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

Proceedings of the Indian Academy of Sciences 
(Animal Sciences) 

Volume 96, 1987 
CONTENTS 

Ovarian polymorphism in relation to reproductive diversity and associated 
histological and histochemical attributes in some sporophagous tubuliferan 
thysanoptera K Dhileepan and T N Ananthakrishnan 1 

Host preferences of some acridids (Insecta: Orthoptera) in relation to some 
biochemical parameters K P Sanjayan and T N Ananthakrishnan 15 

Ecobehavioural and biological studies on two species of seed infesting thrips 
(Thysanoptera: Insecta) A Balu and A Mohan Daniel 23 

Light and electron microscopic observations on lipid droplets of the sebaceous 
flank gland of the shrew, Suncus murinus viridescens (Blyth) M Balakrishnan 33 

Quantitative analysis of carbohydrases in the crystalline style of some intertidal 
bivalve molluscs P Shahul Hameed and A L Paulpandian 41 

Glycogen level and glycogen phosphorylase activity in the eggs of silkworm, 
Bombyx mori L. P M Chandrashekar and Geetha Bali 49 

A shift in metabolic pathway of Sarotherodon mossamhicus (Peters) exposed to 
thiodon (endosulfan) M Vasanthi and M Ramaswamy 55 

Aspects of culture: Acanthamoeba astronyxis (Ray and Hayes 1954) from Bay of 
Bengal coasts, India Mrinal Ghosh and Amalesh Choudhury 63 

Development and survival of Mythimna separata (Wlk.) on some host plants 
during winter at Hisar Ram Singh and J P Chaudhary 71 

Food utilization and surfacing activity of Channa striatus fry in relation to 
quality of food K Sampath and E Vivekanandan 77 

On some plant galls from the Fiji Islands M S Mam and P Jayaraman 81 

Biochemical correlates in Rhipiphorothrips cruentatus-Terminalia catappa inter- 
actions with special reference to leaf infestation patterns 

G Suresh and T N Ananthakrishnan 1 17 

Effect of sublethal doses of dichlorvos and carbaryl on the nervous system of 
Pericallia ricini Fabr. (Lepidoptera: Arctidae) Y N Singh and F Shaheen 129 

Effect of starvation on food utilization in the freshwater snail Plla ylobosa 
(Swainson) M A Haniffa 135 



ii Contents 

Limnology of river Cooum with special reference to sewage and heavy metal 
pollution Bernice Anantharaj^ V Bagyalakshmi and R Lakshmi 141 



Proceedings of the Symposium on the Impact of Nutrition on the Reproductive 
Biology of Insects, Madras, January 1987 

Foreword 151 

Nutritional modulation of reproduction in two phytophagous insect pests 

S S Krishna 153 

Relation between feeding and egg production in some insects 

J Muthukrishnan and T J Pandian 171 

Nutrient modulation of hormone production: Dietary essential 4atty. acids, 
tissue prostaglandins and their probable role in insect reproduction 

V K K Prabhu and Mariamma Jacob 181 

Assessment of neuroendocrine mechanisms of feeding and reproduction in 
phytophagous insects D Muraleedharan 185 

Nutritional value of malvaceous seeds and related life-table analysis in terms of 
feeding and reproductive indices in the dusky cotton bug, Oxycarenus laetus 
Kirby (Hemiptera: Lygaeidae) K Raman 195 

Influence of leaf age on feeding and reproduction in Raphidopalpa atripennis F. 
(Coleoptera: Chrysomelidae) R S Annadurai 207 

Effect of food quality on fecundity of Mylabris pustulata (Coleoptera: 
Meloidae) T Manoharan, S Chockalingam and K P S Jeyachandran 217 

Effect of temperature and host seed species on the fecundity of Callosobruchus 
maculatus (F.) J Chandrakantha, J Muthukrishnan and S Mathavan 221 

Nutritional influence on the growth and reproduction in two species of acridids 
(Orthoptera: Insecta) K P Sanjayan and K Murugan 229 

Correlation of nutritional changes with the reproductive potential of Aphis 
yossypii Glover on egg plant T K Banerjee and D Raychaudhuri 239 

Influence of nutrition on the reproductive biology of sugarcane pests and their 
natural enemies H David, S Easwaramoorthy and K Subadhra 245 

Nutrition and reproduction in haematophagous arthropods R S Prasad 253 

Feeding and reproduction in vector mosquitoes R Reuben 275 

Host preferences in some pentatomids and related impact on the fecundity of 
their parasitoids R Velayudhan 281 

Impact of predation and food utilization on reproduction of Diplonychus 
indicus and Ranatra filiformis P Venkatesan and S Muthukrishnan 293 

Observations on feeding propensities, growth rate and fecundity in mayflies 
(Insecta: Ephemeroptera) -K G Siuaramakrishnan and K Venkataraman 305 



mpact of chemicals on feeding and reproduction in insects Kumuda Sukumar 311 

mpact of differential feeding on the reproduction of tiger beetle Cicindela 
ancellata DeJean (Cicindelidae: Coleoptera) 

T Shivashankar and G K Veeresh 317 

>ome observations on the nutrition-reproduction correlation in grouse locusts 
Orthoptera: Tetrigidae) A M Bhalerao, N M Naldu and S Y Paranjape 323 

nfluence of biochemical parameters of different hosts on the biology of Earias 
nttella (Fab.) (Noctuidae: Lepidoptera) R Sundararaj and B V David 329 

Effects of feeding regime on energy allocation to reproduction in the silkworm 
3ombvx mori S Mathavan, G Santhi and B Nagaraj Sethuraman 333 

nfluence of azadirachtin on insect nutrition and reproduction G K Karnavar 341 

-lost dependency among haematophagous insects: A case study on flea-host 
issociation R S Prasad 349 

Effect of okra fruit blocks, seeds and pericarp on post-embryonic development 
)f Earias vittella (Fab.) in relation to some phytochemicals of selected okra 
genotypes Ram Singh 361 

'n vitro studies on the involvement of brain and gonad in the functional diflfe- 
entiation of prostate gland in Cryptozona belangeri (Deshayes) (Mollusca: 
jastropoda) Umct Natarajan and Vijayam Sriramulu 369 

_ife and fertility tables of Achaea Janata Linnaeus on castor 

A N Byale and G G Bilapate 383 

radmium induced vertebral deformities in an estuarine fish, Ambassis 
.'ommersoni Cuvier 

V Pragatheeswaran, B Loganathan, R Natarajan and V K Venugopalan 389 

Distribution and population dynamics of soil nematodes in a tropical forest 
ecosystem from Sambalpur, India G B Pradhan and M C Dash 395 

Biology of Nyphasia apicalis Gahan (Coleoptera: Cerambycidae) with 
Darticular emphasis on emergence and imaginal life T N Khan and T Khan 403 

Vlicromorphology and cytochemistry of the branchial glands of the freshwater 
nullets, Rhinomugil corsula (Ham.) and Sicamugil cascasia (Ham.) 

Jag dish Ojha and A K Mishra 417 

Mature of resistance in selected rice varieties and population fluctuation of 
*reen leafhoppers, Nephotettix virescens (Distant) and Nephotettix nigropictus 
Stal) Kasi Viswanathan and M B Kalode 425 



Proceedings of the Workshop on Biosystematics of Insects, Madras, April 1987 

Foreword 437 

Multifaceted approach to evaluate the relationship among closely related forms 

)f Drosophila H A Ranganath and A Ushakumari 439 



iv Contents 

Chromosomal basis of raciation in Drosophila: A study with Drosophila nasuta 
and Drosophila albomicana H A Ranganath and N B Ramachandra 451 

Importance of Karyology in aphid taxonomy D Raychaudhuri and P L Das 461 

Recent advances in biosystematics of Trichogramma and Trichogrammatoidea 
(Hymenoptera, Trichogrammatidae) H Nagaraja 469 

Biosystematic studies on Aphytis in India- A promising area of research 

A Uma Narasimhan and M J Chacko 479 

Feeding and behavioural parameters and egg ultrastructure in the biosyste- 
matics of Reduviidae (Insecta-Heteroptera) 

E T Haridass, A Balu and M Noble Morrison 485 

Biological, behavioural and morphological tools in the biosystematics of 
Reduviidae (Insecta-Heteroptera-Reduviidae) Dunston P Ambrose 499 

Some aspects of biosystematics of Bruchidae (Coleoptera) H R Pajni 509 

Newer trends in the biosystematics of Membracidae 

K S Ananthasubramanian 517 

Significance of haemolymph protein patterns in biosystematic studies of some 
grouse locusts (Tetrigidae: Orthoptera) 

S YParanjape, N M Naidu and N N Godbole 527 

Biosystematics of fig wasps (Chalcidoidea: Hymenoptera) 

K J Joseph and U C Abdurahiman 533 

Biosystematics of Chalcididae (Chalcidoidea: Hymenoptera) 

T C Narendran and S Amareswara Rao 543 

Biosystematics of rice brown planthopper and rice green leafhoppers 

U Ramakrishnan 551 

Biosystematics of gall aphids (Aphididae, Homoptera) of western Himalaya, 
India S Chakrabarti 561 

Biosystematic studies on Agromyzidae from India Ipe M Ipe 573 

Biosystematics of Aleyrodidae (Homoptera: Insecta) B V David 583 

Biosystematics of Tingidae on the basis of the biology and micromorphology of 
their eggs David Livingstone and M H S Yacoob 587 

Biosystematics in lepidoptera and its importance in forest entomological 
research George Mathew 613 

Recent trends in the biosystematics of Entognathous Apterygota with special 
reference to Collembola N R Prabhoo 619 

Biosystematics of Culex vishnui and Culex pseudovishnui based on ecobeha- . 
vioural pattern A K Hati and S Bhattacharya 629 

Biosystematic studies of south Indian Leptophlebiidae and Heptageniidae in 
relation to egg ultrastructure and phylogenetic interpretations 

KG Sivaramakrishnan and K Venkataraman 637 



Contents v 

Experimental studies on the hatching rhythm and larval release in Palaemonid 
and Atyid prawns 

R Ponnuchamy, S Ravichandra Reddy and Katre Shakuntala 647 

Population structure and breeding season in Rattus rattus wroughtoni Hinton 

S Keshava Bhat, A Sujatha, R Advani and A S Sukumaran 657 

Feeding and extracellular digestive rhythms in some intertidal bivalve 
molluscs P Shahul Hameed and A L Paulpandian 667 

Terminal velocity of the first instar Ectropis excursaria (Guenee) (Lepidoptera: 
Geometridae) R Ramachandran 673 

Evaluation of brodifacoum against house murids of Garhwal Himalaya (India) 

C Sheikher, N Ahmad and S S Guraya 679 

Qualitative and quantitative changes in lipids along the length of female 
reproductive system of the poultry nematode Ascaridia galli (Schrank 1788) 

D S Jitla, V R Parshad and S S Guraya 683 

Stress induced alterations in the hemocyte population of Periplaneta americana 

(L.) N K More and YS Sonawane 689 

Effect of cruciferous glucosinolates on probing pattern and feed uptake by 
mustard aphid, Lipaphis erysimi (Kaltenbach) V K Dilawari and A S Atwal 695 

Energy utilization and freshwater mullet, Rhinomugil corsula (Hamilton) under 
exercise N Sukumaran and M N Kutty 705 

Wing microsculpturing in two Arabian species of termite genus Amitermes 
(Termitidae, Amitermitinae) M L Roonwal and N S Rathore 715 

Haematological effects of sublethal concentration of formalin on Sarotherodon 
mossambicus (Peters) M Razia Beevi and S Radhakrishnan 721 

Subject index 727 

Author index 739 



Proceedings of the 

Indian Academy of Sciences 

(Animal Sciences/Plant Sciences) 

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Copyright CO 1986 by the Indian Academy of Sciences. All rights reserved. 

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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 1, January 1987, pp. 1-13. 
Printed in India. 



Ovarian polymorphism in relation to reproductive diversity and associated 
histological and histochemical attributes in some sporophagous tubuli- 
feran thysanoptera 

K DHILEEPAN and T N ANANTHAKRISHNAN 

Entomology Research Institute, Loyola College, Madras 600 034, India 

MS received 17 September 1986 

Abstract Ovarian polymorphism involving oviparity, ovoviviparity and viviparity is 
discussed in relation to .sporophagous species such as Tiarothrips subramanii (Ramk.), 
Elaphrothrips denticollis (Bagnall) and Bacterothrips idolomorphus Karny alongside with the 
comparative histology and histochemistry of polymorphic ovaries. 

Keywords. Oviparity; ovoviviparity; viviparity; germarium; vitellogenic zone; pre and post 
vitellogenic zones. 



1. Introduction 

The incidence of ovoviviparity and viviparity in thrips alongside with oviparity is on 
record among some Phlaeothripine mycophagous species (John 1923; Bagnall 1921; 
Hood 1934; Viswanathan and Ananthakrishnan 1973a; Ananthakrishnan et al 
1983b; Dhileepan and Ananthakrishnan 1984). Facultative viviparity was first 
reported in Megathrips lativentris (Heeger) by John (1923), and ovoviviparity in 
species of Anactinothrips and Diceratothrips species by Hood (1935). Hood (1950) 
showed that Megathrips lativentris (Heeger) and Bactrothrips buffai (Karny) 
( = Caudothrips buffai Karny) living in temperate regions are known to be 
ovoviviparous and number of other tropical species of Diceratothrips and 
Anactinothrips may also produce living young ones. Bournier (1951, 1956, 1957) 
made a detailed study on ovoviviparity common in B. buffai in southern France. 
Viswanathan and Ananthakrishnan (1973a) discussed the adaptation for survival in 
extreme conditions in relation to the occurrence of ovoviviparity in Tiarothrips 
subramanii (Ramk.). An analysis of reproductive strategies and behavioural aspects of 
some sporophagous Idolothripinae involving oviparity, ovoviviparity and viviparity 
in T. subramanii, Elaphrothrips denticollis (Bagnall), Elaphrothrips procer Schmutz 
were made by Ananthakrishnan et al (1983a). Ananthakrishnan et al (1983b, 1984) 
further described the behavioural attributes in oviposition in some of the 
mycophagous thrips indicated the mean fecundity rates and incubation periods of 
some sporophagous thrips, and showed that the oviparous forms are more fecund 
than the ovoviviparous and viviparous forms. Ananthakrishnan and Dhileepan 
(1984) also observed the functional diversity in relation to reproductive polymor- 
phism in Bactrothrips idolomorphus Karny. Kiester and Strates (1984) noticed as 
many as 300 eggs in a colony of Anactinothrips gustaviae Bagnall, the eggs hatched 
under field conditions between 10th and 15th day after they were laid. Results herein 
presented relates to the histological and histochemical aspects of polymorphic 
ovaries and associated functional attributes. 



2 K Dhileepan and T N Ananthakrishnan 

2. Materials and methods 

The sporophagous species T. subramanii, B. idolomorphus, E. denticollis, E. procer 
and Adelothrips cracens (Anan.) were collected from their respective fungus infested 
host plants and maintained in a BOD incubator at 271C and 80% RH. 
Individual thrips as well as mated pairs were reared in plastic vials (4-5 x 3-0 cm) 
containing the specific fungal meal. Mouths of the rearing vials were covered with 
muslin cloth for aeration. Cotton plugs soaked in water were kept in each culturing 
vials for moisture, and these cotton buds were moistened periodically. 

For anatomical studies, adult, pupae and larvae were dissected out in insect 
Ringer's solution and the testes and ovaries were fixed in Bouin's or Carnoy'^ fluid, 
dehydrated in the alcohol series, cleared in xylene, stained with Haematoxylin and 
Eosin and mounted in Canada Balsam. Whole-mounts of ovaries, testes and 
spermathecae were studied under Leitz Dialux-20 compound microscope and 
photographs were made using a 'wild' camera and Ilford 35 mm films (ASA 125). 
Numerical variations of oocytes at various stages of development both in the 
ovarioles as well as in the lateral oviducts were also obtained. Diagrams of the whole 
mounts of ovaries, testes and spermathecae were made using a camera lucida and 
measurements were made using a calibrated occular micrometer. 

For histological studies, testes, ovaries and spermathecae were fixed in Bouin's 
fluid, dehydrated through the alcohol series, blocked in moulton paraffin wax and 
sectioned at 7 /*m thickness in a rotary microtome, stained with Haematoxylin- 
Eosin combinations and mounted in Canada Balsam. For a study of histochemical 
analysis (Pearse 1968), the following tests were used: mercuric bromophenol blue test 
for proteins; aqueous bromophenol blue test for basic proteins; toluidine blue test for 
acidic proteins; Best's Carmine test for glycogen and Sudan III method for total 
lipids. 

Mating patterns involving diverse breeding combinations between different sex- 
limited polymorphs were studied by freely allowing individuals to mate and the 
mating pairs carefully transferred to a different vial (4-0 x 3-0 cm). Without distur- 
bing the mating process and reared separately and observations on the oviposition 
behaviour and fecundity were made. 

3. Results 

The ovaries which lie ventrolateral to the hind gut in oviparous females, extend from 
seventh or eighth abdominal segment to third or fourth segment, and from first or 
second abdominal segment and extending upto the prothorax, in view of the very 
long oviduct in ovovivipartms as well as viviparous forms. The lateral and common 
oviducts of the oviparous female is short with 4-8 fully developed oocytes laden with 
yolk, and never retained in the lateral oviducts, the subsequent development taking 
place after oviposition. Individuals with the ovoviviparous ovary have 2-4 oocytes 
partly laden with the yolk, developing in the long lateral oviducts only upto blasto- 
kinesis. Eggs laid in this partly developed condition hatch after a very short 
incubation period. All the 4 basal oocytes mature simultaneously without any yolk 
accumulation in the viviparous ovary. The lateral oviducts are extremely long in 
viviparous forms, the complete development of the pre-vitellogenic oocytes taking 
place in the oviducts, followed by larviposition. 



Ovarian polymorphism in some sporophagous species 3 

The dimensions of the mature basal oocytes of the oviparous ovarioles are 3-5 and 
6-10 times respectively greater than in the ovo viviparous and viviparous conditions 
indicating the amount of yolk deposition. The oviduct of ovoviviparous and vivi- 
parous individuals are 2-10 and 2-20 fold respectively longer than those of the 
oviparous individuals. Enormously long lateral oviducts facilitate the retention of the 
partly vitellogenic oocytes as in ovoviviparous forms till blastokinesis and the 
previtellogenic oocytes as in viviparous forms, till they complete their development 
within the lateral oviducts. The number of oocytes undergoing subsequent develop- 
ment in the lateral oviducts ranges from 9-11 and 5-12 in ovoviviparous and vivi- 
parous ovaries respectively. 



3.1 Histology of oviparous ovarioles 

The terminal filament, germarium, pre-vitellogenic zone with pre-vitellogenic 
oocytes, vitellogenic zone with vitellogenic oocytes, post-vitellogenic zone with fully 
mature ovum and pedicel or calyx opening into the lateral oviduct are typical of 
oviparous ovarioles. The post-vitellogenic zone is absent in the ovoviviparous forms 
with the vitellogenic zone shorter resulting in the shorter length of the ovariole. In 
the viviparous ovarioles only 3 regions are evident viz (i) terminal filament, (ii) ger- 
marium and (iii) pre-vitellogenic zone. Unlike oviparous ovarioles, viviparous 
ovarioles lack the vitellogenic and post-vitellogenic zone. As a result the pre-vitello- 
genic oocytes are ovulated as such, without any yolk accumulation. 

The distal part of the gentnarium of the 4 ovarioles on each side in the oviparous 
individuals fuse together forming one coalescent germarium which tapers smoothly 
with elongated epithelial cells to form the terminal filament attached to the salivary 
glands. Oogonial cells of the germarium are covered by an external sheath and an 
internal tunica propria. Considerable differences do not occur in the number of 
oogonia among oviparous, ovoviviparous and viviparous ovarioles and generally 
the number of oogonia ranges from 8-14 in each ovariole. Each oogonium is a small 
spherical cell with a distinct nucleus and peripheral cytoplasm; however, the oogonia 
lack clear-cut demarcation from each other, these undifferentiated oogonia, mature 
and become fully developed by the time they reach the proximal region as they 
descend down the ovariole. As the oogonia descend down the pre-vitellogenic zone 
they have a squarish or rectangular profile with a centrally placed nucleus, occupying 
most of the oocyte. These pre-vitellogenic oocytes are oriented in a linear fashion 
and surrounded by a thin layer of follicular epithelial cells. As the pre-vitellogenic 
oocytes descend down, they enter the vitellogenic zone, in which continuous yolk 
deposition occurs and resulting in the oocytes with a solid, centrally located nucleus, 
increasing in dimension followed by the stretching of the follicular epithelium so as 
to completely cover the oocyte. It is a major zone occupying more than half the 
entire length of both oviparous and ovoviviparous ovarioles. The post-vitellogenic 
zone is characterised by the proximal, well-developed oocytes with adequate yolk 
reserves, each covered by an inner vitelline membrane. The interfollicular epithelial 
cells are very prominent in between the basal vitellogenic oocytes and the most 
proximal post-vitellogenic ovum. The proximal end of each ovariole opens into the 
distal end of the lateral oviduct through the pedicel, the lumen of which is filled by 
epithelial plug that is removed only during the first ovulation (figure 1). 



4 K Dhileepan and T N Ananthakrishnan 

3.2 Histology of ovoviviparous and viviparous ovarioles 

[n the ovoviviparous ovarioles, due to the absence of the post-vitellogenic zone and 
the relatively shorter vitellogenic zone, the ovariole length is comparatively shorter. 
The terminal oocytes at the proximal end of the ovarioles do not attain the full 
dimension as in oviparous basal oocytes indicating partial yolk accumulation. The 
basal oocyte is oval and externally surrounded by the follicular epithelial cells with a 
distinct, proximally placed nucleus. 

