082 



N 2826d=97 27609C 2268101 208998 244878 269579 2640106 

A 2250110 2200100 172689 160890 1833 104 2149 48 204389 

N 3168118 3090115 245287 2286ll9 262087 2896112 2768122 

A 2742120 2675110216088 192092 228086 250988 2390124 

Fatty acids N 681 78 61072 50542 46346 56232 58440 5753S 

A 52050 48065 38836 32039 40929 440'20 42830 

N 78070 75060 56472 50242 61084 70688 62892 

A 64882 60062 45042 39040 50442 56046 52049 

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 l74 5 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 = -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 = 09493) 

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 L 1 ' 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 (r 2 ) (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 16'01090'5921 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 10-89270'0858 l-l5300'1131 27'2993 0*7517 
15-1-20-0 

cm 149 17-06390'1168 17- 6628 0' 1062 1'34580-1342 86'0302 6-1856 

>20-lcm 131 31-8632M824 34- 5254 1-5788 l'61860-2459 1 155- 6406185- 1899 

Pooled 829 19* 0041 0* 4349 15-7957 0*3864 1'20170'0029 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 L 2 : 5S 


Male 


log W = 8812 4 1 ' 9769 log L 


HP =0- 0013146 L 1 ' 9769 


<l5-0cm 


log W= 1-4208 4 1 -4819 log L 


PF = 0- 037949 L 1 ' 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 L 2 ' 4157 


Pooled 


log ^=2- 9632 4- 1- 9561 log 


PF = 0- 0010884 L 1 ' 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(yr) a 8-7873 


7-9748 


8-8412 


5-0822 


5-9265 


8- 7579 


Z 1 (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- 2 unexp. 1-6427 


1-415 


1-7228 


0-3504 


0-2417 


1-5074 


^ 0-7931 


0- 6512 


0-6607 


0-4443 


0-01532 


0-8999 


r 2 0-7949 


0-6101 


0-6397 


0-3158 


0-0579 


0-8684 



proportion of correlated variance ; <r 2 = 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 (r 2 ) 
(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 

YY- 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 
(HgCl 2 ) 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 

~ 1$ 

I u 



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% , 6-83% , 9'75% , 12*69% , 15'62% ) 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 I 5 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% S i.e., 24-14% of the predator body weight, 
the lowest amount being at 15*62% S i.e., 13*41% of the predator body weight. 
Thus in the *9% -15 *62% 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. T