The germarium and the pre-vitellogenic oocytes in viviparous ovarioles are almost 
identical to those of the oviparous and ovoviviparous ovarioles. Due to the absence 
of both the post-vitellogenic and vitellogenic zones, the ovarioles of viviparous 
ovaries are very much shorter. As the primary oogonia descend down into the pre- 
vitellogenic zone, they are spherical and externally covered only with ovariole sheath 
with a follicular epithelial covering. The pre-vitellogenic oocytes, as they descend 
increased moderately in their dimensions and have a large centrally placed nucleus 
with a very prominent compact centrally placed nucleolus (figure 2). Similarly the 
most proximal oocytes possess densely stained ooplasm and open into the lateral 
oviduct through a short pedicel. Since both vitellogenic and post-vitellogenic zones 
which form two-thirds of the entire length of the oviparous ovarioles are lacking, the 
viviparous ovarioles are 2-3 times shorter than the oviparous ones. The pre- 
vitellogenic oocytes without any traces of yolk ovulate into the lateral oviducts, 
where the subsequent .embryogenesis takes place during which time they derive 
nutrients from maternal resource. 

3.3 Numerical variations of oocytes in polymorphic ovaries 

Considerable variations occur in the number of vitellogenic oocytes that are in the 
process of accumulating yolk in the ovarioles, post-vitellogenic oocytes (the most 
basal ovum fully laden with yolk in the ovariole), and the number of oocytes or 
embryos undergoing subsequent development in the lateral oviducts. 

Studies on 5 species of sporophagous Tubulifera viz, B. idolomorphus, T. subramanii, 
E. procer, E. denticollis and Ethirothrips agasthya (Bagnall) indicate that the number 
rf oogonial cells and the pre-vitellogenic oocytes does not differ among polymorphic 
) varies, ranging from 46-59 and from 11-17 in each ovary. However, the number of 
/itellogenic oocytes in each ovary is higher (10-12/ovary) in oviparous forms, 
comparatively less (6-9/ovary) in ovoviviparous forms and are lacking in the 
Oviparous ovaries. Mature ova are evident only in the oviparous forms (3-4/ovary). 
fhe number of embryos undergoing development in each lateral oviduct is higher in 
viviparous ovaries (10-12/each lateral oviduct) than in ovoviviparous ovaries 
6-10/each lateral oviduct). A positive correlation is evident between the number of 
>ocytes/embryos developing in the lateral oviduct and the length of the lateral 
>viduct, in which the latter increases proportionately with increase in the number of 
ieveloping embryos. 

-.4 Histology of oviducts 

listological studies of both lateral and common oviducts of T. subramanii of 
viparous ovaries, revealed the presence of single layer of secretory columnar 



Ovarian polymorphism in some sporophagous species 




D 



Figure 1. A. Oviparous ovary of Bactrothrips idolomorphus (x 35). B. L. S. of lateral 
oviduct showing developing embryos (xJ25). C. Pre-vitellogenic oocytes (xlOOO). 
D. Spherical basal pre-vitellogenic oocytes ( x 780). 



K Dhileepanand TN Ananthakrishnan 




Figure 2. Histology of oviparous ovary of T. subramanii. A. L. S. of the oviparous ovary 
( x 300). B. Pre-vitellogenic oocytes ( x 1250). C. Early vitellogenic oocytes ( x 600). 
D. Vitellogenic oocytes ( x 60(5). E. L. S. of lateral oviduct ( x 300). F. L. S. of common 
oviduct ( x 360). 



epithelial cells resting on a basement membrane. These secretory epithelial cells are 
apocrine in nature with a very large centrally placed nucleus and a prominent, but 
eccentrically placed nucleus. Varying number of vacuoles are evident near the 
basement membrane. At the distal free margins of the secretory cells, a large number 
of secretory vesicles occur. The presence of secretory granules, dense cytoplasm and 
numerous vacuoles suggest the secretory nature of the epithelial cells. 

The lateral oviducts of ovoviviparous ovaries exhibit secretory cells only at the 
proximal one-third of the lateral oviducts, while the rest of the distal two-thirds 
region lack secretory cells. In the lateral oviducts of the ovoviviparous ovaries, the 



Ovarian polymorphism in some sporophagous species 1 

secretory regions at the proximal end of the oviducts are highly variable in length, 
depending upon the number of oocytes/embryos undergoing development in these 
oviducts, as well as on the degree of the ovoviviparity, especially the gestation period 
of the embryos and the stage at which the embryos are laid. In ovoviviparous 
oviducts the secretory cells lining the proximal region of the lateral oviduct and 
common oviduct are tall, columnar with centrally placed nucleus and are apocrine. 
The secretory nature of the cells are evident only in the lateral oviducts where the 
embryos are in an advanced stage of development. In those lateral oviducts with 
embryos in the early stage of development, the cells are inactive with dense 
cytoplasm and without any secretory granules. With the ovulation of the mature 
ovum with partly deposited yolk, the subsequent development of the oocyte takes 
place in the distal region of the lateral oviducts which are devoid of any secretory 
cells. In the viviparous ovaries both the lateral and common oviducts do not have 
any secretory cells and consist only of the basement membrane. 

3.5 Histochemistry of polymorphic ovaries 

Histochemical studies involving total proteins, acidic proteins, basic proteins, 
glycogen and lipids indicated the diversity in yolk deposition in polymorphic ovaries. 
Pre-vitellogenic oocytes in oviparous, ovoviviparous and viviparous ovaries show a 
protein positive reaction, where acidic proteins are abundant in the nucleus and 
perinuclear cytoplasm. Pre-vitellogenic oocytes of all the 3 types of ovaries showed a 
negative reaction to the glycogen and a weak lipid reaction. In the vitellogenic 
oocytes of both oviparous and ovoviviparous forms the intensity of protein and 
lipids increase both in nucleus and in ooplasm (figures 3 and 4). 

Histochemical observations of the yolk granules of polymorphic ovaries indicate 
the incidence of increased total proteins as well as both basic and acidic protein yolk 
granules evenly dispersed in the ooplasm of the vitellogenic and post-vitellogenic 
oocytes. In the oviparous ovarioles the protein yolk granules, especially the acidic 
ones are more dense than the basic protein granules. In addition, both glycogen and 
lipid yolk granules are evenly distributed. In the vitellogenic oocytes of 
ovoviviparous ovarioles the glycogen granules are more abundant than in the ovi- 
parous vitellogenic oocytes. Moreover the ooplasm also has protein and lipid yolk 
evenly distributed in the peripheral ooplasm. In viviparous ovarioles with only pre- 
vitellogenic oocytes no yolk granules were evident, indicating the absence of yolk 
deposition. 

3.6 Embryogenesis in polymorphic ovaries 

In oviparous ovaries the fully mature eggs with yolk reserves are laid with a 
protective chorion and the embryogenesis takes place only after they are laid. In the 
ovoviviparous ovaries, mature oocytes in partly yolk-accumulated condition are 
ovulated into the lateral ovidiict, where embryogenesis continues upto blastokinesis. 
There is a positive correlation between the increase in the size of the embryos in the 
lateral oviducts and the distance traversed by the embryos in the lateral oviducts. 
This correlation suggests that there is a quantitative increase in the size of the 
embryo as it descends down the lateral oviducts, indirectly indicating the nutrient 



K Dhileepan and T N Ananthakrishnan 



B 




Figure 3. Histochemistry of polymorphic ovaries of T. subramanii. A. L. S. of oviparous 
basal oocyte showing positive reaction to total proteins ( x 400). B. Dense acidic protein 
evenly distributed in the ooplasm of oviparous basal oocytes ( x 400). C. Oviparous basal 
oocyte showing basic protein granules ( x 350). D. Ovoviviparous basal oocyte showing 
basic protein positive reaction ( x 400). 



Ovarian polymorphism in some sporophagous species 




Figure 4. Histochemistry of polymorphic ovaries of T. subramanii. A. Oviparous basal 
oocyte showing glycogen spheres ( x 650). B. Ovoviviparous basal oocyte showing acidic 
protein positive reaction ( x 400). C L. S. of ovoviviparous embryo in the lateral oviduct 
with glycogen granules ( x 300). D. Ovoviviparous basal oocyte with lipid yolk granules 
( x 350). E. Oviparous basal oocyte showing protein yolk granules ( x 350). 



10 K Dhileepan and T N Ananthakrishnan 

intake from maternal resource. The remaining embryonic development takes place 
after they are laid. In viviparous ovaries the pre-vitellogenic oocytes without any 
yolk reserves are ovulated into the lateral oviduct where the complete embryonic 
ievelopment takes place and fully developed larvae are laid as such. Histology of the 
lateral oviducts with developing embryos whose stage of development increased 
towards the distal most region of the lateral oviduct which opens into the common 
oviducts. Embryos at the proximal end of the lateral oviduct are only at initial stage 
of embryogenesis and smaller in size. As they move down, there are large number of 
embryos at various developmental stages. Those at the distal end of the lateral 
oviduct or in the common oviducts are in the stage of completing the embryogenesis 
or as fully formed larvae. Since the lateral oviducts of the viviparous ovaries contain 
many embryos, histological studies revealed all the stages of embryonic development 
in a sequence. Further there is a significant, proportionate increase in the size of the 
embryos as they descend down in the lateral oviduct. 



4. Discussion 

Both the ovoviviparous and viviparous ovaries differ from each other, as well as from 
oviparous ovaries in structure and function. Statistical analysis of 5 species of diversely 
reproducing idolothripines like B. idolomorphus, T. subramanii, E. procer, E. denticollis 
and M. menoni indicated a significant reduction in the ovarioles of both ovoviviparous 
and viviparous forms with corresponding elongation of the lateral oviducts. The 
oviparous ovarioles are longer due to long vitellogenic zone with post-vitellogenic 
zone containing larger basal oocytes. The viviparous ovarioles are shorter due to the 
absence of vitellarium. However, in ovoviviparous ovarioles the vitellarium is short 
with partly developed basal oocytes, resulting in shorter size. Since the vitellarium 
occupies 2/3 of the entire ovariole, their absence or reduction significantly alters the 
ovariole length. Similar reduction of vitellarium was also reported in ovoviviparous 
and viviparous Blattaria (Roth 1964). 

The enormously long lateral oviducts facilitate the retention and nourishment of 
the embryos. The elongation is pronounced as early as the pupal stage when 
ovulation has not yet commenced. This increased length of the lateral oviducts is due 
to continuous ovulation and retention of oocytes/embryos. This is supported by the 
positive correlation between the increase in the lateral oviduct length and increase in 
the number of embryos in the lateral oviduct. Bournier (1957, 1962, 1966) noticed the 
retention of eggs in the long lateral oviducts of ovoviviparous B. buffai, but their 
structural adaptations in relation to egg retention were not revealed. Similar long 
lateral oviducts retaining embryos were also reported in ovoviviparous T. subramanii 
'Viswanathan and Ananthakrishnan 1973) and B. brevitubus (Haga 1975). 

As a result of structural variations among polymorphic ovaries their relative 
position in the females were also considerably altered. In both ovoviviparous and 
viviparous forms the elongated lateral oviducts resulted in the shifting of the 
ovarioles towards the anterior side of the abdomen, near the metathorax. In 
oviparous ovaries, due to the short lateral oviducts, the long ovarioles emerged from 
the posterior portion of the abdomen and extended upto metathorax. Haga (1975) has 
also mentioned about the relative position of ovaries of the oviparous and 
ovoviviparous B. brevitubus. 



Ovarian polymorphism in some sporophagous species 1 1 

Both ovoviviparous basal oocytes with partial yolk deposition and viviparous 
basal oocytes without any yolk are not covered with chorion, before ovulation. John 
(1923) also reported in M. lativentris the absence of chorion when larvae were laid 
and the presence of a distinct chorion when the insects laid eggs. A similar 
phenomenon is also observed in the polyctenid Hesperoctenes fumarius West wood 
and several other viviparous species (Hagan 1951). The most important role of 
follicular epithelium during insect oogenesis has been considered to be synthesis and 
deposition of egg envelope during choriogenesis (Mahowald 1972). However, in the 
gall midge Heteropeza pygmaea Winnertz, which exhibits paedogenesis, the chorion 
is not formed at the end of oogenesis and the growing embryos remain enveloped by 
the folliculae epithelium (Junquera 1983). Hansen (1894) and Heymons (1909, 1912) 
noticed that the eggs of viviparous Hemimerus taploides Walker has no chorion and 
contains little yolk and the thickened follicular epithelium provides nutrients for the 
growing embryo. 

4.1 Numerical variation of oocytes in polymorphic ovaries 

In addition to anatomical and histological variations, numerical variation of oocytes 
and embryos in the polymorphic ovaries also occur. The higher number of 
vitellogenic and post-vitellogenic oocytes in oviparous forms and comparatively 
lesser number of only vitellogenic oocytes in ovoviviparous forms appear to be due 
to the long vitellarium as in oviparous forms and shorter in ovoviviparous species. 
Incidence of post-vitellogenic ovum only in oviparous forms suggests that complete 
maturation of the ovum occurs only in oviparous forms. Correspondingly the 
number of embryos in the lateral oviducts of the viviparous ovaries are higher than in 
ovoviviparous ovaries. This is because of the prolonged gestation period of the 
viviparous embryos than the ovoviviparous embryos. Haga (1974) reported as many 
as 24 embryos, 1 1 in the right and 1 3 in the left lateral oviducts of B. brevitubus. As 
more number of pre-vitellogenic oocytes undergo vitellogenesis to become 
vitellogenic oocytes, the number of pre-vitellogenic oocytes are retained as such 
resulting in their increased numbers. Moreover the frequency of ovulation of pre- 
vitellogenic oocytes is low in viviparous forms, as compared to the ovulation of 
mature ova and partly developed ova in oviparous and ovoviviparous ovaries 
respectively. 

4.2 Histochemistry of polymorphic ovaries 

Histochemical analysis revealed no yolk granules in basal oocytes of viviparous 
ovarioles indicating the absence of vitellogenesis. On the contrary in both 
ovoviviparous and oviparous ovaries, protein, lipid and glycogen yolk granules were 
evident substantiating the occurrence of vitellogenesis. Bonhag (1958) reported the 
incidence of protein, lipid and glycogen particles as the main deutoplasmic 
substances of oocytes, where the protein yolk are numerous while the glycogen may 
not be present in all insect eggs. The intensity of these yolk granules was dense in 
oviparous forms, and less dense in ovoviviparous basal oocytes. Though the 
incidence of glycogen yolk granules is not very common among insects (Bonhag 
1958), present observations indicate the presence of glycogen granules in both 



12 K Dhileepan and T N Ananthakrishnan, 

oviparous and ovoviviparous oocytes, their intensity being higher in the latter. 
Instances of glycogen deposition in eggs during vitellogenesis are reported in 
Anoplura (Ries 1932), Bumble bees and Anisolabis sp. (Bonhag 1958). 

Structural and functional modifications involving the histology of the lateral and 
common oviducts, to suit the type of reproduction, vary among polymorphic ovaries. 
In both complete ovoviviparous and viviparous ovaries the lateral oviduct wall is 
without any secretory cells and are stretched into a thin membrane. The developing 
embryos without chorionic covering lie close to the wall of the lateral oviducts and 
derive nutrients through thin membranous part. Haga (1975) also reported similar 
thin and transparent lateral and common oviduct walls in ovoviviparous B. hrevitubus. 
Thus, the absence of secretory cells facilitates the embryonic nourishment through 
the lateral oviduct walls. However, in partial ovoviviparous ovaries, the secretory 
cells at the distal end of the lateral oviducts alone are concerned with the embryonic 
nourishment, whereas those at the proximal secretory region are responsible for the 
chorion secretion, for the eggs that are laid. Thus the length of the secretory region of 
the oviduct is inversely proportional to the stage of the embryonic development at 
the time of laying. Greater the secretory region, earlier is the stage of the embryo laid, 
and when the secretory cells are absent, fully developed larvae are laid. Though in 
general, the chorion is secreted by the follicular epithelium in the ovariole 
(Mahowald 1972), in both ovoviviparous and viviparous ovaries presently observed, 
the oocytes that are ovulated are without chorion. Since only embryonated eggs are 
laid in ovoviviparous forms, it is assumed that the chorion might be secreted by the 
cells lining the lateral and common oviducts. However, in viviparous forms showing 
direct larviposition secretory cells are absent. 

References 

Ananthakrishnan T N, Padmanaban B and Dhileepan K 1983a Gut spore composition and the influence 

of fungal host on the rate of mortality and post-embryonic development in Tiarothrips subramanii 

(Ramk.); Proc. Indian Acad. Sci. (Anim. Sci.) 92 11-18 
Ananthakrishnan T N, Dhileepan K and Padmanaban B 1983b Reproductive strategies and behavioural 

attributes in some sporophagous Idolothripinae (Tubulifera : Thysanoptera); Proc. Indian Acad. Sci. 

(Anim. Sd.) 92 95-1 08 
Ananthakrishnan T N, Padmanaban B,'Dhileepan K and Suresh G 1984 Ecological interactions, species 

dynamics and reproductive biology of r some mycophagous Thysanoptera,; Occt. Publ. No. 5, 

Entomology Res. Inst.^ Loyola College, Madras, p 37 
Ananthakrishnan T N and Dhileepan K 1984 Thrips-Fungus association with special reference to the 

sporophagous Bactrothrips idolomorphus (Karny) (Tubulifera : Thysanoptera); Proc. Indian Acad. Sci. 

(Anim. Sci.) 93 243-249 
Bagnall R S 1921 On Thysanoptera from the Seychelles Islands and Rodrigues; Ann. Mag. Nat. Hist. 1 

257-293 

Bonhag 1958 Ovarian Structure and Vitellogenesis in Insects; Ann. Rev. Entomol. 3 137-160 
Bournier A 1951 Entomologie un deuxieme cas.d' ovoviviparite chez les Thysanopteres Caudothrips\ 

buffai (Megathripinae) Exhait de; C. R. Seances Acad. Sci. Roum. 506-508 
Bournier A 1956 Contribution a 1' etude de la parthenogenise des thysanopteres et de sa cytologie; Arch. 

Zool Exp. Gen. 93 135-141 
Bournier A 1957 Un deuxieme cas d' ovoviviparite chez les Thysanopteres Caudothrips buffai Karny; C. R. 

Acad. Sci. 244 506-508 
Bournier A 1962 L' appareil general femelle de Caudothrips buffai Karnet et sa Pumpa Spermatique; Ann. 

Soc. Entomol. Fr. (N. S.) 67 203-207 
Bournier A 1966 L' Embryogenese de Caudothrips buffai Karny (Thysanoptera : Tubulifera); Ann. Soc. 

Entomol. Fr. (N. S.) 2 



Ovarian polymorphism in some sporophagous species 1 3 

Haga K 1974 Post-embryonic development of Megathripine species Bactrothrips brevitubus 

(Thysanoptera : Insecta). Bull. Sugalaira Biol. Lab. Tokyo, Kyoto Univ. No. 611-31 
Haga K 1975 Female reproductive systems of Megathripine species Bactrothrips brevitubus 

(Thysanoptera : Insecta); Bull. Suyalaira. Biol. Lab. Tokyo, Kyoto Univ. No. 7 13-24 
Hagan H R 1951 Embryology of viviparous insects; (New York: The Ronald Press Company) 
Hansen H J 1894 On the structure and habits of Hemimerus talpoides Walk.; Entomol Tidskr. 15 65-93 
Heymons R 1909 Fine Plazenta bei cinern insekt (Hemimerus); Varhandl d. deut. Zoo/. Gesellsch. 19 97- 

107 
Heymons R 1912 Uber den genital apparat und die Entwicklung von Hemimerus talpoides Walk.; Zoo/. 

Jahrb. Suppl. 15 141-184 

Hood J D 1934 New Thysanoptera from Panama; J. N. Y. Entomol. Soc. 41 407-434 
Hood J'D 1935 A note on Heterogony in the Thysanoptera with description of two new species from 

Tanganyika; Stylops 4 193-201 

Hood J D 1950 Thrips that Talk; Proc. Entomol. Soc. Washington 52 42-43 

John O 1923 Fakultative viviparat bei Thysanopteran; Entomol. Mitt. Zoo/. Mus. Hambarg 12 227-232 
Junquera P 1983 The role of follicular epithelium in-growing eggs of a Dipteran insect during late 

oogenesis and devage; J. Morphol. 178 303-312 

Kiester A R and Strates E 1984 Social behaviour in a Thrips from Panama; J. Nat. Hist. 18 303-314 
Mahowald A P 1972 Oogenesis; in Developmental systems: Insects (eds) S J Counce and C H Waddington 

(New York: Academic Press) Vol. 1 1, pp 1-47 

Pearse ACE 1968 Histochemistry: Theoretical and applied. 3rd edition (J A Churchill Ltd) Vol. I, p 758 
Ries E 1932 Die prozesse der eibildung und des eiwashstums bei pediculiden und mallophagen; Z. Zellforsch. 

Mikrosk. Anat. 16 314-388 
Roth L M 1964 Control of reproduction in female cockroaches with special reference to Nauphoeta 

cinerea I. Pre-oviposition period; J. Insect Physiol. 10 915-945 
Viswanathan and Ananthakrishnan T N 1973 On partial ovoviviparity in Tiarothrips subramanii (Ramk.) 

(Thysanoptera); Curr. ScL 42 649-650 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 1, January 1987, pp. 15-21. 
(C) Printed in India. 



Host preferences of some acridids (Insecta: Orthoptera) In relation to 
some biochemical parameters 

K P SANJAYAN and T N ANANTHAKRISHNAN 

Entomology Research Institute, Loyola College, Madras 600 034, India 

MS received 9 October 1986 

Abstract. Biochemical analysis of host plants of 6 species of acridids for their total protein, 
carbohydrates, phenols, free aminoacids, water and nitrogen content revealed significant 
correlation in regard to their host preferences, wherein the leaf water-nitrogen index as well 
as the total phenol composition appeared to play an important role. 

Keywords. Biochemical parameters; host preferences; acridids. 



1. Introduction 

The presence or absence of phagostimulants/deterrents in the right proportion to 
facilitate optimal food utilization is of significance in host selection in acridids 
(Mulkern 1967). Less pre'ferred/non-host plants exhibit varied resistance mechanisms 
in terms of such chemical parameters as their total nitrogen, protein, aminoacids and 
phenols (Chapman and Bernays 1977; Miller and Strickler 1984). Specialist and 
generalist feeders are equally common among acridids, some exclusively 
graminaceous feeders, others only on dicots and yet others on both (mixed feeders). 
In terms of the quantitative intake of leaves which influences the biotic potential of 
the insect (Ananthakrishnan et al 1985a,b), an analysis of the chemical factors of the 
host plants appear relevant for a basic understanding of the nature of host prefe- 
rences of such acridids as Oxya nitidula (Walker) (Oxyinae), Truxalis indica Boliver 
(Truxallnae), Orthacris maindroni Boliver (Pyrgomorphidae), Atractomorpha 
crenulata (Fabricius) (Pyrgomorphidae), Aiolopus thalassinus (Fabricius) (Oedipo- 
dinae) and Cyrtacanthacris ranacea Stoll (Cyrtacanthacridinae). 



2. Materials and methods 

Different species of acridids were collected from the fields of Oryza saliva, Panicum 
maximum and Gossypium hirsutum and mass reared in the laboratory on the various 
host plants in wooden cages measuring 25 x 25 x 30 cm. The duration of develop- 
ment of the acridids on the various host plants was observed and this was taken as 
an index for the host preferences of the species. 

The host leaves were subjected to biochemical estimation for the total proteins 
(Lowry et al 1951), carbohydrates (Dubois et al 1965), nitrogen (Vogel 1963), phenol 
(Hori 1974) and total free amino acids (Moore and Stein 1948). The moisture content 
was estimated by weighing accurately 2 g of the sample and incubating at 37C till a 
constant weight was obtained. The difference in weight was calculated and the 
moisture content represented as percentage. 

15 



16 K P Sanjayan and T N Ananthakrishnan 

3. Results 

Food preference in terms of the rate of development on various host plants indicated 
faster development when reared on the preferred host, the quickest rate being that of 
A. crenulata when fed on Ricinus communis (37-7 5-9 days), and the slowest when fed 
on Solanum torvum (994 10-7 days). In the case of 0. maindroni the longest nymphal 
duration was observed when fed on Clerodendron sp and shortest on Dolichos 
lablab. Faster post embryonic development of C. ranacea occurred when fed on 
Gossypium hirsutum (50-0 0-94 days) as against the malvaceous weed Abutilon 
indicum (62-0 db 1-66 days). Panicum maximum appeared to facilitate faster nymphal 
development of Aiolopus thalassinus and T. indica and Cyperus rotundus for 0. 
nitidula (Ananthakrishnan etal 1985a,b). 

0. nitidula feeds on poaceous and cyperaceous weeds and graminaceous crops 
avoiding dicotyledons. Likewise, Truxalis indica and A. thalassinus feed exclusively 
on graminaceous plants, whereas A. crenulata is a mixed feeder, feeding both on 
monocots and dicots. C. ranacea was observed to have a comparatively narrow 
feeding range, specifically confined to feeding on malvaceous plants thereby earning 
the 'specialist' status. Individuals of 0. maindroni feed on a wide spectrum of dicots 
exhibiting greater preference towards R. communis and to a lesser extent on D. lablab. 

Quantitative biochemical analysis of the leaves of the host plants in terms of water, 
nitrogen, proteins, carbohydrates, aminoacids, phenols and silica content revealed 
striking correlation with the food preference of these acridid species (table 1). 
Acridids feeding exclusively on monocots generally preferred host with low water 
content with the exception of A. thalassinus. The dicot as well as mixed feeders pre- 
ferred hosts with high water content. An estimation of the water content of the host 
plants of 0. nitidula indicated that P. maximum, one of the natural hosts with higher 
water content (77-29%) to be the least preferred. Though the water content of C. 
rotundus was less than that of P. maximum, the leaves of the former were readily 
consumed by all the stages of 0. nitidula. Although the percentage of water in 
0. saliva (60-77%) and C. dactylon (61-97%) was nearly the same, C. dactylon was 
preferred to 0. saliva, presumably due to the effect of the other chemical stimulants of 
the host. Similarly for T. indica, the most preferred host C. dactylon had a lesser 
water content A. crenulata and 0. maindroni preferred D. lablab with a higher water 
content (83%). For the specialist, C. ranacea, Abelmoschus esculentus with a higher 
water content (80-49%) was preferred less to G. hirsutum. Although the susceptibility 
of the host to insect attack is known to depend on the nitrogen content of the plant, 
the water-nitrogen ratio provided a better insight of the resistance of plants to 
acridids, showing a distinct correlation of their relative preference with the water- 
nitrogen index of the host, preferring the host with a low index. However, the mixed 
feeder, A. crenulata and T. indica preferred hosts with a high water-nitrogen index 
(figure 1). 

As the total free amino acid content of the monocots is quantitatively lower than 
the dicots, an analysis of the comparative feeding preferences of the acridids indi- 
cated a preference for plants with higher amino acid content. The monocot feeding 
acridids generally showed a greater preference for feeding on higher amino acid 
content plants. All the acridids studied here appeared to show greater preference 
to leaves with high carbohydrate content, proteins being generally known to be 
essential for the growth and development of the insect. A comparative assessment of 



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Figure 1. Comparative preference of different acridid species in relation to total phenols 
and water-nitrogen index of host plants. 



the host plants for the total protein content indicated that the mixed as well as dicot 
feeders preferred hosts with high proteins, even though the monocots feeders were 
capable of thriving on plants with low protein content. The phenolic compounds are 
considered to act as deterrents to feeding in grasshoppers is evident in that the most 
preferred host plants of 0. nitidula, A. crenulata and 0. maindroni seemed to have a 
lesser phenol content as compared with the lesser preferred hosts (figure 1). G. 
hirsutum, the preferred host of C. ranacea has a less phenol content (232-5 mg/g) in 
comparison to A. esculentus (245-0 mg/g). However, a reverse trend was observed in 
the case of A. thalassinus and T. indica, which preferred hosts with higher phenol 



Host preferences of acridids 1 9 

content and this is presumably be due to their capacity to convert phenols into 
phenylalanine as proposed for Schistocerca gregaria by Bernays and Woodhead 
(1982). 

4. Discussion 

The choice of plants by the acridids seem to reveal that these may be based on the 
absence of feeding deterrents or their presence in smaller amounts in the host plants. 
For instance 0. nitidula totally rejected all forbes, perhaps due to the presence of one 
or more compound deterrent to the graminivore (Bernays and Chapman 1977). 
General selection of food by an adult acridid appears to have a greater chemical 
basis (Bernays and Chapman 1970). 0. nitidula, T. indica and A. thalassinus are found 
to select and feed constantly on the mojnocots only. This is possibly due to the 
favourable sensory inputs from the sensilla of the mouth parts and preoral cavity 
(Haskell and Schoonhoven 1969). Though the feeding activity could be initiated by 
the sensory receptors of the insect, the activation of these receptors requires a 
stimulatory factor to evoke normal feeding behaviour. The leaf surface chemicals 
play an important role in determining the feeding activity and acridids have been 
known to identify the phagostimulatory ones from the deterrents by palpation and 
recognise the leaf form from the leaf surface attributes (Chapman 1977). 

Some hosts are rejected because of the presence of chemicals deterrent to feeding 
and such deterrent chemicals seem to play an important role in the feeding 
behaviour of acridids. Further, some secondary compounds that are deterrent for 
Locusta are ineffective for Schistocerca and some which are deterrent at high 
concentrations stimulate feeding at lower concentrations (Bernays and Chapman 
1978). The substances which may stimulate feeding are specific and the occurrence of 
suitable phagostimulants may, in part, effect feeding. Some amino acids (proline and 
serine), hexose sugar and disaccharides are found to be phagostimulatory for 
L. migratoria (Cook 1977). Acridid species have evolved a diversity to recognise only 
small number of phagostimulants rather than a large number of feeding inhibitors. 
Hence feeding depends on the balance between the phagostimulants and the feeding 
deterrents and the response varies in different species so that a plant may be 
acceptable to some, but not to other species, or the rejection of hosts by these insects 
may be owing to the presence of one or more chemicals in amounts which inhibit 
feeding; some host plants are accepted only because of the absence of deterrent 
chemicals in sufficient quantities to limit feeding (Bernays and Chapman 1977; 
Bernays 1978). 

Acridids are generally observed to take larger meal on lush grass with high water 
content (Chapman and Bernays 1977). Although Bernays and Chapman (1970) felt 
that moisture content may not play any part in the food selection of Chortippus 
parallelus, it cannot be ignored in the acridids studied here as the food plant provide 
water in excess of the requirements of the insect. Scriber (1984) suggested that the 
post ingestional growth performance in phytophagous insects is determined to a 
large extent by plant chemical quality and can be indexed by leaf water-nitrogen 
composition. All the acridids studied here appeared to show greater preference for 
host plants with low water-nitrogen index. 

Total phenol content of the host plant appears to interfere with the food selection 
of the acridids. The high phenol content makes D. lablab, the least palatable, while 



20 K P Sanjayan and T N Ananthakrishnan 

R. communis with least phenol content is most preferred for A. crenulata. The feeding 
preferences of C. ranacea only attest the fact that food selection depends not so much 
on specific phagostimulants in the acceptable plants as in the presence of inhibitory 
chemicals in the non-acceptable plants (Thorsteinson 1960). A very high concen- 
tration of sugar usually limits the amount of food taken (Cook 1977). However, 
A. crenulata, 0. maindroni and T. indica appear to show greater preference for host 
with high sugar content. Hence, food selection of these acridids presumably involves, 
besides many physical and chemical factors, the balance between deterrent chemical 
and phagostimulants (Chapman and Bernays 1977), the inhibitory factors very often 
overriding the stimulatory ones. 



Acknowledgement 

This work was supported by a grant from the Department of Science and Techno- 
logy, New Delhi. 

References 

Ananthakrishnan T N, Dhileepan K and Padmanaban B 1985a Behavioural responses in terms of feeding 

and reproduction in some grasshoppers (Orthoptera: Insecta); Proc. Indian Acad. Sci. (Anim. Sci.) 94 

443-461 
Ananthakrishnan T N, Sanjayan K P and Suresh Kumar N 1985b Host plant preferences in 

Cyrtacanthacris ranacea Stoll in some malvaceous hosts in terms of food utilization; Proc. Indian Natl 

Sci. Acad. (in press) 

Bernays E A 1978 Tanin an alternative view point; Entomol. Exp. Appl. 24 244-253 
Bernays E A and Chapman R F 1970 Food selection by Chorthippus parallelus (Zetterstedt) (Orthoptera: 

Acrididae) an the field; J. Anim. Ecol. 39 383-394 
Bernays E A and Chapman R F 1977 Deterrent chemicals as a basis of oligophagy in Locusta migratoria 

(L); Ecol. Entomol. 2 1-18 
Bernays E A and Chapman R F 1978 Plant chemistry and a'cridid feeding behaviour; in Biochemical 

aspects of plant and animal co-evolution (ed) Harbone (Academic Press) pp 99-141 
Bernays E A and Woodhead S 1982 Plant phenols utilized as nutrients by a phytophagous insect; Science 

216 201-203 
Chapman R F 1964 The structure and wear of the mandibles in some African grasshoppers; Proc. Zool. 

Soc. London 142 107-121 
Chapman R F 1977 The role of the leaf surface in food selection by acridids and other insects; Colloq. Int. 

C. N. R. S. 265 133-149 
Chapman R F and Bernays E A 1977 The chemical resistance of plants t.o insect attack; Pontif. Acad. Sci. 

Script. Varia 41 603-643 
Cook A G 1977 Nutrient chemicals as phagostimulant for Locusta migratoria (L); Ecol. Entomol. 2 

113-121 
Dubois M, Gilles K A, Hamilton J K, ReberfrP A and Smith F 1956 Colorimetric determination of sugars 

and related substances; Anal. Chem. 28 351-356 
Haskell P T and Schoonhoven L M 1969 The function of certain mouthparts receptors in relation to 

feeding in Schistocerca gregaria and Locusta migratoria migratoria; Entomol. Exp. Appl. 12 423-440 
Hori K 1974 Studies on the feeding habits of Lygus disponsi Linn (Hemiptera : Miridae) and the injury to 

its host plant. V. Phenolic compounds, acid phosphatase and oxidative enzymes in artificially infested 

tissues of sugar beet leaf; Appl. Entomol. Zool. 9 225-230 
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurements with Folin Phenol 

reagent; J. Biol. Chem. 193 265-275 
Miller J R and Stockier K L 1984 Finding and Accepting host plants; in Chemical Ecology of Insects (ed) 

W J Bell and R T Carde (London: Chapman and Hall Ltd) pp 127-157 



Host preferences of acridids 21 

Moore S and Stein W H 1948 Photometric ninhydrin method for use in chromatography of amino acid; 

/. Biol Chem. 176 367-388 

Mulkern G B 1967 Food selection by grasshoppers; Annu. Rev. Entomoi 12 509-523 
Scriber J M 1984 Plant-herbivore relationships: Host plant acceptability; in The Chemical Ecology of 

Insects (ed) W Bell and R Garde (London: Chapman and Hall Ltd) pp 159-202 
Thorsteinson A J 1960 Host selection in phytophagous insects; Annu. Rev. Entomoi. 5 193-218 
Vogel I A 1963 Determination of nitrogen by KjeldahFs method; in A text book of quantitative inorganic 

analysis including elementary instrumental analysis, (ed) I A Vogel (London: ELBS) pp 256-257 



Proc. Indian Acad. Sci. (Anim. SdL), Vol. 96, No. 1, January 1987, pp. 23-32. 
Printed in India. 



Ecobehavioural and biological studies on two species of seed infesting 
thrips (Thysanoptera : Insecta) 

A BALU and A MOHAN DANIEL 

Entomology Research Institute, Loyola College, Madras 600 034, India 

MS received 11 August 1986; revised 9 October 1986 

Abstract. Ecobehavioural and biological aspects of two species of seedy infesting thrips, viz 
Chirothrips mexicanus Crawford and Chiridothrips indicus Ramk. and Marg. are discussed 
with particular reference to their reproductive biology, population dynamics on different 
host plants and their damage potential to seed production of their primary host plants. 

Keywords. Seed-infesting thrips; Chirothrips mexicanus; Chiridothrips indicus; damage 
potential. 

1. Introduction 

Loss in seed production due to thrips infestation on graminaceous plants is attri- 
buted to Chirothrips hamatus Trybom (Vappula 1965; Hukkinen 1936), Chirothrips 
mexicanus Crawford, (Roney 1949; Riherd.1954; Ananthakrishnan and Thirumalai 
1977, 1978), C. pallidicornis Priesner (Doull 1956) and C.falsus Priesner (Watts 1965). 
Frankliniella spp. (Bailey 1948) and Kakothrips robustus (Uzel) (Bhul 1973; Franssen 
1960) are also known to reduce seed production in Some dicots. Being seed infesting 
species, both C. mexicanus and Chiridothrips indicus Ramk. and Marg. considerably 
reduce seed production, controlling the propagation of the weed host. Results 
presented here attempt to discuss the reproductive biology of the species, their 
population trends on different host plants and their damage on seed production. 

2. Material and methods 

The inflorescence of Chloris barbata Sw., Eleusine aegyptiaca Desf. and Eleusine 
indica Gaertn. along with the adults and immature stages of C. mexicanus^ were 
collected in plastic bags and subsequently transferred to a collecting chamber comp- 
rising a chimney with a split cork at the bottom and with a polythene bag fitted with 
a rubber band at the top. The flower stalks were inserted through the split cork and 
the cut ends were kept in water in a wide mouthed bottle. The emerging adults that 
always moved upwards, were collected from the polythene bag using a camel hair 
brush (No. 0). 

The inflorescence of Achyranthes aspera L. along with the varying stages of 
C. indicus, a seed infesting tubuliferan were brought to the laboratory in a tightly 
closed polythene bag. After gentle tapping on to a white pad, the thrips were trans- 
ferred to a vial of 70% alcohol with a camel hair brush. For biological studies, the 
methods followed by Ananthakrishnan and Daniel (1981) were adopted. For popu- 
lation studies of C. indicus the dry count technique of delayed counting method as 
well as direct counting method (Irwin et al 1979) and for C. mexicanus the common 

23 



24 A Balu and A Mohan Daniel 

beating technique (Ananthakrishnan 1969) were followed. Population studies were 
carried out twice a month for a period of 3 years. For assessing the parthenogenetic 
reproduction of C. rnexicanus^ the male prepupae were removed and the female 
prepupae cultured on the 3 different host plants. In C. indicus mating was observed 
by releasing males and females in solidified, paraffin-filled petriplates with a shallow 
moat (Varadarasan 1979). In order to establish a possible relationship between the 
number of ovaries damaged by feeding and oviposition, the correlation coefficient V 
was computed. 

3. Observations 

3.1 Sexual dimorphism and parthenogenesis 

Females of C. rnexicanus are macropterous with an average life span of 17-2 days and 
reproduce both sexually and parthenogenetically. The smaller apterous males exhibit 
an average life span of 7-3 days. In C. indicus that reproduce only sexually, both the 
sexes are macropterous, and do not exhibit variation in their life span of 15-20 days. 
The number of eggs laid by C. rnexicanus vary with different host plants, fewer eggs 
resulting from parthenogenesis. On C. barbata, the parthenogenetic females lay on an 
average 26-0 3-62 eggs throughout their life span, the fecundity index (number of 
eggs laid/preoviposition period) being 7-77 1-64. On E. aegyptiaca and E. indica an 
individual female produces an average of 29-5 4-89 and 14-92 3-09 eggs, the fecun- 
dity index being 8-39 2-47 and 3-59 1-02 respectively (table 1). The preoviposition 
period in parthenogenetically reproducing individuals also varies on different host 
plants. 

3.2 Mating, oviposition and fecundity 

Mating of adult males with premature females (pupae) is noticed in C. rnexicanus 
(Ananthakrishnan and Daniel 1981) without subsequent mating after metamorphosis 
into adults. Males are polygamic and resort to multiple mating with several female 
pupae. After mating for 1-3 min the males move to new spikelet in search of another 

Table 1. Comparison of sexual and parthenogenetic reproduction of C. rnexicanus on 
different host plants. 



Host plant 




Life span of 
adults (days) 


Preoviposition 
period (days) 


Fecundity" 


Fecundity 
Index" 


Mortality' 


C. barbata 


S 
P 


21 -00 2-33 
17-20 2-01 


2-36 0-50 
3-50 0-52 


38-23 7-46 
26-00 3-62 


16-10 5-82 
7-77 1-64 


' 35% 


E. aegyptiaca 


S 
P 


24-82 3-10 
18-43 2-43 


2-40 0-50 
3-67 0-65 


40-96 5-87 
29-50 4-89 


17-78 4-41 
8-39 2-47 


23% 


E. indica 


S 
P 


18-801-71 
15-60 2-63 


3-08 0-62 
4-25 0-62 


27-24 5-75 
14-92 3-09 


9-44 2-86 
3-59 1-02 


58% 



Mean Standard deviation; S, Sexual reproduction; P, parthenogenetic reproduction. 

"Number of eggs laid during the life span; 6 Fecundity/preoviposition period; Total percentage of 

mortality from egg to adult. 



Studies on seed infesting thrips 25 

pupae. Already mated female pupae mate again with several males that visit the same 
spikelet. On the contrary, only fully matured adults of C. indicus mate for 20-50 s 
and their males are also polygamic. When pairing, the male grasps the pterothorax of 
the female with its forelegs, twists its abdomen around 180 beneath the female and 
moves its copulatory organ till it encounters the genitalia of the female. 

In C. mexicanus, fecundity is comparatively higher and the reproducing days are 
more in sexually reproducing individuals. In the laboratory, parthenogeneticaily and 
sexually reproducing females lay everyday 2-3 and 3-4 eggs respectively. While the 
total number of eggs laid by single sexually reproducing female is 38-23 7-46 on 
C. barbata, it is 40-96 5-86 on E. aegyptiaca and 27-24 5-75 on . indica, the fec- 
undity index (total number of eggs laid/preoviposition period) being 16-1 5-82, 
17-78 4-41 and 9442-86 respectively (table 1). C. indicus females oviposit among 
the floral parts of A. aspera, either within the corolla or calyx. Under laboratory 
conditions these females lay an average of 2-5 eggs per day, with a fecundity of 
56 3-05. The oval eggs of C. indicus, with hexagonal chorionic reticulations, 
measure 144-5 11-52 \JL wide and 384-l223-59 p long. 

3.3 Life cycle, sex ratio and mortality rates 

Studies on the post embryonic development of C. mexicanus were made during 
February, May, August and November on 3 different host plants, C. barbata, 
E. aegyptiaca and E. indica. While the time required for the completion of post- 
embryonic development is shorter in May in all the 3 host plants, the high fecundity 
and short life cycle in all the seasons is observed in these thrips on C. barbata than in 
the other host plants. Unlike C. mexicanus, C. indicus does not show seasonal vari- 
ation in the life cycle and the total duration ranges from 14-19 days, the duration of 
incubation for I larva, II larva, I pupa and II pupa being 2*57, 3-57, 4-71, 1-00, 2-28 
and 1-57 days respectively. Soon after hatching the I instar larvae move from flower 
to flower to feed on the ovarian tissues of the spikelets of A. aspera. After completion 
of the larval period they conceal themselves within the closed flowers and pupate 
(figure 1). The duration of each instar of C. mexicanus is provided in table 2. 

While the sex ratio (female : male) in C. mexicanus is 1:1 when inhabiting -C. barbata 
and E. aegyptiaca, it is 3 : 2 in E. indica. More males are produced during the months 
of May and August, the sex ratio being 1 : 4 in all the 3 host plants and fewer males 
are produced in November changing the 'sex ratio to 1:2. In the months of April and 
May, a higher population of males is evident in the field followed by a subsequent 
decline. Population analysis of C. indicus reveals that the females are often predo- 
minant and outnumber the males. The sex ratio which remained as 2:1 (female : 
male) throughout the study period does not get affected by any seasonal variation or 
biotic influence. 

Individual cultures of C. mexicanus on C. barbata, E. aegyptiaca and E. indica in- 
dicate a higher mortality on E. indica (58%) than on C. barbata (35%) and E. aegyptiaca 
(23%). In C. indicus which infest only A. aspera mortality rate is 28% (table 1). 

3.4 Population trends of C. mexicanus and C. indicus 

Under natural conditions though C. mexicanus infests all the 3 host plants, E. aegyptiaca 
alone harbours a large population throughout the period of occurrence. The popu- 



26 



A Balu and A Mohan Daniel 




Figure 1. A. Adults of C. indicus on the inflorescence of A. aspera. B. Eggs of C. indicus 
inside the flowers of A. aspera. C. Pupa of C. indicus inside the flower of A. aspera. 

D. Enlarged view of the eggs of C. indicus. E. An adult of C. mexicanus feeding on the 
ovary of C. barbata. F. Egg of C. mexicanus partially inserted into the ovary of 

E. aegyptiaca. G. Egg of C. mexicanus partially inserted into the ovary of C. barbata. 
e, Egg; o, Ovary. 



lation generally increases with an increase in temperature and decrease in humidity 
and in the absence of rainfall. While the population peak of C. mexicanus on C. barbata 
is completed by March, on E. indica and E. aegyptiaca infestation commences only 
by August and continues till May, the population of males increasing more than 5 
folds during March and April on all the 3 hosts. High temperature and low humi- 
dity during May initiates the decline of the population. Since the flowers of E. aegyptiaca 
and E. indica dry and wither by the end of May, the population of the thrips on these 
host plants declines. Even though the flowers of C. barbata are available throughout 
the year, the population of C. mexicanus disappears by December, but reappears on 
E. aegyptiaca and E. indica at a very low level. Rainfall profoundly affects the popu- 
lation of C. mexicanus on all the host plants (figure 2). 

C. indicus is highly seasonal occurring for about 8 months (October-April). Their 
incidence on the inflorescence of A. aspera is noticed by October and is maintained 



Studies on seed infesting thrips 

Table 2. Life-cycle of C. mexicanus on different host plants during different months. 



27 



Host plant 


Month 


Incubation* 


I instar* 


II instar* 


Prepupa* 


Pupa* 


Total* 


C. barbata 


Feb. 


5-8 0-79 


3-5 0-54 


740-51 


1-1 2 0-09 


3-5 0-54 


2l-394-ll 




May 


4-4 0-51 


2-5 0-54 


5-5 0-54 


0-88 0-1 3 


2-7 0-48 


15-98 0-95 




Aug. 


5-3 0-41 


34 0-51 


6-3 048 


0-96 0-03 


2-6 0-51 


1846H7 




Nov. 


7-5 0-64 


5-1 0-89 


7-4 0-51 


1-65 0-03 


3-8 0-79 


2545 2- 11 


E. indie a 


Feb. 


7-4 0-51 


4-5 0-54 


7-8 0-79 


1-00 0-00 


3-9 0-73 


24-60 1-55 




May 


5-1 0-25 


3-8(M3 


6-7 0-19 


0-91 0-00 


3-7 0-16 


20-04 0-57 




Aug. 


5-8 0-25 


4-0 0-25 


7-4 0-1 6 


0-98 0-01 


4-6 0-22 


22-78 0-60 




Nov. 


740-16 


5-6 0-1 6 


8-3 0-1 6 


1-69 0-03 


54 022 


28-49 0-44 


E. aegyptiaca 


Feb. 


6-5 0-51 


4-2 0-25 


7-8 0-79 


1-00 0-00 


44 0-51 


23-90 1-67 




May 


5 ; 40-51 


3-5 0-54 


6-1 0-89 


0-97 0-03 


3-5 0-5 1 


1946 1-86 




Aug. 


5-7 0-66 


4-6 0-79 


7-1 0-73 


1-00 0-00 


4-7 0-79 


23-102-56 




Nov. 


7-9 0-74 


5-5 0-54 


7-7 0-82 


1-63 0-09 


4-7 0-57 


27-36l-ll 



Mean Standard Deviation *Duration in days. 



at a low level till January. In the following months, the population gradually incre- 
ases and attains peak by April, but declines and disappears by May. Increase in 
temperature and decrease in the humidity enhances the population build-up of 
C. indicus but a high temperature with low humidity appears to have an adverse 
effect on the population, showing the preference of C. indicus for the warmer months 
(January-April) to build-up their population. Rainfall adversely affects their popu- 
lation. The availability of the host plant is also an important factor for the 
population fluctuations of C. indicus, the survival of the former to a great extent is 
affected by the rainfall (figure 3). 



3.5 Damage to seeds of E. aegyptiaca and A. aspera 

Based on the observations on the duration of lifecycle, mortality, average life span 
and population intensity, E. aegyptiaca appears to serve as the primary host for 
C. mexicanus that not only feeds but also inserts its egg into the ovarian tissues. An 
attempt has therefore been made to study the extent of seed damage in E. aegyptiaca 
by feeding and oviposition of C. mexicanus. Adults feed on the milky contents of the 
3-6 days young ovaries preventing seed formation. Eggs are laid in the ovarian 
tissues of 9-14 days old inflorescence, the developing larvae feeding on the ovarian 
tissues of the spikelet from which they emerged. As many as 9 ovaries/day are 
consumed by an individual adult with an average consumption of 223-2 ovaries 
throughout its life span. In the laboratory, individual females lay 2-3 eggs per day 
producing an average of 38-23 746 eggs during their life time. As a result, during its 
life span, a female damages as many as 261-43 7-46 ovaries of E. aegyptiaca both by 
feeding and oviposition. The adults cause more damage by feeding than the larvae. 
The overall damage is 70-80% in the laboratory and 20-30% in the field. The assess- 
ment of the damage in the field is based on the number of ovaries of the spikelet of 
. aegyptiaca containing eggs and immature stages of thrips. A significant positive 
correlation (r = 0-87) is obtained between the total number of ovaries in an inflore- 
scence and the number of ovaries damaged by C. mexicanus (figure 4). 
Both adults and larvae of C. indicus feed on the outer surface of the ovarian tissues 



28 



A Balu and A Mohan Daniel 



'! 20 




IP 35 

~2O 

6OO 
57O 
45O 

4OO 



280 



ex 

200 

CO 

m 



CL 
"51 



o 
6 



150 
1OO 

5O 







Male 
Female 



J JASONDJFMAMJJASONDJFMAMJJASONDJFMA 

198O 1981 1982 1983 



Figure 2. Population dynamics of C. mexicanus on 3 different host plants of E. aeyyptiaca 
(Ea), C. harbutti (Cb) and E. indica (Ei). 



Studies an seed infesting thrips 



29 




200 



f-150 
o 



100 



50 
o 







OU 



NO JFMAM 

1980 1981 



OND JFMA M 
1982 



ONDJF 
1983 



Figure 3. Population dynamics of C. indicus on A. aspera. 



95 



80 



.? GO 



o 



40 



Eleusine oegyptiaca 



r = 0-78 
y on x 




U 



J_ 



J_ 



55 75 100 125 150 175 200 225 
No- of ovaries on the spike 



Figure 4. Ovarian damage due to C. mexicanus infestation. 



30 



A Balu and A Mohan Daniel 



of A. aspera preventing subsequent seed production. As many as 5 individuals are 
found inside single spikelet and before they get enclosed by periants the first larvae 
move to fresh non-infested flowers to continue their feeding activity, affecting more 
and more ovaries of the spikelets. The randomly feeding adults damage 46-53% of 
ovaries of A. aspera. Statistical analysis reveals a significant positive correlation 
(r = 0*82) between the total number of ovaries in spike and the number of ovaries 
damaged by C. indicus (figure 5). 

4. Discussion 

Sexual and parthenogenetic reproduction appear to be typical of C. mexicanus, and 
arrhenotoxy is the principal mode of parthenogenetic reproduction, 'resulting in a 
dense population of males. More males are produced during summer when the 
temperature is high. Loan and Holdaway (1955) have also reported initiation of 
parthenogenesis in Haplothrips niger (Osborne) only at temperatures above 25C and 
not below. These observations reveal that some thrips species resort to partheno- 
genetic reproduction at higher temperatures. In C. indicus however, the females 
always outnumber the males due to the absence of parthenogenesis and do not 
exhibit any such variation in sex ratio with respect to seasonal changes. As observed 
in the case of Limothrips denticornis Haliday (Pussard-Radulesco 1930) adult males 
of C. mexicanus also mate with premature females. But unlike in L. denticornis, 
C. mexicanus neither exhibit hibernation nor store the sperms in prepupal 
spermatheca, but exhaust all the sperms once they attain maturity. As has been 
reported in many terebrantians (Riherd 1954; Doull 1956; Ananthakrishnan and 
Thirumalai 1978), C. mexicanus oviposit their eggs inside the ovarian tissues of the 
host plants. Due to lack of ovipositor, C. indicus always lays its eggs only on the 
host surfaces. It is well known that, the rate of development of insects does not 
increase proportionately with the rise in temperature throughout the range suitable 



250 - 




100 



150 200 250 300 

No- of ovaries on the spike 



350 400 



Figure 5. Ovarian damage due to C. indicus infestation. 



Studies on seed infesting thrips 31 

for development, but is faster at the optimum temperature, any deviation slowing 
down the rate of growth (Lewis 1973). It is also well recognised that in thrips not 
only seasonal temperature changes but also the constant and fluctuation tempera- 
tures affect the development of the species. In Anaphothrips obscurus (Muller), the 
duration of life cycle varies from 12 days in summer to 30 days in spring. Thrips 
tabaci Lindeman takes 11-2 days to complete its life cycle (from egg to adult) at a 
constant temperature of 30C whereas under fluctuating temperature with a mean of 
3O8C, it takes 13-9 days (Lall and Singh 1968). In C. mexicanus, which reproduces 
throughout the year, summer with high temperatures appears to be a favourable 
season for development. 

Increasing temperatures and decreasing relative humidity enhance the population 
build-up in C. indicus. Similar observations have also been made in C. mexicanus 
(Ananthakrishnan and Thirumalai 1978). As the flowers of E. aegyptiaca and 
E. indica dry up by the end of May, no further populations of C. mexicanus are 
observable. Inspite of the occurrence of flowers of C. barbata throughout the year, 
the prevalence of low temperature and high humidity during rainy season cause the 
insects to disappear on this host. This rainfall appears to have a profound and 
adverse effect on C. mexicanus and C. indicus populations. 

Host plants have a significant influence on the biology of C. mexicanus, a seed 
infesting species, whereas this is not so in the monophagous C. indicus. Though 
Ananthakrishnan and Thirumalai (1977) have recorded C. barbata as the primary 
host for C. mexicanus, comparative studies on the population and reproductive 
biology of this thrips on the 3 different host plants show E. aegyptiaca to be the most 
suitable and preferred host of C. mexicanus, on which it exhibits high fecundity, long 
life span and low mortality rate (table 1). C. barbata and E. indica may be considered 
as secondary and tertiary hosts respectively. 

Different species of Chirothrips are known to feed on the ovarian tissues of many 
grass species destroying a large number of seeds (Lewis 1973). The feeding of 
C. indicus larvae on the ovarian tissues restricted to the oviposited sites and that of 
the adults on these tissues at very many places results in extensive loss of seed 
production to about 70-80% in E. aegyptiaca. Similarly C. mexicanus- is reported to 
cause 60% and 5-10% on C. barbata and on Pennisetum typhoides, respectively 
(Ananthakrishnan and Thirumalai 1977). C. hamatus and C. pallidicornis species 
closely related to C. mexicanus, have also been reported to cause 25 and 30% seed 
loss in meadow foxtail grass and cocksfoot grass respectively (Hukkinen 1936; Doull 
1956). Statistically there is a significant and direct correlation between the number of 
ovaries in an inflorescence and the number of ovaries damaged by C. mexicanus on 
E. aegyptiaca. 

The fecundity of sexually and parthenogenetically reproducing individuals of C. 
mexicanus is always higher when they feed and develop on E. aegyptiaca than on 
C. barbata and E. indica. These thrips also complete their development faster, with 
low mortality rate, always maintaining a high populations on E. aegyptiaca than the 
other hosts. Hence E. aegyptiaca appears to be a more suitable and preferred host. 
Based on the assessment of these parameters it can also be said that E. indica is a less 
preferred host than C. barbata. But these hosts enable the thrips to maintain their 
population at a very low level throughout their infestation. 



32 A Balu and A Mohan Daniel 

Acknowledgement 

The authors are grateful to Prof. T N Ananthakrishnan, under whose guidance this 
work was carried out. 

References 

Ananthakrishnan T N 1969 Indian Thysanoptera; CSIR Zoo. Monogr. No. 1 p 171 

Ananthakrishnan T N and Daniel A M 1981 Behavioural components in feeding, reproduction and 

dispersal of the grass seed-feeding thrips Chirothrips mexicanus Crawford; Curr. ScL 50 733-735 
Ananthakrishnan T N and Thirumalai G 1977 The grass-seed infesting thrips Chirothrips mexicanus 

Crawford on Pennisetum typhoides and its principal alternative host Chloris barbata; Curr. ScL 46 193- 

194 
Ananthakrishnan T N and Thirumalai G 1978 Population fluctuations of three species of anthophilous 

Thysanoptera with notes on the biology of the seed feeding species Chirothrips mexicanus Crawford; 

Bull. Zool. Surv. India I 197-201 

Bailey S F 1948 Grain and grass infesting thrips; J. Econ. Entomol. 41 701-705 
Bhul C 1973 Beitrage Zur Kenntnis der Biologie, Wirtschaftliche Bedeutung and Bekeampfung Von 

Kakothrips robustus Uz.; Z. Angew. Entomol. 23 65-113 
Doull K M 1956 Thrips infesting cocks foot in New Zealand II. The biology and economic importance of 

the cocksfoot thrips Chirothrips manicatus Haliday; N. Z. J. ScL Technol. A38 56-65 
Franssen C J H 1960 Biology and control of the pea thrips; Versl. Landbouwkd. Onderz. 66 1-36 
Hukkinen Y 1936 Investigation on the seed pests of the meadow foxtail grass, Alopecurus pratensis. I. 

Chirothrips hamatus; Vail. Maatalouskoetoiminnan. Julk. 81 1-132 
Irwin M E, Kenneth V Y and Norman L M 1979 Spatial and seasonal patterns of phytophagous thrips on 

soybean fields with comments on sampling techniques; Environ. Entomol. 8 131-140 
Lall B S and Singh L, M 1968 Biology and control of onion thrips in India; J. Econ. Entomol. 61 676-679 
Lewis T 1973 Thrips, their biology, ecology and economic importance; (London: Academic Press) p 349 
Loan C and Holdaway F G 1955 Biology of the red clover thrips Haplothrips niger (Osborne) 

(Thysanoptera: Phlaeothripidae); Can. Entomol. 87 210-219 
Pussard-Radulesco E 1930 Quelques observations biologiques sur Parthenothrips dracaenae Heeg. et. 

Aptinothrips rufus Gmel. Van connaticornis Uzel; Rev. PathoL Veg. Entomol. Agric. Fr. 17 24-28 
Riherd P T 1954 Thrips as a limiting factor in grass seed production; J. Econ. Entomol. 47 709-719 
Roney J N 1949 Bermuda seed grass insects in Arizona; J. Econ. Entomol. 42 555 
Vappula N A 1965 Pests of cultivated plants in Finland; Ann. Agric. Fenn. (Suppl. 1) 1 1-239 
Varadarasan S 1979 Bioecological investigations on some gall-inhabiting Thysanoptera (Insecta : Arthro- 
pods); Ph.D. Thesis, University of Madras, Madras 
Watts J G 1965 Chirothrips falsus on black gamma grass; Agric. Exp. Stn. Bull. 499 1-20 



Proc. Indian Acad Sci. (Anim. Sci.), Vol. 96, No. 1, January 1987, pp. 33-40. 
Printed in India. 



Light and electron microscopic observations on lipid droplets of the 
sebaceous flank gland of the shrew, Suncus murinus viridescens (Blyth) 

M BALAKRISHNAN 

Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695 581, India 

MS received 13 October 1986 

Abstract. Light and electron microscopic investigations have been made on the lipid 
droplets of the sebaceous flank gland alveoli of the Indian musk shrew, Suncus murinus 
viridescens. The undifferentiated sebaceous cells can be distinguished from the differentiating 
and differentiated cells by the absence of lipid droplets in the former. Adjacent droplets in 
the differentiating cells fuse to form lajrger droplets. Lipid droplets are also synthesized in 
the differentiating cells. Large accumulation of lipid droplets are seen in well differentiated 
cells. The cells of the inner layer of "the sebaceous alveoli disintegrate and the holocrine 
products are liberated into the lumen. The probable role of mitochondria and polyribo- 
somes in the synthesis and development of lipid droplets in the sebaceous cells of the flank 
gland is discussed. 

Keywords. Indian musk shrew; Suncus murinus viridescens; flank gland; sebaceous alveoli; 
lipid droplets; light and* electron microscopic investigations. 



1. Introduction 

Although extensive investigations on specialized integumentary glands of mammals 
have been carried out during the past few years (for review, Miiller-Schwarze 1983), 
skin glands of only a few of the Indian mammals have been studied so far (for review, 
Balakfishnan and Alexander 1985). Balakrishnan et al (1986) have studied the 
structure of the specialized cutaneous glands of the Indian musk shrew, Suncus 
murinus viridescens in detail. Sebaceous glandular alveoli form the bulk of the flank 
gland of this species with a few sudoriferous tubules located mainly at the glandular 
periphery. Each alveoli has a central lumen where the glandular secretory materials 
are temporarily stored and later discharged to the exterior, especially during the 
process of scent marking (Balakrishnan and Alexander 1985). 

Lipids form the major component of this glandular secretion of the musk shrew 
(Balakrishnan and Alexander 1985). These substances have specific behavioural 
relevance during social interactions. The present report deals with light and electron 
microscopic observations on the formation and development of the lipid secretory 
droplets in the sebaceous alveoli of the flank gland of S. m. viridescens. 

2. Material and methods 

Adult shrews (9 males and 8 females) were used for the present investigation. They 
were trapped live on the University Campus, Kariavattom, and were kept in indi- 
vidual wire-mesh cages for 2-3 days on a regular supply of minced beef sprinkled 
with shark liver oil and tap water, ad libitum. Fur covering the flank glands was 
shaved off using a fine blade and the glands were dissected out for processing, for 
histological, histochemical and ultrastructural studies. Glands from one side from 5 



34 M Balakrishnan 

males and 5 females were fixed in Bouin's fluid, processed, paraffin sections were cut 
at 5 /an and stained in haematoxylin and eosin. The glands from the other side were 
fixed in 10% formalin, gelatin blocks were prepared and the sections were cut at 
8-10 /an on a Model CTD International Harris Cryostat at -20C. These sections 
were stained with Sudan black-B. 

For electron microscopic investigations, the glands from 4 males and 3 females 
were dissected out under ether anaesthesia. Thin slices of the glandular tissues were 
fixed in 2-5% glutaraldehyde in 0-1 M phosphate buffer (pH 7-2-7-3). The tissues 
were washed in the buffer for 2 h and post fixed in 1% osmium tetroxide in 0-1 M 
phosphate buffer. The samples were then carried to the USSR in the same buffer and 
dehydrated using alcohol and acetone in the ascending grades ancl embedded in 
Epon 812." Ultrathin and 1 jum sections were cut using glass knives in LKBUltro- 
tomes III and IV. Ultrathin sections wei:e mounted on formvar-coated grids and 
stained with uranyl acetate and lead citrate (Reynolds 1963) and examined under a 
JOEL JEM 100 C electron microscope at an accelerating voltage of 80 kV. One 
microne section was prepared and stained with toluidine blue and observed under a 
light microscope. 

3. Results 

Each of the sebaceous alveoli of the flank gland consists of about 4-6 layers of cells 
(figure 1). The peripheral layer of the alveoli consists of undifferentiated cells. The 
middle layers consist of differentiating cells and the inner layers consist of well differ- 
entiated and disintegrating cells. 

Light microscopic observations revealed that the cells in the outer-most layer of 
the sebaceous alveoli are devoid of lipid droplets. Lipid droplets are seen in large 
numbers in the cells of the middle and inner layers of the alveoli (figure 2). The cells 
of the inner-most layer can be well distinguished by their holocrine secretory activity; 
these cells disintegrate in situ and discharge their contents into the lumen of the 
sebaceous alveoli in the form of large number of well developed droplets. During 
development and differentiation the lipid droplets enlarge with their opaque con- 
tents. Cells in the inner layers of the alveoli contain large number of such droplets. 
The glandular secretion is eliminated to the exterior through the special ducts of the 
alveoli. 

Histochemical studies have revealed that the secretory products of the sebaceous 
cells of the flank gla^d are predominantly lipoid in nature. The secretory products in 
general stained deeply with Sudan black-B. Lipids were not detected in appreciable 



Figures 1-4. L Light micrograph of the sebaceous flank gland alveoli of a male shrew. 
Note the arrangement of cells in the alveoli. 5 /mi haematoxylin x eosin ( x 100), 2. One 
microne section of the flank gland of a male shrew showing the pattern of distribution of 
lipid droplets in the sebaceous alveoli. Note the greater accumulation of lipid droplets in the 
inner cells and in the lumen of sebaceous alveoli. Capillaries ending in the epithelial wall 
(indicated by arrows) are seen with erythrocytes ( x 1000). 3. Flank gland alveoli of a male 
shrew showing the absence of lipids in the connective tissue envelope of the alveoli (arrows) 
The peripheral cells contain less amount of lipids as compared to the inner cells Sudan 
black-B (x400). 4. Part of a sebaceous alveoli of the flank gland of a female shrew 
showing the undifferentiated cell on the left half with dense cytoplasm when compared to 
that of the differentiated cell on the right ( x 22,000). 



Shrew flank gland lipid droplets 




36 



M Balakrishnan 




Shrew flank gland lipid droplets 37 

quantity in the outer covering and in the peripheral layer of the alveoli. Heavy lipid 
accumulation was noted in the lumen and in the cells of the sebaceous alveoli (figure 

3). 

No difference in the histochemical and ultrastructural features of the sebaceous 
cells in the flank gland between males and females could be detected. 

The undifferentiated cells of the sebaceous alveoli can also be distinguished from 
the differentiated cells by the difference in the intensity of the cytoplasm. The 
cytoplasm of undifferentiated cells are denser as compared to that of the differentia- 
ted cells (figure 4). The differentiating cells have lipid droplets in various stages of 
development. Adjacent smaller droplets fuse to form larger droplets during cellular 
differentiation and maturation (figures 5 and 6). 

Large number of mitochondria are observed around the lipid droplets both in the 
differentiating and in the well differentiated sebaceous cells of the flank gland (figures 
6-8). Most of the mitochondria in the differentiating cells are large, round and 
swollen (figure 7), whereas those of the differentiated and disintegrating cells are 
smaller and oval or elongated (figures 8 and 9). Polyribosomes are also seen 
accumulated around lipid droplets (figures 6-9). In the well developed cells, lipid 
droplets occupy most of the area in the cytoplasm (figure 9). 



4. Discussion 

The sebaceous alveoli of the flank gland of S. m. viridescens show cells in different 
stages of development, which can be identified on the basis of presence and larger 
accumulation of lipid droplets. New layers of secretory cells grow from the periphery 
of the alveoli, develop, differentiate and subsequently disintegrate and discharge their 
contents into the lumen by holocrine secretion (Kurosumi 1961). 

In the course of lipogenesis in the mouse Meibomian gland, Gorgas and Volkl 
(1984) have demonstrated 4 phases, (i) formation of large number of small, spherical 
lipoid droplets, (ii) enlargement of these droplets, (iii) further expansion in the 
volume and accumulation of an electron opaque material in it and (iv) the lethal 
phase. It is also reported that the composition of cellular lipids changes during the 
development and differentiation of sebaceous cells (Potter et al 1979; Wheatley et al 
1979). In the present investigation, it was noticed that during cellular differentiation, 
smaller lipid droplets fused together to form larger droplets (figures 5 and 6). 
However, in differentiating and well differentiated sebaceous cells, both small and 
large lipid droplets were noticed. This may be due to the fact that new lipid droplets 



Figures 5-7. 5. Part of a sebaceous alveoli of the flank gland of a female shrew showing a 
differentiating and a differentiated cells. The adjacent smaller lipid droplets synthesized 
during the process of differentiation are fused to form larger droplets of the differentiated 
cells ( x 22,000). 6. Part of a differentiating cell of the sebaceous alveoli of a female shrew 
showing the process of fusion of lipid droplets. Large, round and swollen mitochondria are 
seen near the developing lipid droplets. Polyribosomes are accumulated on the periphery of 
the lipid droplets ( x 22,000). 7. Part of a differentiating cell of the sebaceous alveoli of the 
flank gland of a female shrew showing the close association of large, round and swollen 
mitochondria with developing lipid droplets ( x 22,000). 



38 



M BaUikrishmtn 




Figures 8-9. 8. Part of the well differentiated cell of the sebaceous alveoli of a female 
shrew flank gland showing large number of polyribosomcs attached to the periphery of the 
lipid droplets. The mitochondria in the developed cells are comparatively smaller, and of 
oval, elongated or of irregular shapes ( x 22,000). 9. Part of a disintegrating cell of the 
sebaceous alveoli of the flank gland of a female shrew. Lipid droplets occupy most of the 
area in the cytoplasm of the developed and disintegrating cells. The mitochondria are small 
and oval, elongated or irregular in shape ( x 22,000). 



Shrew.jhink gland llpid droplets 39 

are continuously synthesized by these cells as observed by Wooding (1980) in the 
sebaceous cells of the Hardarian glands of rabbit. 

The close association of lipid droplets with'mitochondria having the characteristic 
electron dense inclusions in the sebaceous flank gland cells of the shrew has already 
been reported (Balakrishnan et al 1986). It is also suggested that the mitochondria 
are engaged in lipid metabolism in specialized glandular cells (Bjersing 1967; Fawcett 
1981; Jenkinson et al 1985). Balakrishnan et al (1986) have also observed that 
polyribosomes are situated around the periphery of the lipid droplets which may 
contribute necessary enzymes for the synthesis and development of lipid droplets. 

Well developed capillaries in the peripheral cells of the sebaceous alveoli of the 
shrew flank gland (figure 2) facilitate the flow of raw materials from the surrounding 
media, and the finger-like protrusions of the cellular periphery at the level of 
capillaries (Balakrishnan et al 1986) provide additional surface for absorption and 
ingestion of more raw materials into these secretory cells. 

Since steroid hormones have profound influence on the structure and function of 
the sebaceous glands in a number of species of mammals (Bell 1974; Strauss et al 
1,976), u.ltrastructural investigations on the sebaceous flank gland tissues of castrated 
and hormone administered shrews are likely to yield valuable information on the 
lipogenesis in the sebaceous cells. 



Acknowledgement 

I am grateful to the Indian National Science Academy, New Delhi and the USSR 
Academy of Sciences, Moscow for the award of an Academic Exchange Fellowship 
during the period of which part of this investigation was made. I am also grateful to 
Academician V E Sokolov of the A N Severtzov Institute of Evolutionary Animal 
Morphology and Ecology, USSR Academy of Sciences for offering me necessary 
facilities and to Dr O A Basurmanova of the above institute for her constant help 
and stimulating discussions during this investigation. I am indebted to Prof. 
C J Dominic, Department of Zoology, Banaras Hindu University, for his critical 
review of this paper and suggesting several improvements. Thanks are also due to 
Prof. K M Alexander, University of Kerala ibf-encouragement. Financial assistance 
from the Department of Science and Technology, New Delhi is also acknowledged. 



References 

Balakrishnan M and Alexander K M 1985 Sources of body odour and olfactory communication in some 

Indian mammals; Indian Rev. Life Sci. 5 277-313 
Balakrishnan M. Sokolov V E and Basurmanova O A 1986 Further observations on the ultrastructure of 

the flank gland of the musk shrew, Suncus murinus viride&cens (Blyth); Proc. Indian Acad. Sci. (Anim. 

Sci.} 95 667-675 
Bell M 1974 Effect of androgen on the ultrastructure of the sebaceous gland in two species; J. Invest. 

Dermatol. 62 202-210 
Bjersing L 1967 On the ultrastructure of granulosa lutein cells in porcine corpus luteum with social 

reference to endoplasmic reticulum and steroid hormone synthesis; Z. Zettforsch. 82 187-211 
Fawcett D W 1981 The Cell (Philadelphia: Saunders Company) 2nd edition 
Gorgas K and Volkl A 1984 Peroxisornes in sebaceous glands IV. Aggregates of tubular peroxisomes in 

the mouse Meibomian gland; Histochem. J. 16 1079-1098 



40 M Balakrishnan 

Jenkinson M D, Elder H Y, Mongomery J and Moss V A 1985 Comparative studies on the infrastructure 

of sebaceous gland; Tissue Cell 17 683-698 

Kurosumi K 1961 Electron microscopic analysis of the secretion mechanism; Int. Rev. Cyiol. 11 1-124 
Muller-Schwarze D 1983 Scent glands in mammals and their functions; in Advances in the study of 

Mammalian 'Behaviour (eds) J F Eisenberg and D G Kleiman; Spec. Publ. Am. Soc. Mamm. 1 150-197 
Potter J E R, Prutkin L and Wheatley V R 1979 Sebaceous gland differentiation 1. Separation, 

morphology and lipogenesis of isolated cells from the mouse preputial gland tumor; J. Invest. Dermatol. 

12 120-127 
Reynolds E S 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy; 

J. Cell. Biol. 17 208-212 
Strauss J S, Pochi P E and Downing D T 1976 The sebaceous glands: Twenty five years of progress; 

J. Invest. Dermatol. 67 90-97 
Wheatley V R, Potter J E.R and Lew G 1979 Sebaceous gland differentiation II. The isolation, separation 

and characterization of cells from the mouse preputial gland; J. Invest. Dermatol. 73 291-296 
Wooding F B P 1980 Lipid droplet secretion by the rabbit Hardarian gland; J. Ultrastruct. Res. 71 68-79 



Abbreviations used in the figures: SA, Sebaceous alveoli; Lu, lumen of the sebaceous alveoli; L, lipid 
droplets; e, erythrocyte; N, nucleus; M, mitochondria; Ifd, lipid droplets in the process of fusion and 
development; pr, polyribosomes.. 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 1, January 1987, pp. 41-47. 
Printed in India. 



Quantitative analysis of carbohydrases in the crystalline style of some 
intertidal bivalve molluscs 

P SHAHUL HAMEED*f and A L PAULPANDIAN 

Centre of Advanced Study in Marine Biology, Parangipettai 608 502, India 

* Post-Graduate Department of Zoology, Jamal Mohamed College, Tiruchirapalli 620020, 

India 

MS received 21 November 1986 

Abstract. Amylase activity in different pH ranging from 3-6 to 8-0 and activity of sucrose, 

maltose, trehalase, raffmase, lactase, glycogenase, pectinase, chitinase, carboxy methyl 

cellulase and cellulase at their respective optimum pH were quantitatively estimated in the 

crystalline style of intertidal bivalves? Crassostrea madrasensis, Meretrix meretrix, Meretrix 

casta, Katelysia opima and Donax cuneatus. Amylase was the most active among all enzymes 

tested. A maximum activity of 137-5 jug/mg/h was recorded in the style of Katelysia opima 

while the activity was minimal in Donax cuneatus (39-3 //g/mg/h). Two pH optima were 

recorded for style amylase of Crassostrea madrasensis (6-0 and 6-8) and Katelysia opima (5-6 

and 6-8). But the activity was higher at higher optimum pH. The enzymes hydrolysing 

oligosaccharides were weak in most cases or absent. The enzymes acting on reserve 

polysaccharides such as amylase and glycogenase showed maximum activity in the species 

tested. However the activity of enzymes degrading structural polysaccharides such as 

cellulase, carboxy methyl cellulase, pectinase and chitinase was either at a low level or 

absent/ The role of the style enzymes iri the extracellular digestion of bivalves is discussed. 

Keywords. Bivalve molluscs; crystalline style; carbohydrases; Crassostrea; meretrix; 
katelysia; donax. 

1. Introduction 

Consequent to the loss of salivary gland in the evolution of Bivalvia, crystalline style 
might have developed as a source of supply of starch splitting enzyme (Yonge 1923). 
Two groups of digestive enzymes, carbohydrases and lipases have been identified in 
the crystalline style of bivalves (Owen 1966, 1974; Morton 1983). The presence of 
amylase in the style of bivalves was reported by Morton (1983). Other carbohydrases 
like glucosidases and galactosidases and structural polysaccharases like cellulase, 
carboxy methyl cellulase and chitinase were also shown to be present in the style of 
many bivalves and this subject has been reviewed by van Weel (1961), Owen (1966, 
1974) and Morton (1983). However, quantitative measurements on the activity of 
carbohydrases of the style were limited to the work of Horiuchi and Lane (1966), 
Kristensen (1972), Wojtowics (1972) and Langton and Gobbott (1974). The present 
work was undertaken to quantify the activity of style carbohydrases and to under- 
stand the nature of the process of digestion in some intertidal bivalves. 

2. Materials and methods 

Crassostrea madrasensis (Preston), Meretrix meretrix (LinnaeuS), Meretrix casta 
(Chemntiz) and Katelysia opima (Gmelin) were collected from the mud flats of Vellar 



42 P Shahul Hameed and A L Paulpandian 

estuary joining Porto Novo sea shore (1129' N lat. and 7946' E long.) and Donax 
cuneatus (Linnaeus) from the marine intertidal zone. 

2.1 Preparation of style extract 

Aqueous extract of the style was prepared by homogenising it with distilled water 
using an all glass homogeniser. After centrifugation for 15 min at 20,000 g the super- 
natant was used as enzyme extract. The strength of the extract was 3% . 

2.2 Quantitative estimation ofamylase activity in different pH 

A reaction mixture of 1 ml of 1% starch, 1 ml of style extract and 2 ml of buffer was 
incubated for 2 h at different pH ranging from 3-6-8-0. Two drops of toluene were 
added to cover the surface of the reaction mixture in test tube to prevent contami- 
nation from air. The amount of reducing sugars released by amylase activity wa$ 
determined by Somogyi-Nelson's photometric method (Nelson 1944). The extinction 
was measured at 540 nm using distilled water as reference in a spectrophotometer 
(Spectronic-20). The amount of glucose released was calculated from the calibration 
curve constructed with standard D-glucose. The protein of the style extract was 
determined colorimetri'cally by the method of Raymont et al (1964). The enzyme 
activity was expressed as /zg glucose/mg protein/h (^g/mg/h). Control experiments 
were run simultaneously with heat inactivated style extract. Three estimations were 
made for each species and the mean values used. 

2.3 Quantitative estimation of activity of other carbohydrases 

The substrates used in these experiments were 2% sucrose, 2% maltose, 1% 
trehalose, 2% raffinose, 2% lactose, 1% glycogen, 1% pectin, 1% chitin, 1% sodium 
carboxy methyl cellulose and 1% cellulose. The activity of the different carbohy- 
drases was estimated by the method described above. However, the reaction mix- 
tures were incubated for 8 h and at the optimal pH of the enzyme in question as 
described by Horiuchi (1963). 

2.4 Buffers 

0-2 M acetate buffer was used to maintain the pH i'n the range of 3-6-5-6 and 0-2 M 
sodium phosphate buffer in the range of 6-0-8-0. 



3. Results 

The amylase activity at different pH and activity of other carbohydrases in their 
respective optimum pH in the style extract of 5 bivalves species are presented in 
figures 1 and 2. The activity profile of amylase and other carbohydrases are 
described. 



Carbohydrases in the crystalline style of bivalve molluscs 



43 



150 



C. modrasensis 
ML nneretrix 

M. costa 
K. opimo 

D. cuneatus 




Figure 1. Amylase activity in the crystalline style of the tested bivalve species in relation 
to pH. 



3.1 C. madrasensis 

Considerable amylase activity was recorded in the style extract of this oyster in the 
pH range between 5-6 and 7-0 with peak activity at pH 6-0 (62-15 jug/mg/h) and at 
pH6-8 (64-5 jug/mg/h). The style extract of this oyster was found to hydrolyse 
oligosaccharides like sucrose, trehalose, raffinose and polysaccharides like carboxy 
methyl cellulose and glycogen. The hydrolysis of glycogen was faster (9-6 jug/mg/h) 
than the hydrolysis of other carbohydrates. Sucrose activity was observed only in C. 
madrasensis and absent in others. 



3.2 M. meretrix 

The optimum amylolytic activity of the style extract of this clam was found at pH 5*6 
(64-0 /zg/mg/h) and at pH 6-8 (91-0/zg/mg/h). However, the hydrolysis of the starch 
was spread over the pH range 5-6-8-0. In M. meretrix style extract hydrolysed two 
oligosaccharides, maltose and trehalose and two polysaccharides, glycogen and 
carboxy methyl cellulose. 



44 



P Shahul Hameed and A L Paulpandian 



J 

*0 



. 
x 



50 
10 
8 
6 

4 
2 



150 
11O 
70 

30. 

1O" 
8 
6 
4 



sp Donox cuneotus 




O 



11O 
7O 

10 
8 
6 
4 
2 





Crossostreo modrosensis 



Sucrose 

Maltose 

Trehalose 

Raffinose 

Glycogen 

Starch 

Carboxymethyl cellulose 

Cellulose 



Kotelysia opima 




Meretrix meretrix 




Figure 2. Histograms showing the activity of carbohydrases in the crystalline style of the 
tested bivalve species. 



3.3 M. casta 

The activity profile for amylase and other carbohydrases of M. casta was almost 
similar to that of M. meretrix', however there is a single pH optimum in M. casta. 



3.4 K. opima 

There were two pH optima (5-6 and 6-8) in which maximal amylase activity was 
measured. Of all the bivalves tested the highest rate of amylolysis (137-5 /ig/mg/h) 
was recorded at the optimum pH of 6-8 and at the lower pH optimum of 5-6, the 



Carbohydrases in the crystalline style of bivalve molluscs 45 

style extract released 75-1 ^g/mg/h. Only two polysaccharides, carboxy methyl 
cellulose and glycogen were also hydrolysed. 



3.5 D. cuneatus 

Amylase of D. cuneatus style was observed to have a lowest level of activity 
(39-3 jug/mg/h) at the optimum pH of 6-8 when compared with the activity of the style 
amylase of other species. Donax style extract also hydrolysed 3 oligosaccharides, 
maltose, trehalose and raffinose and 3 polysaccharides, cellulose, carboxy methyl 
cellulose and glycogen, as in other species the rate of glycogen hydrolysis 
(5-8 ^g/mg/h) was significantly at a higher level. 



4. Discussion 

Styles of all the tested bivalves maintained a high level of amylase activity but the rate 
of activity varied among the tested species. A maximum activity of 137-5 /xg/mg/h was 
recorded in the style extract of K. opima. The activity was minimal in D. cuneatus 
(39-3 /zg/mg/h). In general the result indicated that the activity of the style amylase 
was always higher in venerids (M. meretrix, M. casta and K. opima) than in ostreid 
(C. madrasensis) or in donacid (D. cuneatus). van Weel (1961) and Owen (1966, 1974) 
also showed variation in amylase activity among some bivalve species. Wojtowics 
(1972) quantified the amylase activity in the style and digestive diverticula and 
showed that the activity was higher in the style than in digestive diverticula. 

Two pH optima were recorded for the style amylase of C. madrasensis (6-0 and 6-8) 
and K. opima (5-6 and 6-8). The occurrence of two pH optima is not uncommon. 
Hashimoto and Kijima (1956) in Mactra veneriformis, Dinamani (1957) in Villorita 
cyprinoides, Black and Pengelley (1964) in C. virginica and Horiuchi and Lane (1966) 
in Strambus gigas also reported two pH optima for the style amylase. Black and 
Pengelley (1964) showed the presence of an amylase with a single pH optimum in the 
style of oyster larva but the style of the adult contained a second amylase with 
different pH optimum. In all the species tested with two pH optima for amylase, the 
style amylase was more active always at higher pH optimum (6-8). Similar observa- 
tion was also reported by Horiuchi and Lane (1966). Our results indicate that 
hydrolysis of oligosaccharides in most cases were weak or absent. But the style 
contained more active enzymes for the hydrolysis of reserve polysaccharides such as 
starch and glycogen than for structural polysaccharides like cellulose, pectin and 
chitm. Next to amylase, glycogenase showed a higher rate of hydrolysis in all the 
species analysed. The glycogenase is one of the earliest known style enzymes and 
reported to be present in the style of many bivalves (Horiuchi and Lane 1966; Kris- 
tensen 1972; Mathers 1973). Mathers (1973) was of the opinion that the style amylase 
which hydrolysed glycogen was probably the same that also hydrolysed starch, 
amylase and amylopectin. 

The present study also indicates the distribution pattern of cellulolytic enzymes. A 
true cellulase was recorded only in D. cuneatus while the poly-glucosidase capable of 
digesting carboxy methyl cellulose was recorded in all the species analysed. A similar 
distribution pattern for cellulolytic enzymes was also reported in literature (Payne et 



46 P Shahul Hameed and A L Paulpandian 

al 1972; Morton 1978). The study of the style carbohydrases of the bivalve species 
throw some light on the basic nature of digestion in general and extracellular diges- 
tion in particular. Since these bivalves largely feed on phytoplankton and organic 
particulate materials of plant origin (Purchon 1968; Crosby and Reid 1971), they are 
by and large herbivores. Herbivorous animals generally have more active carbohyd- 
rases than carnivores have (Prosser and Brown 1961). As the salivary glands do not 
exist in filter feeding Bivalvia ( Yonge 1923) a compensatory source of carbohydrases 
is inevitable. Adaptively the development of crystalline style in bivalves provides a 
rich source of carbohydrases which initiate the extracellular digestion in the stomach. 
Further, the more active style enzymes are amylase, glycogenase, cellulase and poly- 
glucosidase (present study) and laminarinase (Sova etal 1970; Wojtowics 1972) all of 
which are designed to degrade sugars of high molecular weight. The situation implies 
that the crystalline style initiates extracellular digestion through the mechanical tritu- 
ration of food and release of enzymes for the degradation of high molecular weight 
polysaccharides in the stomach lumen while the final break down and absorption 
take place in the digestive diverticula. 

Acknowledgements 

The authors sincerely thank Dr R W Langton, Bureau of Marine Science, Marine 
Resources Lab., West Boothabay Harbor, Maine, USA for valuable suggestion and 
helpful criticism and University Grants Commission, New Delhi for the award of a 
Fellowship to one of them (PS). 

References 

Black R E and Pengelley E T 1964 Alpha amylase development in the embryos of Crassostrea virginica; 

Biol Bull Woods Hole 126 199-294 
Crosby H D and Reid G B 1971 Relationship between food, phylogeny and cellulose digestion in the 

Bivalvia; Can. J. Zool 49 617-622 
Dinamani P 1957 On the stomach and associated structures in the backwater clam Villorita cyprinoides 

(Gray); Bull. Cent. Res. Inst. Univ. Kerala Trivandrum Ser. C 5 123-148 
Hashimoto Y and Kijima S 1956 Studies on the crystalline style of Molluscs III Amylolytic enzyme; Bull. 

Jpn. Soc. Sci. Fish. 21 1034-1040 
Horiuchi S 1963 On the nature of carbohydrases of digestive diverticula in the marine lameliibranch 

Venerupis philippinarum; Sci. Rep. Tokyo Kyoiku Daiqaku Bll 133-152 
Horiuchi S and Lane C E 1966 Carbohydrases of the crystalline style and hepatopancreas of Strombus 

gigas L.; Comp. Biochem. Physioi 17 1189-1197 
Kristensen J H 1972 Carbohydrases of some marine invertebrates with notes on their food and on the 

natural occurrence of the carbohydrates studied; Mar. Biol. 14 130-142 
Langton R W and Gobbott P A 1974 The tidal rhythm of extracellular digestion and response to feeding 

' in Ostrea edulis; Mar. Biol. 24 181-187 

Mathers N F 1973 Carbohydrate digestion in Ostreai Proc. Malacoi Soc. London 40 3-J9-367 
Morton B S 1978 Feeding and digestion in shipwbrms; Oceanogr. Mar. Biol. Annu. Rev. 16 107-144 
Morton B S 1983 Feeding and digestion in Bivalvia; Mollusca 5 65-147 
Nelson N 1944 A photometric adaptation of Somogyi method for determination of glucose: J. Bio. Chem. 

153 375-380 

Owen G 1966 Digestion; Physioi. Mollusca 2 53-96 

Owen G 1974 Feeding and Digestion in Bivalvia; Adv. Comp. Physioi. Biochem. 5 1-35 
Payne D W,Thrope M A and Donaldson E M 1972 Cellulolytic activity and the study of bacterial popu- 
lation in the digestive tract of Scrobicularia plana (Da Costa); Proc. Malacoi. Soc. London 40 147-160 



Carbohydrases in the crystalline style of bivalve molluscs 47 

Prossor C L and Brown FA 1961 Comparative Animal Physiology; (Philadelphia: Saunders) 

Purchon R D 1968 The Biology of Mollusca; (Oxford: Pergamon Press) 

Raymont J E G, Austin J and Linford E 1964 Biochemical studies on the marine zooplankton I. The 

biochemical composition of Neomysis integer; J. Cons. Perm. Int. Explor. Mer. 28 354-363 
Sova V V, Elyakova L A and Vaskovsky V E 1970 The distribution of laminarinase in the marine 

invertebrates; Comp. Biochem. Physiol. 32 459-464 

van Weel P B 1961 The comparative physiology of digestion in molluscs; Am. Zool I 245-252 
Wojtowics M B 1972 Carbohydrases of the digestive gland and the crystalline style of Atlantic deep sea 

scallop Placopecten mayellanicus (Gmelin); Comp. Biochem. Physiol. 43 131-141 
Yonge C M 1923 Studies on the comparative physiology of digestion. The mechanism of feeding, digestion 

and assimilation on the lamellibranch Mya\ Brit. J. Exp. Biol I 15-63 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 1, January 1987, pp. 49-54. 
Printed in India. 



Glycogen level and glycogen phosphorylase activity in the eggs of 
silkworm Bombyx mori L. 

P M CHANDRASHEKAR and GEETHA BALI 

Department of Zoology, Bangalore University, Bangalore 560 056, India 

MS received 21 February 1986; revised 22 December 1986 

Abstract. The glycogen content of the eggs of silk worm 'Bombyx mori L. was analysed at 
intervals during the course of embryonic development and diapause. Changes in the 
glycogen levels were also examined in acid treated eggs in which diapause has been broken. 
These studies showed significant differences in glycogen utilisation pattern in the diapause 
and non-diapause eggs. The activity of the enzyme glycogen phosphorylase was also found 
to exhibit different patterns in the two types of eggs. Our studies indicate that this enzyme 
plays an important role in the metabolic changes which occur during diapause in silkworm 
eggs. 
Keywords. Bombyx mori; glycogen; glycogen phosphorylase. 



1. Introduction 

Glycogen is stored in the eggs of the silkworm Bombyx mori L. and is made use of as 
and when energy is required during the course of diapause as well as embryonic 
development (Chino 1957). In many other insects also utilisation of glycogen has 
been found to exhibit a definite pattern which is indicative of metabolic regulation 
corresponding to the needs of growth and activity (Karlson and Sekeris 1964). 

The embryonic development in B. mori is interesting because in certain races the 
embryonic development proceeds uninterrupted while in some, it is characterized by 
diapause and the diapause eggs can be artificially treated to continue embryonic 
development without interruption. Therefore, silkworm eggs provide an ideal mate- 
rial for examining the adaptability of glycogen metabolism and its role in diapause. 
There have been some studies earlier, demonstrating the changes in the glycogen 
content in silkworm eggs during diapause (Chino 1957, 1958). During the present 
studies, changes in the glycogen levels during the embryonic development in locally 
available non-diapause eggs have been examined in detail. Changes in glycogen level 
in diapause eggs during the course of diapause and also following acid treatment to ; 
break diapause have been studied. These studies would explain whether differences, if 
any, in the pattern of glycogen utilisation in diapause and non-diapause eggs are due 
to the phenomenon of diapause or due to any inherent differences in these two types 
of eggs. 

The initial and key control step in the utilisation of glycogen is a phosphorolytic 
reaction catalysed by the enzyme glycogen phosphorylase. Glycogen phosphorylase 
is an allosteric enzyme capable . of occurring in two forms namely an active a- 
phosphorylase and an inactive b-phosphorylase which are interconvertible (Steele 
1982). The vertebrate phosphorylases have been extensively studied (Graves and 
Wang 1972; Busby and Radda 1976). Similar studies have been carried out in a- 
number of insects and these studies have been reviewed by Steele (1982). However, 

49 



50 P M Chandrashekar and Geetha Bali 

not much is known about this enzyme in silkworms. The activity of glycogen 
phosphorylase has been studied at intervals in all the 3 types of eggs to examine 
whether the enzyme activity can always account for changes in glycogen levels and to 
see how the activity of the enzyme differs in diapause and non-diapause eggs. 



2. Material and methods 

2. 1 Materials 

Bivoltine (NB 4 D 2 ) and multivoltine (Pure Mysore) silkworm races were maintained 
under standard conditions. Diapause eggs of the bivoltines and non-diapause eggs of 
multivoltines were used for the experiments. Loose eggs were prepared on polythene 
sheets and kept at 25C with a relative humidity of 75%. 



2.2 Breaking of diapause 

For breaking diapause, 20 h old diapause eggs were treated with HC1 solution of 
specific gravity 1-075 at 46-TC for 3-4 min, washed thoroughly with water and dried. 
The eggs were then kept at 25 2C. 



2.3 Determination of glycogen in eggs 

Glycogen was determined by the method of Hassid and Abraham (1957) with slight 
modification. 



2.4 Enzyme preparation 

A 10% (w/v) homogenate of the eggs were prepared in ice cold distilled water with a 
glass homogenizer fitted with teflon pestle. The homogenate was filtered through a 
cotton pad and centrifuged at 5500 g for 15 min at 0C. The supernatant was 
filtered through Whatman No. 1 filter paper and the resultant filtrate was used as the 
enzyme source. 



2.5 Determination of enzyme activity 

The activity of phosphorylase was assayed according to the procedure of Cori et al 
(1973). The inorganic phosphate was estimated by Fiske and Subba Row (1925) 
method. The enzyme activity is expressed as ju mol P^/min/mg protein. Protein con- 
centration was determined according to the method of Lowry et al (1951) using 
Bovine serum albumin as standards. 

All the above experiments were conducted in 7-8 samples and the mean values 
expressed along with the standard deviation. 



Carbohydrate metabolism in silkworm eggs 



51 



3. Results 

3.1 Studies on glycogen levels 

At the time of egg laying, the level of glycogen was found to be more in diapause eggs 
than in non-diapause eggs (figures 1 and 2). 

In non-diapause eggs, the glycogen level decreased very gradually during the first 3 
days following oviposition and on the 3rd day, the glycogen content of the eggs was 
not much different from that of the newly laid eggs. Following this period, there was 
a more significant decrease and glycogen decreased to 60% of the initial content 
around the 9th day. 

In diapause eggs after acid treatment, with the onset of development glycogen level 
followed a pattern very similar to that observed in non-diapause eggs (figure 2). In 
diapause eggs, a pattern altogether different from the above two types was seen 
(figure 3). The glycogen content started decreasing markedly and suddenly 1 day 
after oviposition and it was reduced to 25% of the initial level within 4 days after 
oviposition. The glycogen level decreased to about 17% of the initial level by the 
10th day and it remained at this level for a long time (figure 4). 



- 25 - 



o 5 




0-07: 



0-05 1 

.5 



2468 
Duration in days after oviposition 



10 



0-01 .y 

I 



Figure 1. Changes in the glycogen level and glycogen phosphorylase activity during 
embryonic development in non-diapause eggs. 



35 



r - 



5 ~ 




J L 



0-07 



0-05 



0-03 



0-01 



2468 
Duration in days after ovipositiorv 



10 



Figure 2. Changes in the glycogen level and the glycogen phosphorylase activity during 
embryonic development in bivoltinc eggs after acid treatment. 



52 



P M Chandrashekar and Geetha Bali 



- 0-07- 




2 46 8 

Duration in days after oviposition 



10 



Figure 3. 

diapause 



Changes in the glycogen level and the glycogen phosphorylase activity during 
in bivoltine eggs. 



35 




10 2O 30 40 

Duration in days after oviposition 



50 



60 



Figure 4. Changes in the glycogen content of the bivoltine eggs during diapause. 



3.2 Studies on phosphorylase activity 

In non-diapause eggs, the phosphorylase. activity was low to start with after egg 
laying (figure 1). The- level started increasing about 3 days after oviposition and was 
highest at the time of hatching. 

In diapause eggs after acid treatment, the total phosphorylase activity was 
comparable to that observed in non-diapause eggs. There was an increase in the level 
of activity about 5 days after acid treatment and a maximum level was obtained 
around the 8th day after oviposition (figure 2). 

In diapause eggs, the level of activity soon after oviposition was slightly higher 
than that observed in non-diapause eggs. But the activity suddenly increased 1 2 h 
after oviposition to reach a peak on the 2nd day following which the activity 
decreased to 74% of the peak level which was more than the initial level and around 
this increased level it was maintained over a long period (figure 3). 



4. Discussion 

It is evident from our studies that the glycogen content of the diapause eggs at the 
time of oviposition is much 1 higher than that of non-diapause eggs. This may be an 



Carbohydrate metabolism in silkworm eggs 53 

adaptive phenomenon. Because, in non-diapause eggs the embryonic development 
proceeds and completes in about 10 days while in diapause eggs, considerably long 
period precedes the development and a demand for energy can be expected to occur 
during diapause even though to a smaller degree compared to that during embryonic 
development. 

The glycogen level gradually decreased in non-diapause eggs and similar decrease 
in glycogen level was also observed in diapause eggs after acid treatment, showing 
that glycogen may be an important source of energy during growth and differenti- 
ation in silkworm eggs. During the early phase of embryonic development, the non- 
diapause eggs provide a marked contrast to diapause eggs. While in non-diapause 
eggs, the glycogen level decreases very gradually, in diapause eggs rapid fall in 
glycogen level is observed. Thus, in diapause eggs though growth and development 
are temporarily suspended, glycogen is being broken down for some purpose. This 
marked decrease in glycogen level also coincides with the onset of diapause. These 
observations agree with those of Chino (1957) and Yamashita et al (1975). 

The activity of the enzyme phosphorylase shows a reciprocal relationship with the 
level of glycogen in all the 3 types of eggs. In diapause eggs phosphorylase activity 
reaches a very high level 2 days after oviposition explaining the rapid rate of 
glycogen break down associated with the initiation of diapause. 

Newsholme and Start (1973) have shown that glycogen phosphorylase plays an 
important role in the metabolism of glycogen in many species of animals. Our studies 
also indicate that glycogen phosphorylase plays an important role in the utilization 
of glycogen at the initiation of diapause. 

The pattern exhibited by phosphorylase activity, wherein it increases accompanied 
by decrease in glycogen level seems to be a general pattern in silkworms since the 
same was also reported in the eggs of Philosamia ricini (Pant and Nautiyal 1974). 
Interestingly, when non-diapause eggs of silkworm were subjected to anoxia, the 
glycogen phosphorylase activity was found to be abruptly elevated (Yamashita et al 
1981), showing that there is always a rapid break down of glycogen associated with 
the onset of diapause and that phosphorylase acts as a key enzyme involved in this 
glycogen utilisation. Hence, it is suggested that glycogen phosphorylase plays a key 
role in regenerating the glycosyl units either as a source of energy or as building 
blocks for biosynthetic reactions in silkworm eggs during the embryonic develop- 
ment and during diapause. 



Acknowledgements 

Thanks are due to the University Grants Commission, New Delhi for financial 
assistance to PMC under the FIP scheme. Thanks are also due to Dr K P Rajashekar 
and Miss D Manjula Kumari for technical help. 

References 

Busby S J W and Radda G K 1976 Regulation of the glycogen phosphorylase system From physical 

measurements to biological speculations; Curr. Top. Cell ReguL 10 89-160 
Chino H 1957 Carbohydrate metabolism in diapause eggs of the silkworm Bombyx mori. I. Diapause and 

the change of glycogen content; Embryologia 3 295-316 



54 P M Chandrashekar and Geetha Bali 

Chino H 1958 Carbohydrate metabolism in the eggs of the silkworm Bombyx mori. II. Conversion of 

glycogen into sorbitol and glycerol; J. Insect Physiol 2 1-12 

Cori G T, Illingworth B and Keller P J 1973 Muscle phosphorylase; Methods Enzymoi I 200-205 
Fiske C H and Subba Row Y 1925 The colorimetric determination of phosphorus; J. Biol. Chem. 66 375- 

400 
Graves P J and Wang J H 1972 Glucan phosphorylases chemical and physical basis of catalysis and 

regulation; Enzymes 7 435-482 

Hassid and Abraham 1957 Chemical procedures for analysis of polysaccharides; Methods Enzymoi. 3 34 
Karlson P and Sekeris C E 1964 Comparative Biochemistry (New York: AC AD Press) Vol. 6 pp. 180-220 
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement by the Folin phenol 

reagent; J. Biol. Chem. 193 265-275 

Newsholme E A and Start C \913Regulation in metabolism (London: John Wiley awd Sons) pp 80-98 
Pant R and Nautiyal G L 1974 Changes in protein, glycogen, free sugar content and active phosphorylase 

activity during embryogenesis of Phiiasamia ricini; Proc. Indian Acad. Sci. B80 121-126 
Steele J E 1982 Glycogen phosphorylase in insects; Insect Biochem. 12 131-147 
Yamashita O, Suzuki K and Hasegawa K 1975 Glycogen phosphorylase activity in relation to diapause 

initiation in Bombyx eggs; Insect Biochem. 5 7Q7-718 
Yamashita O, Yaginuma T and Hasegawa K 1981 "Hormones and metabolic control of egg diapause of 

the silkworm Bombyx mori (Lepidoptera: Bombycidae); Entomol. Gener. 7 195-211 



Proc. Indian Acad. Sci. (Anim. Sci.). Vol. 96, No. 1, January 1987, pp. 55-61. 
Printed in India. 



A shift in metabolic pathway of Sarotherodon mossambicus (Peters) 
exposed to thiodon (endosulfan) 

M VASANTHI and M RAMASWAMY 

Department of Zoology, Government Arts College, Coimbatore 641 018, India 

MS received 4 July 1986; revised 23 October 1986 

Abstract. Total oxygen consumption, glycogen content and succinic dehydrogenase levels 
in liver, muscle and heart in normal and thiodon-exposed (to sub-lethal concentration for 
48 h and to lethal concentration for 6h) Sarotherodon mossambicus (Peters) were studied. 
While oxygen consumption of the fish dropped to 43 and 35% respectively under sub-lethal 
and lethal exposures, the magnitude of decrease in tissue glycogen content as well as tissue 
succinic dehydrogenase level followed a uniform order of liver > muscle > heart in both the 
exposures. Reduction in oxygen uptake and tissue glycogen levels were indicative of the 
.onset of hypoxia involving utilization of stored glycogen by the fish under thiodon 
exposure. The inhibition of succinic dehydrogenase levels in the 3 tissues of thiodon-exposed 
Sarotherodon mossambicus, while indicating the impairment of aerobic metabolism, is also 
suggestive of a shift from aerobic to anaerobic metabolism in the fish under pollution stress. 

Keywords. Sarotherodon mossambicus', thiodon; succinic dehydrogenase; aerobic 
metabolism; anaerobic metabolism. 



1. Introduction 

Pesticides are known to affect the oxygen consumption and metabolic pathways of 
non-target species like fish in freshwater ecosystems. Effect of pesticides on the 
oxygen consumption of fish has been studied in Mystus vittatus (Reddy and 
Gomathy 1977) and Colisa lalia (Reddy et al 1977; Uthaman 1977). Utilization of 
stored glycogen by the fish under pesticide stress was reported in Heteropneustes 
fossilis (Qayyam and Shaffi 1977), Labeo rohita, Ophiocephalus punctatus and 
Clarias batrachus (Shaffi 1979), Channa punctatus (Shah and Dubale 1983) and 
Tilapia mossambica (Vasanthi 1983) exposed to different pesticides. Inhibition of 
succinic dehydrogenase (SDH) (EC 1-3-99-1) enzyme activity has also been uniformly 
observed in Tilapia mossambica (Koundinya and Ramamurthi 1978), Channa striatus 
(Natarajan 1981), H. fossilis (Dubale and Mohini Awasthi 1982) and T. mossambica 
(Kabeer et al 1983) under pesticide-exposed conditions. While the effect of pesticide 
on oxygen consumption, stored glycogen content and SDH activity levels of tissues 
were studied in different fishes exposed to different pesticides (providing only 
fragmentory informations), the present paper provides a comprehensive information 
on the effects of a particular pesticide on the metabolic pathway of a particular fish 
by studying total oxygen uptake, stored glycogen levels and SDH activity levels of 
liver, muscle and heart of normal S. mossambicus (Peters) and of those exposed to an 
organochlorine pesticide, thiodon (endosulfan). 

2. Materials and methods 

Thiodon, an organochlorine as well as organic sulphite pesticide, is in wide usage in 

55 



56 M Vasanthi and M Ramaswamy 

the eradication of cotton pest in Coimbatore District The pesticide washes from 
cotton fields were observed to pollute 4 major lakes in and around Coimbatore City 
which are the chief sources of inland fisheries. S. mossambicus, locally called as Jilebi 
Kendai, is the major fish variety available from these lakes throughout the year. 

Samples of S. mossambicus (5-9 g), obtained from 4 major lakes in and around 
Coimbatore City, were maintained in cement tanks at 28 1C and fed with cooked 
rice regularly. Feeding was discontinued one day before the experiment. 

A stock solution of technical grade thiodon (supplied as 'Hochesf product by 
Parry Limited, Bombay) in acetone was used to prepare different concentrations used 
in the static bioassay study. Sub-lethal (0-001 ppm) and lethal (OO05 ppm) concen- 
trations were determined employing repeated exposure experiments and Probit 
analysis method (Finney 1964). 

Fishes were exposed (in glass jars of 3 1 capacity) to 0-001 ppm thiodon for 48 h 
and to 0-005 ppm thiodon for 6 h. The pesticide water containing 0-001 ppm thiodon 
was renewed every 12 h to maintain the dissolved oxygen content and pesticide 
concentration constant throughout the period of exposure. Throughout this 
investigation, control fishes were also treated with 0-25 ml acetone/1 which formed 
the largest aliquot of stock used in the pesticide water. 

2. 1 Measurement of oxygen consumption 

Oxygen consumption of control and thiodon-exposed (TE) fish was measured using 
a simple respiratory chamber by measuring the loss of oxygen content (due to the 
respiration of the fish) of water in the respiratory chamber. Oxygen content of water 
sample was estimated using Winkler's method (Welsh and Smith 1960). The oxygen 
consumption of the fish was expressed in ml/kg/h. 

Weighed samples of liver, muscle and heart tissues were dissected out from the 
control and TE fish by keeping the stunned (by a blow on the head) fish in an iced 
trough and used for estimation of glycogen content and SDH activity levels, 

2.2 Estimation of glycogen content 

The glycogen content of liver, muscle and heart was estimated by employing the 
method of Kemp and Kits (1954). The weighed tissue sample was homogenized in 
5 ml of 80% methanol and centrifuged. The residue was collected and treated with 
5 ml of 5% tricarboxylic acid (TCA) (w/v). The mixture was boiled for 5 min in a 
water bath, again made upto 5 ml by adding 5% TCA and centrifuged. To 2 ml of 
the supernatant, 6 ml of concentrated sulphuric acid (Analar grade) was added and 
heated in a boiling water bath for exactly 6-5 min. The final mixture was cooled and 
the colorimetric reading was obtained at 530 p using a photoelectric colorimeter 
(Erma, AE-11. model, Japan). Using the colorimetric readings, glycogen values were 
obtained from a standard graph already prepared using known glucose standard 
solutions. The glycogen contents of tissues were expressed in mg/g of tissue. 

2.3 Estimation of SDH activity 

SDH enzyme activity level of the 3 tissues were estimated using the method of 



S. mossambicus exposed to thiodon 57 

Nachlas et al (1960). For the assay of SDH activity, a 5% homogenate of the tissue 
was prepared with 0-25 M cold sucrose solution. The homogenate was centrifuged at 
3000 rpm for 1 5 min to remove the cell debris. Enzyme reaction mixture was 
prepared with 0-1 ml of 0-5 M sodium succinate; 1-0 ml of phosphate buffer (pH 74); 
1-0 ml of INT solution [(2-p-iodophenyl)-(3-/?-nitrophenyl-5-phenyl tetrasolin 
chloride)]; 0-5 ml of 5% homogenate supernatant and 04 ml of distilled water. The 
reaction was stopped after 30 min of incubation at 37C by adding 6-0 ml of acetic 
acid. The formazon formed was extracted into 6*0 ml of toluene after the mixture was 
kept in freeze for 12 h. The optical density (OD) of pink colour formed was read at 
495 jU using a photoelectric colorimeter (Erma, AE-11 model, Japan). A reagent 
blank (to set the colorimeter) was prepared as above except 0*5 rnl of supernatant. 
Instead, 0-5 ml of distilled water was added. From a standard graph of formazon 
formed, /zmol of formazon formed in the enzyme reaction mixture was obtained. 
SDH activity levels of the tissues were given as jumol formazon/mg tissue/h. 

Changes in oxygen consumption, tissue glycogen content and SDH activity levels 
of TE fish from that of control fish were expressed in percentages and tested for 
statistical significance using Student's V test. 

3. Results 

Oxygen consumption of control and TE S. mossambicus are presented in table 1. 
Table 2 provides the glycogen content in liver, muscle and heart of control and TE 
fish. Levels of SDH activity in liver, muscle and heart of control and TE fish are 
given in table 3. 

S. mossambicus showed a 43% reduction in oxygen consumption when exposed to 
sub-lethal concentration (0-001 ppm) of thiodon for 48 h. When exposed to lethal 
concentration (0-005 ppm) of thiodon for 6 h, the fish also showed a significant drop 
in oxygen uptake to the tune of 35% (table 1). 

Of the 3 tissues examined, liver appears to be the major site of stored glycogen 
content. Exposure of S. mossambicus to thiodon concentrations caused significant 
drop in glycogen content of all the 3 tissues studied. Under sub-lethal exposure, the 
drop in glycogen content of liver, muscle and heart were 78, 73 and 67% respectively. 
When exposed to lethal concentration of thiodon, the liver showed a minimum 9% 
drop in its glycogen content which was statistically not significant. This indicates 



Table 1. Oxygen uptake of control and thiodon-exposed 5. 
mossambicus expressed in ml/kg/h S.E. 



Thiodon-exposed (ppm) 


Control 


0-001 


0-005 


2 10-00" 20- 12 


H8-90 b 16-38 
(-43) 
P<0-01 


1 37-20 17-91 
(-35) 
?<0-05 



"Mean value of 6 observations; *Mean value of 5 observations; c Mean 
value of 7 observations. 

'-' denotes % decrease from control level. (% Changes from control 
levels are given in parentheses). 



58 M Vasanthi and M Ramaswamy 

Table 2. Glycogen levels in liver, muscle and heart of control and 
thiodon-exposed S. mossambicus expressed in mg/g of tissue S.E. 



Thiodon-exposed (ppm) 


Tissues 


Control 0-001 


0-005 


Liver 


4-69 0-38 1-05 0-10 
(-78) 
P<0-01 


4-28 0-66 
(-9) 

NS 


Muscle 


2-33 0-04 0-63 0-10 
(-73) 
P<0-05 


0-48 0-05 
(-79) 
P<0-01 


Heart 


2-400-10 0-79 0-12 
(-67) 
P<0-01 


0-55 0-05 . 
(-77) 
P<0-01 



NS, Not significant statistically (P>0-05). 

'-' denotes % decrease from control level. 

Values are means of 7 observations (% changes from control levels 

are given in parentheses). 



Table 3. SDH activity in liver, muscle and heart of control and 
thiodon-exposed S. mossambicus, expressed in /rniol formazon/mg 
tissue/h. 



Thiodon-exposed (ppm) 


Tissues 


Control 0-001 


0-005 


Liver 


0-570-17 0-180-02 
(-68) 
P<0-01 


0-37 14 
(~35) 
P<0-01 


Muscle 


0-190-03 0-17 0-01 
(-11) 
P<0-01 


0-1 7 0-03 
(-11) 
P<0-01 


Heart 


0-29 0-02 0-26 0-06 
(-10) 
P<0-01 


0-28 0-08 
(-4) 

NS 



NS, Not significant statistically (P>0-05). 

'.- 1 denotes % decrease from control level. 

Values are means of 6 observations S.E. (Changes from control 

levels are given in parentheses). 



that utilization of stored liver glycogen occurs only upon prolonged exposure. On 
the other hand, muscle and heart tissues showed increased magnitudes of drop in 
glycogen contents to about 79 and 77% respectively (table 2) which implies that 
utilization of stored glycogen content increased with increase in concentration of 
pesticide irrespective of the period of exposure. This variation in the drop in 
glycogen content of the 3 tissues indicates that different tissues behave differently 
under pesticide exposure. 

The data on tissue SDH activity levels clearly indicate an inhibition of SDH 
activity to about 68, 11 and 10% respectively in liver, muscle and heart tissues of 



S. mossambicus exposed to thiodon 59 

S. mossambicus exposed to sub-lethal concentration of thiodon (table 3). However, 
exposure of fishes to lethal concentration of thiodon elicited only a lesser magnitude 
of drop in SDH activity level in the liver tissue (35%). Muscle tissue showed a similar 
drop in SDH level as that of 0-001 ppm TE fishes under lethal exposure (11%). The 
non-significant minimum drop (4%) in SDH activity level of heart tissue under lethal 
exposure of the fish is noteworthy for discussion (table 3). 

4. Discussion 

A perusal of table 1 clearly indicates that exposure of fish to sub-lethal and lethal 
concentrations of thiodon caused a definite reduction in oxygen uptake of the fish. A 
similar depression in oxygen uptake has been reported in C. lalia (Reddy et al 1977; 
Uthaman 1977) and Mystus vittatus (Reddy and Gomathy 1977) exposed to different 
pesticides. The decreased oxygen uptake in TE S. mossambicus (in the present study) 
could be suggested as a sequel to gill damage upon pesticide exposure as reported in 
C. striatus (Natarajan 1981) or due to hypochromic microcytic anaemia caused due 
to pesticide toxicity as reported in S. mossambicus (Koundinya and Ramamurthi 
1979), C. striatus (Natarajan 1981) and C. batrachus (Goel et al 1984). The drop in 
oxygen uptake of TE S. mossambicus, observed in the present investigation, indicates 
the onset of a severe hypoxia in the fish following pesticide exposure which will 
trigger on some biochemical changes in different tissues of the fish body. 

Anoxia or hypoxia is known to increase carbohydrate consumption (Dezwaan and 
Zandee 1972). The unequivocal reductions in stored glycogen content in liver, muscle 
and heart of 0-001 ppm TE fish and in muscle and heart of 0-005 ppm TE fish 
indicate the utilization of stored glycogen possibly through anaerobic glycolysis to 
meet the energy requirement under hypoxia caused due to pesticide stress. A similar 
reduction in stored tissue glycogen content has been reported in H.fossilis exposed 
to mercuric nitrate (Qayyam and Shaffi 1977), L. rohita, 0. punctatus and C. batrachus 
following copper sulphate intoxication (Shaffi 1978), A. scandens exposed to zinc 
sulphate (Natarajan 1981), T. mossambica exposed to lindane (Vasanthi 1983) and 
methyl parathion (Siva Prasada Rao and Ramana Rao 1979) and C. punctatus 
exposed to malathion (Shah and Dubale 1983). 

The data on tissue SDH activity levels, obtained in the present study (table 3), 
clearly indicate an inhibition of SDH activity in liver, muscle and heart of 0-001 ppm 
TE fish and in liver and muscle of 0-005 ppm TE fish. A similar drop iirSDH activity 
levels of different tissues has also been reported in S. mossambicus exposed to lindane 
(Lakshmi 1984), C. striatus exposed to metasystox (Natarajan 1981), H. fossilis 
(Dubale and Mohini Awasthi 1982) and T. mossambica under malathion stress 
(Kabeer et al 1983). SDH being a key enzyme in TCA cycle, its inhibition under toxic 
condition, as observed in liver, muscle a&d heart of S. mossambicus exposed to sub- 
lethal concentration of thiodon (in the present study) and also reported in other 
fishes exposed to different pesticides could be considered as indication of the fish 
showing a shift towards anaerobic metabolism (utilizing stored metabolites like 
glycogen) under toxic conditions. Based on SDH and LDH studies, Koundinya and 
Ramamurthi (1978) suggested the operation of anaerobic glycolysis in different 
tissues of T. mossambica exposed to sumithion. Exposure of fish to pesticide, thus, 
causes impairment of an important enzyme system (SDH activity) besides other toxic 
effects. 



60 M Vasanthi and M Ramaswamy 

The observed insignificant 4% drop in SDH activity level of heart tissue under 
lethal exposure of the fish, inspite of a significant 77% reduction in stored heart 
glycogen content under similar exposure, indicates a continued operation of aerobic 
breakdown of glycogen in the heart tissue to meet energy demand during early 
periods of exposure. This further indicates that the heart is aerobic possibly due to 
increased oxygen supply to the heart in order to prevent the onset of lactic acidosis 
in the heart tissue which will occur normally during hypoxic conditions. Thus the 
heart tissue is found adaptive in maintaining itself physiologically fit for effective 
cardiac function during early periods of pesticide exposure. 

In conclusion, it could be stated that exposure of S. mossambicus to thiodon 
elicited a severe hypoxia resulting in the utilization of stored glycogen by way of 
anaerobic glycolysis to meet the energy demand during pesticide stress. The 
inhibition of SDH activity levels in different tissues of TE S. mossambicus, while 
suggesting the failure of aerobic metabolic pathway, also hints upon the possibility of 
a shift from aerobic to anaerobic mode of energy metabolism in tissues. 

Acknowledgements 

The authors are grateful to Dr V Subramaniam and to Prof. S Sambandam, Depart- 
ment of Zoology, Government Arts College, Coimbatore for providing facilities. 

References 

Dezwaan A and Zandee D I 1972 The utilization of glycogen and accumulation of some intermediates in 

anaerobiosis in Mytilus edulis L; Comp. Physioi Biochem. B43 47-54 
Dubale M S and Mohini Awasthi 1982 Biochemical changes in the liver and kidney of a cat fish, 

Heteropneustesfossilis exposed to dimethoate; Comp. Physioi. EcoL 7 111-114 
Finney D J 1964 Probit analysis; 2nd edition (London: Cambridge University Press) p 20 
Goel K A, Kalpana Sandhya and Agrawal U P 1984 Alachlor toxicity to freshwater teleost, Clarias 

batrachus; Curr. Sci. 33 1050-1052 
Kabeer Ahamed Sahib I, Sambasiva Rao K R S and Ramana Rao K V 1983 Dehydrogenase systems of 

the teleost, Tilapia mossambica under augmented sublethal malathion stress; J. Anim. Morphol Physioi 

30 101-106 
Kemp A and Kits Vanheijnigen AIM 1954 A colorimetric micromethod for the determination of 

glycogen in .tissues; J. Biochem. 56 646-648 
Koundinya P R and Ramamurthi R 1978 Effect of sumithion (fenitrothion) on some selected enzyme 

systems in the fish Tilapia mossambica (Peters); Indian J. Exp. Biol 16 809-811 
Koundinya P R and Hamamurthi R 1979 Haematological studies in Sarotherodan (Tilapia) mossambicus 

exposed to lethal (Lc50/48 hrs) concentration of sumithion and sevin; Curr. Sci. 48 877-879 
Lakshmi D 1984 Effect of Undone (y-BHC) on the SDH activity of different tissues of Sarotherodon 

mossambicus (Peters), M. Sc., Dissertation, Bharathiaf University, Coimbatore 
Nachlas M M, Margulies S I and Seligman A M 1960 Sites of electron transfer to tetrazolin salts in the 

succin-oxidase systems; J. Biol. Chem. 235 2739 
Natarajan G M 1981 Effect of lethal (Lc50/48 hrs) concentration of metasystox on selected oxidative 

enzymes tissue respiration and histology of gills of the freshwater air-breathing fish, Channa striatus 

(Bleeker); Curr. Sci. 50 985-989 
Qayyam M A and Shaft! S A 1977 Changes in tissue glycogen of a freshwater cat fish, Heteropneustes 

fossilis (Bloch) due to mercury intoxication; Curr. Sci. 46 652-653 
Reddy T G and Gomathy S 1977 Toxicity and respiratory effects of the pesticide, thiodon on catfish, 

Mystus vittatus; Indian J. Environ. Health. 19 360-363 
Reddy T G, Palaniappan S P and. Pillai K P S 1977 Respiratory and histopathological effects of the 

pesticide, disyston on the anabantid fish, Colisa laliai All India Symposium on Environmental Biology, 

University of Kerala, 27-29 December 1977 



S. mossambicus exposed to thiodon 61 

Shaffi S A 1979 Acute toxicity of Heptachlor for freshwater fishes; Toxicol Lett. 4 31 

Shah P H and Dubale M S 1983 Biochemical changes induced by malathion in the body organs of 

Channa punctatus; J. Anim. Morphol. Physiol. 30 107-1 18 
Siva Prasada Rao K and Ramana Rao K. V 1979 Effect of sublethal concentration of methylparathion on 

selected oxidative enzymes and organic constituents in the tissues of the freshwater fish Tilapia 

mossambica (Peters); Curr. ScL 4$ 526-528 
Uthaman M 1977 Toxicity and respiratory effects of the pesticide BHC in an anabantid fish, Colisa lalia, M. 

Sc. Dissertation, Annamalai University, Annamalainagar 
Vasanthi R 1983 Effect of lindane (y-BHC) on the tissue alycogen content of a freshwater teleost, Tilapia 

mossambica (Peters), M. Sc. Dissertation, University of Madras, Madras 
Welsh J H and Smith R I 1960 Laboratory Exercise in Invertebrate Physiology (Minneapolis: Burgess 

Publishing Company) 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 1, January 1987, pp. 63-69. 
Printed in India. 



Aspects of culture: Acanthamoeba astronyxis (Ray and Hayes 1954) 
from Bay of Bengal coasts, India 

MRINAL GHOSH* and AMALESH CHOUDHURYt 

*Ecology Division, Zoological Survey of India, 8, Lindsay Street, Calcutta 700 087, India 
*tSD Marine Biological Research Institute, Sagar Island, West Bengal, India 
*fDepartment of Marine Science, University of Calcutta, 35 Ballygunge Circular Road, 
Calcutta 700 019, India 

MS received 3 May 1986 

Abstract. Acanthamoeba astronyxis strains, isolated from intertidal belts of Sagar Island 
(2130' to 2T50' N and 8804' to 88()8' E) facing Bay of Bengal, were cultured in saline 
distilled water agar medium supplemented with Escherichia coli as food bacterium. Popu- 
lation growth in bay water agar, cer.ophyl bay water agar and soil extract distilled water 
agar were compared with special reference to devised saline distilled water agar medium. 
The overall growth showed little variation over ranges of media salinity (5-30% ). However, 
greater values were encountered in media of intermediate range. Isolate from mangrove 
litter-soil of highest high tide belt (strains) 'grew more efficiently in cerophyl bay water agar 
and soil extract distilled water agar media while mid-littoral and lowest low tide isolates 
preferred bay'water agar and saline distilled water agar media more. 

Keywords. Acanthamoeba astronyxis; growth comparison. 



1. Introduction 

Free-living small naked amoebae are ubiquitous in their distribution and Singh 
(1973) contends that soil constitutes a reservoir for them to disseminate in other 
environments. Bovee and Sawyer (1979) put forward the idea that their wide distri- 
bution in coastal and estuarine benthic habitats is owing to wave action and ocean 
currents. 

The maintenance of a continuous culture for amoebae is a prerequisite for any 
planned study of amoeba biology as conventional standard methods are lacking to 
preserve them in their typical locomotive forms. Therefore, samples must be cultured 
to determine their growth under varying conditions. Several workers have proposed 
and successfully used different media to culture amoebae isolated from all probable 
sources of natural environments (Adams 1959; Bovee and Sawyer 1979; Chang 1958; 
Davis et al 1978; Dixon 1937; Fulton 1970; Menapace et al 1975; Page 1967, 1970; 
Prescott 1956; Prescott and James 1955; Sawyer 1971, 1975; Singh 1945, 1955). 

One of the most predominant genera of naked amoebae, Acanthamoeba is 
relatively tolerant of high osmolarities and has often been found in salt water (Page 
1981). Species belonging to this genus have repeatedly been isolated as a predomi- 
nant microbenthic component from Gangetic estuary facing bay (Bhattacharya et al 
1985; Choudhury and Ghosh 1985; Ghosh and Choudhury 1985a, b, c, 1986; Ghosh 
et al 1985). The present investigation has been envisaged to culture and study the 
growth of A. astronyxis strains, isolated from intertidal flats of bay coast in media of 
different salinities and also of different ingredients. 

63 



64 Mrinal Ghosh and Amalesh Choudhury 

2. Materials and methods 

2.1 Sampling 

Samples upto 3 cm depth from different littoral belt areas during the monsoonal 
period (July to October) were collected aseptically and transported to the laboratory 
and were processed soonafter. 

The highest high tide samples were typically litter-soils with decaying leaves of 
Excoecaria agallocha L., Avicennia sp., Phoenix paludosa Roxb, Acanthus ilicifolius L. 
etc. The mid-littoral belt was characterized by hard loamy clay with dead and 
decaying roots of the recently deforested 'Mangal'. The lowest low tide samples were 
typically sand-silt type. 

2.2 Isolation and identification of A. astronyxis strains 

Samples from the 3 zones were inoculated on Escherichia coli patch in respective soil 
extract distilled water agar (SEDWA) petri dishes. Establishment of A. astronyxis 
clones corroborated the observations of Singh and Hanumaiah (1979). 

23 Food source 

Laboratory maintained E. coli was used as food. The ingredients of the culture media 
were: 1 1 of glass distilled water (pH 64), 20 g NaCl, 20 g Bacto Agar (Difco Labs., 
USA), 2-60 g Na 2 HPO 4 , 2 g glucose and 20 g Bacto Tryptone (Difco Labs., USA). 

2.4 The supporting, media 

2.4a Media of different salinity gradients: Saline distilled water agar (SDWA) media: 
Each of the 3 sets of experiments require 6 media of different salinity strengths 
ranging from 5 - 30%o (w/v) at 5% intervals. To each of the 6 containers, 1 1 of glass 
distilled water was taken and pH was adjusted to 8*3 which was found to be the 
approximate mean value of bay water during monsoon. NaCl of the quantities 5, 10, 
15, 20, 25 and 30 g were then added separately to each container along with 15 g 
agar. They were boiled, autoclaved and dispensed in sterile 7-5 cm dia petri dishes in 3 
replicates for each medium. 

2.4b Bay water agar (BWA) medium: 15 g of agar was added to 1 1 of filtered bay 
water, boiled and autoclaved. pH was adjusted as before. 

2.4c Cerophyl bay water agar (CBWA) medium: This was prepared after Page (1970) 
with minor modifications. 1 g of cerophyl (Cerophyl Lab. Inc., USA) was boiled in 
1 1 of filtered bay water, filtered thrice through glass wool. 15 g of agar was then 
added, boiled and autoclaved. pH was adjusted as before. 

2.4d Soil extract distilled water agar (SEDWA) medium: This was prepared after 



Growth of saline isolate of A. astronyxis in different media 65 

Singh (1975) with minor modifications. 400 g of littoral belt samples were boiled in 
1 1 of glass distilled water for 30 min, extracts were decanted, filtered and finally made 
to 1 1. 15 g of agar was then added to each, boiled and autoclaved. pH was adjusted 
as before. 

2.5 Modified Page's amoeba saline (PAS) 

For organisms growing in SDWA media the chemical ingredients of the PAS were 
same as in Page (1966) except NaCl which was adjusted as per the salinity of the 
respective fluid base of SDWA medium. To 1 1 of glass distilled water the following 
components were added :MgSO 4 .7H 2 O, 0-004 g; CaCl 2 .2H 2 O, 0-004 g; Na 2 HPO 4 , 
0-142 g and KH 2 PO 4 , 0-136 g. For the organisms from all other media, chemicals 
other than NaCl were added to respective fluid bases. 

2.6 Experimental methodology 

E. coll from young cultures were used as food. They were suspended in as many tubes 
as the sets of experiments containing respective sterile modified PAS. After 
centrifugation and triple wash, the pellets were resuspended to make a final wet 
weight suspension qf 0-05 g/ml. 

To 3 replicate plates for each experiment, E. coli suspensions were added, so that 
each received about 0-01 g wet weight of microorganisms. Excess fluid was allowed 
to absorb. Cyst from each clonal culture was then inoculated on media plates and kept in 
BOD incubator at 25 1C. Efficacy of tfye media used, to support the growth of 
A. astronyxis strains under study was assessed as follows. 

The medium was considered to be non-efficient if the mean number of organisms 
in culture of 6 fields of 2 replicate plates is less than 25 per microscopic field of 100 x 
after desired periods of 3, 7, 14, 21, 28, 35 and 42 days. One moderately efficient 
medium would show a value ranging between 25 and 100 under such condition. 
While a highly efficient medium exhibits more than 100 organisms per field. The 
overall growth was enumerated after 100 days of culture using haemocytometer 
(mean of 3 replicate plates). Some organisms may have been lost while harvesting. 



3. Results 

The efficiency of different SDWA media to support the growth of A. astronyxis 
strains is shown in figure 1. Within 3 days in media having salinities 10, 15 and 25%o 
the amoebae showed moderately efficient growth . for lowest low tide (LL) strains 
while the 20%o medium exhibited highly efficient growth. Poor growth for the same 
strain was observed in 5 and 30% salinity media. 

Mid-littoral (ML) strains showed very efficient growth in media ranging from 
10-20% salinities. Moderate growth was observed in media of 5 and 25%o salinities. 
Within the same period poor growth was encountered in 30%o saline medium. All the 
media except 30%o promoted efficient growth within 3 days for high tide belt (HH) 
strains. 30%o saline medium showed poor growth during this period. Within 7 days, 
most of the media promoted highly efficient growth. The exceptions were 5% 



66 



Mrinal Ghosh and Amalesh Choudhury 



LJLHIHH 
^trains 



"300 




Figure 1. Efficiency of the media of different salinity gradients to support growth of 
A. astronyxis strains. NE (Non-efficient media): Average count in 6 fields (IQOx) of 2 
replicate plates upto 25 organisms; ME (Moderately efficient media): This value lies between 
25 and 100; HE (Highly 'efficient media): More than 100 organisms. 



30.0 




Strains 



Figure 2. Overall growth of A. astronyxis strains after 100 days of culture in media of 
different salinity strengths. 



medium for LL strains, and 30%o medium for fcoth ML and HH strains which show 
moderately efficient growth. Then onwards all the media supported highly efficient 
growth for all the investigated strains. The growth efficiency in 4 selected media viz 
SDWA (20%o), BWA, CBWA and SEDWA were also observed. It is evident that all 
the media initiated highly efficient growth within 3 days. Two bar diagrams are 
shown in figures 2 and 3. The former depicts growth in SDWA media. Average mean 
values range between 0-2 to 1-3 x 10 6 . Media of intermediate salinities supported 
maximum growth. The latter presents growth in 4 selected media. Average growth 



Growth of saline isolate of A. astronyxis in different media 



67 




LLMLHH 
Strains 

Figure 3. Growth of A. astronyxis strains in 4 selected media: SDWA, BWA, CBWA and 
SEDWA. 



ranges between 1-1 to 2-1 x 10 6 . Maximum values for HH strain were obtained in 
CBWA and SEDWA media while ML and LL strains grew better in BWA and 
SDWA media. 



4. Discussion 

Amoebae are frequent in marine neuston (Sieburth et al 1976; Sieburth 1979) and 
benthic situations (Sawyer 1980) and are important epibiotic fauna preying upon 
phytoplankton and bacteria (Bamforth 1981). Their importance in the oceanic eco- 
system has only recently begun to be assessed as Bovee and Sawyer (1979) point out 
that bactivorous and the scavenging forms probably maintain the cleanliness of 
shallow inshore water and the surface water of open ocean. Page (1980) points to the 
paucity of data concerning the ecological role of naked amoebae as marine and 
estuarine benthos. 

The media described in the present investigation have been used to culture 
amoebae isolated from diversified samples, including typical litter-soil, hard loamy, 
silt-clay and the sand types. Comparison of population growth shows that they do 
not encyst on challenge with 'salt stress' but adjust and quickly cope up with the new 
environment. These observations are in accord with a general impression that 
intertidal fauna, experiencing estuarine fluctuating environment are in need to 
acquire a certain degree of euryhalinity as an insurance against such conditions. A 
harmonious ecological balance thus is required between environmental conditions 
and the tolerance of organisms to variations in one or more of the said conditions to 
successfully colonize and maintain a population. 

After studying in detail the growth of A. astronyxis strains in media of different 
components and proportions, we would like to propose finally that, since little 
variations in growth were exhibited by different media, those of use should initiate 
highly efficient growth within the shortest period. Moreover, it would certainly be a 



68 Mrinal Ghosh and Amalesh Choudhury 

good practice to avoid media from either of the extremities, atleast for typical deltaic 
samples, like the ones in the present investigation. 

Acknowledgements 

MG is grateful to Dr B K Tikader, Zoological Survey of India, for laboratory faci- 
lities and the award of a DOEn fellowship. 

References 

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Bamforth S S 1981 Protist Biogeography; J. Protozooi. 28 2-9 

Bhattacharya A, Ghosh M and Choudhury A 1985 Seasonal population dynamics of Acanthamoeba 

rhysodes (Singh 1952) in a mangrove litter-soil ecosystem of lower deltaic Bengal, India; Abstr. Vll Int. 

Cong. Protozooi,, Nairobi, Kenya 
Bovee E C and Sawyer T K 1979 Marine Flora and Fauna of the Northeastern United States. Protozoa: 

Sarcodina: Amoebae; NOAA Technical Report, NMFS Circular 419 
Chang S L 1958 Cultural, cytological and ecological observations on the amoeba stage of Naeyleria 

yruberi; J. Gen. Microbioi 18 565-578 
Choudhury A and Ghosh M 1985 Benthic gymnamoebae as micropredator components in a -non- 

reclaimed mangrove swamp of Gangetic-estuary, India; Abstr. Vll Int. Cong. Protozooi., Nairobi, 

Kenya 
Davis P G, Caron D A and Sieburth J Me N 1978 Oceanic amoebae from the North Atlantic. Culture, 

Distribution and Taxonomy; Trans. Am. Microsc. Soc. 97 73-88 

Dixon A 1937 Soil protozoa; their growth on various media; Ann. Appl. Biol. 24 442-456 
Fulton C 1970 Amoebo-flagellates as research partners: the laboratory biology of Naegleria and 

Tetramims; in Methods Cell PhysioL (ed) D M Prescott (New York: Academic Press) 4 341-476 
Ghosh M and Choudhury A 1985a Cultiiriny gymnamoebae from saline environments of Hooghly estuary, 

India; Abstr. II Nati Seminar on Mar. Intertidal EcoL, Waltair 
Ghosh M and Choudhury A 1985b Acanthamoeba rhysodes (Singh, 1952) (Protozoa: Gymnamoebia) 

from mangrove environment as model organism in systems ecology: I. Population growth kinetics in 

relation to media salinity; Abstr. I Natl. Symp. on Biol. Utiliz. andConser. of Mangroves, Kolhapur 
Ghosh M and Choudhury A 1985c Acanthamoeba rhysodes (Singh 1952) (Protozoa: Gymnamoebia) from 

mangrove environment as model organism in systems ecology: II. 'Food' as it modulates the 

population growth kinetics; Abstr. I Natl. Symp. on Biol. Utiliz. and Conser. of Mangroves, Kolhapur 
Ghosh M and Choudhury A 1986 Growth of Acanthamoeba culbertsoni (Singh and Das 1970) and 

A. rhysodes (Singh 1952) (Protozoa: Gymnamoebia) under simplified co-cultural condition; Proc. 

Indian Acad. Sci. (Anim. Sci.) 95 469-473 
Ghosh M, Bhattacharya A and Choudhury A 1985 Competition amongst intertidal benthic 

Acanthamoeba spp. of Hooghly estuary, India; Abstr. Vll Int. Cony. Protozooi., Nairobi, Kenya 
Menapace D, Klein D A, McClellan J F and Mayeux J V 1975 A simplified overlay plaque technique for 

evaluating responses of small free living amoebae in grassland soil; J. Protozooi. 22 405-410 
Page F C 1966 Crypt odiffluyia operculata n. sp. (Rhizopodea: Arcellinida, Cryptodifflugiidae) and the 

status of the genus Cryptodiffluyiai Trans. Am. Microsc. Soc. 85 506-515 
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and 3 of Vahlkampfia: J. Protozooi. 14 499-521 

Page F C 1970 Two new species of Paramoeba from Maine; J. Protozooi. 17 421-427 
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Page F C 1981 A light and electron microscopical study of Protacanthamoeba caledonica n. sp., Type- 
species of Protacanthamoeba n. g. (Amoebida, Acanthamoebidae); J. Protozooi. 28 70-78 
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Carlsbery Ser. Chim. 30 1-12 
Prescott D M and James T W 1955' Culturing of Amoeba proteus on Tetrahymena; Exp. Cell Res. 8 256- 

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Growth of saline isolate of A. astronyxis in different media 69 

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95 159-188 
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Maryland; Trans. Am. Microsc. Soc. 90 43-51 
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and nine new species within the family Mayorellidae, Flabeliulidae and Stereomyxidae; Trans. Am. 

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III, Johnson P W and Davis P G 1976 Dissolved organic matter and heterotrophic microneuston in 

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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 1, January 1987, pp. 71-76. 
(D Printed in India. 



Development and survival of Mythimna separata (Wlk.) on some host 
plants during winter at Hisar 

RAM SINGH and J P CHAUDHARY 

Department of Entomology, Haryana Agricultural University, Hisar 125 004, India 

MS received 18 January 1986; revised 25 October 1986 

Abstract. Survival and post embryonic development of the oriental armyworm, Myttiimna 
separata (Wlk.) was studied on 5 host plants, viz sorghum, maize, sugarcane, Johnson grass 
and napier grass during winter months (15 November to 7 March) under laboratory condi- 
tions. Variations in different parameters like larval survival (10-3-60-3%), size of larvae 
(7-33-29-67 mm), larval weight (25-5-493-1 mg for 26 days old larva), size of pupa 
(15-0-1 6-49 mm), pupal weight (184-9-249-5 mg for 2-4 days old pupa), pupation 
(3-3-38-9%), developmental period (74-8-109-8 days) and moth emergence (3-4-50-0%) were 
significant on different foods. Low temperature and humidity conditions during experimen- 
tal period resulted in complete mortality of female pupae reared on napier grass, Johnson 
grass and maize whereas in case of males 60-100% moths were crippled winged. However, 
adults emerged on sorghum and sugarcane foods were normal except 28-7% males in 
sorghum were crippled. Total growth index was also higher on sugarcane (0-45) and 
sorghum (0-24) indicating their suitability in carrying over of winter pest population. 

Keywords. Armyworm; Mythimna separata; host plant; growth index. 

1. Introduction 

The oriental armyworm, Mythimna separata (Wlk.) (Lcpidoptera:Noctuidae) is a 
ubiquitous pest of cereal crops and grasses in Asia and Australia. About 16 serious 
outbreaks of the pest have been recorded in India from 1924-1981 (Sharma and 
Da vies 1983). In northern part of the country it has been reported to cause consi- 
derable damage intermittently to many graminaceous crops (Chaudhary and 
Ramzan 1967; Sarup et al 1969; Verma and Khurana 1971; Bindra and Singh 1973; 
Butter et al 1979; Chaudhary and Singh 1980; Singh and Manchanda 1981). This 
armyworm in recent years has started assuming serious proportions quite frequently 
and little information is available on the extent of survival of winter generation of 
this pest on various host plants. Keeping this in view, the present investigations were 
carried out. 



2. Material and methods 

Studies were carried out on 5 host plants, viz Sorghum (Sorghum bicolor (L.) 
Moench), maize (Zea mays L.), sugarcane (Saccharum officinarum L.), Johnson grass 
(Sorghum halepense (L.) Pers) and napier grass (Pennisetum purpureum Schumach) 
from 15 November 1984 to first week of March 1985 under fluctuating laboratory 
conditions at the University. Freshly hatched larvae of armyworm were fed on fresh 
young leaves of these grasses. First instar larvae were taken from the mass culture 
maintained in laboratory on leaves of pearl millet and maize. Small pieces of fresh 
leaves of each grass were put in glass jars (20 x 15cm). In each jar, 100 freshly 

71 



72 Ram Singh and J P Chaudhary 

hatched larvae for each test plant in 3 replications were released. These jars were 
covered with muslin cloth and placed under laboratory conditions. Temperature and 
humidity prevailing in the laboratory were recorded daily with the help of 
maximum-minimum, dry and wet bulb thermometers, respectively. The fresh food 
was provided daily. Various developmental parameters, viz larval survival, dura- 
tions, size and weight; pupation, pupal weight, size and durations; pre-pupal 
duration, moth emergence, adult longevity etc were recorded for each food separa- 
tely. Sexing was done in pupal stage as suggested by Avasthy and Chaudhary (1964). 
The growth indices on different foods were calculated by the following formulae 
(Sharma et al 1982). 

Pupation (%) 
Larval growth index = 



Total growth index = 



Mean larval period (days) 

Moth emergence (%) 



Mean total developmental period (days) 

3. Results and discussion 

From the data summarised in table 1 , larval survival when recorded after 5 days of 
release, ranged from 74-7-97-0% on different foods without any significant difference 
among them. Thereafter, it decreased gradually except in case of sugarcane where 
only 10-3% survival was observed on 22nd day of feeding. Sugarcane though a 
potential host plant of armyworm (Butani 1955; Chaudhary and Ramzan 1967), 
appeared to be less acceptable by first instar larvae probably due to hard leaf tissues. 
Kalode et al (1971) also reported 100% mortality of young larvae of Cirphis compta 
Moore on sugarcane leaves. 

Maximum (60-3%) larvae survived on napier followed by Johnson grass (54-3%) 
and sorghum (48-7%) without any significant difference among them. On maize 
leaves, only 36-7% larvae could survive. Under varying conditions of temperature 
and humidity larval survival of this pest on maize had been reported to vary from 
12-0-60-0% (Bindra and Singh 1973; Dhaliwal and Bains 1978; Alam and Khatri 
1980). 

Table 1. Effect of feeding on different host plants on the larval survival, size and weight 
of M. separata under laboratory conditions. 

Larval survival (%) (days) Size of larva Larval weight (mg) (days) 



Test plant 


5 


10 


15 


(mm) (20. days 
22 old) 


14 


23 


26 


Napier grass 


97-0 


77-7 


71-3 


60-3 


29-67 


56-3 


358-5 


493-1 


Sorghum 


74-7 


56-0 


49-7 


48-7 


25-49 


54-0 


339-5 


482-5 


Johnson grass 


76-3 


66-0 


59-7 


54-3 


25-00 


59-3 


284-0 


306-6 


Maize 


94-0 


61-7 


47-7 


36-7 


22-33 


36-9 


254-0 


344-2 


Sugarcane 


90-7 


27-7 


17-3 


10-3 


7-33 


5-5 


25-4 


25-5 


Average 


86-5 


57-8 


49-1 


42-0 


21-96 


42-4 


252-3 


330-4 


SEM 


9-8 


4-8 


5-5 


5-9 


0-80 


4-6 


19-4 


62-9 


CD. at 5% 


NS 


11-1 


12-7 


13-7 


1-86 


10-6 


44-8 


128-1 



Mean temperature = 1 8-6 3-2C, Mean relative humidity = 55 5%. 
* Based on 300 larvae. 



Development and survival of armyworm in winter 



73 



Size of the larvae compared after 20 days of release ranged from 7-33 (sugarcane) 
to 29-67 mm (napier) indicating the slow growth of larvae on sugarcane. Larval 
weights after 14 days of feeding showed considerable differences on different foods. It 
ranged from 5-5 mg on sugarcane to 56-3 mg on napier grass. After 26 days of 
feeding, the range (25-5-493-1 mg) was still wider. Larvae fed on napier gained 
maximum weight (493-1 mg) followed by sorghum (482-5 mg) without any significant 
difference between them. On maize and Johnson grass, larva weighed 344-2 and 
306-6 mg, respectively -and were also significantly heavier than sugarcane (25-5 mg). 

Pupal size on different foods ranged from 15-0-16-91 mm (table 2). Avasthy and 
Chaudhary (1965) and Alam and Khatri (1980) also reported similar pupal sizes on 
sorghum, maize and sugarcane. Pupal weight of 2-4 days old pupa varied between 
184-9 mg (sugarcane) and 249-5 mg (sorghum) at 18-23-9C and 63 4-0% relative 
humidity. However, Tripathi et al (1982) recorded a higher range (319-5-340-7 mg) of 
pupal weight at 27C and 85% relative humidity. A considerable decrease in pupal 
weight was recorded after 20-22 days of pupal formation on different foods. On all 
foods, male pupa (111-9-153-6 mg) was heavier than female pupa (98*5-127-5 mg). 
Inspite of highest larval weights on napier grass, pupae of both sexes were lighter in 
weight in comparison to that of others. However, maximum pupation (38-9%) was 
recorded on napier followed by sorghum (30-3%), Johnson grass (25-0%) and maize 
(19-7%). On sugarcane only 3-3% larvae pupated. Under favourable conditions more 
than 80% pupation was recorded on maize and sorghum (Dhaliwal and Bains 1978; 
Alam and Khatri 1980). Ratios of males and females at pupal stage were almost 
equal on different foods. 

As is evident from table 3, shortest larval period (28-2 days) was recorded on 
napier grass followed by sorghum (28-3 days) and Johnson grass (29-3 days) without 
any significant difference among them whereas, it was longest (49'8 days) on 
sugarcane at prevailing temperature (18-63-2C) and relative humidity (54 5%). 
During colder months (October hatching) larval stage had been observed to go upto 
100 days on maize (Bindra and Singh 1973), but under favourable conditions it was 
15-11-15-18 days on Johnson grass and maize (Kushwaha and Bhardwaj 1977), 
19-3-20-75 days on sorghum and maize (Alam and Khatri 1980) and 14-22 days on 



Table 2. Effect of feeding on some grasses on pupal parameters of M. separata under 
laboratory conditions. 

Pupal weight (mg) 





Size of pupa 




20-22 days old 




Sex ratio 


Larval 


(mm) (2-4 days 2-4 days 


Pupation 




growth 


Test plant 


old) 


old 


Female 


Male 


(%) 


Male: Female 


index 


Napier grass 


16-49 


236-2 


98-5 


111-9 


38-9 


1:0-82 


1-38 


Sorghum 


15-66 


249-5 


127-5 


153-6 


30-3 


1:1-10 


1-07 


Johnson grass 


15-24 


193-7 


112-4 


127-1 


25-0 


1:1-10 


0-85 


Maize 


16-91 


229-4 


111-3 


157-0 


19-7 


1:0-85 


0-61 


Sugarcane 


15-00 


184-9 


101-2 


131-1 


3-3 


1:1-00 


0-06 


Average 


16-07 


218-7 


110-2 


136-1 


23-5 


1:0-97 




SEM 


0-28 


13-7 


12-1 


13-8 


3-7 






C.D. at 5% 


0-68 


31-5 


NS 


31-8 


8-4 







Mean temperature = 18-2 3-9C. Mean relative humidity = 63 4%. 



74 



Ram Singh and J P Chaudhary 





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Development and survival of armyworm in winter 75 

sugarcane (Avasthy and Chaudhary 1965). The larval durations in the present studies 
were 10-12 days longer than that reported by various workers. This may be because 
of low temperature and humidity. Pre-pupal period ranged from 2-8-10-3 days. 
Bindra and Singh (1973) also reported prolonged pre-pupal period of 11 days in 
January on maize due to low temperature. Pupal period (35-8 days) was shortest on 
Johnson grass and longest (49-7 days) on sugarcane. Due to low temperature condi- 
tions pupal stage of 36-48 days was also reported by Bindra and Singh (1973) and 
Dhaliwal and Bains (1978). However, under favourable temperature conditions, it 
ranged from 8-13 days only on different crops (Avasthy and Chaudhary 1965; 
Bindra and Singh 1973; Kushwaha and Bhardwaj 1977; Singh and Rai 1977; Alam 
and Khatri 1980). 

Contrary to larval survival, moth emergence was maximum (50-0%) on sugarcane 
and minimum (3-4%) on napier grass. Higher pupal mortality on napier grass was 
probably because of higher rate of loss' of weight in pupa at 18-23-9C and 63 4% 
relative humidity in comparison to sugarcane and sorghum. In addition to poor 
moth emergence, male adults showed crippling of wings in napier and Johnson 
grasses. However, in sugarcane not a single crippled moth was recorded. It was 
probably due to prolonged larval and pupal durations which resulted in moth 
emergence in the end of February when the laboratory temperature became more 
suitable for moth emergence. On tfie other hand due to short larval period on some 
grasses, the pupae were exposed to low temperature for a longer time, which was 
unfavourable for the emergence of moths. Female pupae were observed to be affected 
more adversely by low temperature (16-0 1-8C) and relative humidity (66-03%) 
as not even a single female emerged on napier, Johnson grass and maize. As a result 
total growth index was poor on these foods. Crippled moths were found to be short 
lived without participating in reproductive process. Females emerged on sugarcane 
and sorghum laid 400-900 eggs when provided with 10% sugar solution. Hence 
sugarcane and sorghum were found better food plants of this pest for survival during 
winter. 



Acknowledgement 

Authors are grateful to Dr N P Chopra for providing necessary facilities. 

References 

Alam M A and Khatri A K 1980. Effect of different host plants on the growth, development and survival of 

Mythimna separata Walk. (Lepidoptera: Noctuidae); JNKV Res. J. 14 1-6 
Avasthy P N and Chaudhary J P 1964 Morphology of pupa of the green borer of sugarcane, 

Raphimetopus ablutellus Zell; Indian J. Sugarcane 8 271-273 
Avasthy P N and Chaudhary J P 1965 Biology and control of armyworm, Pseudatetia unipuncta Haw; 

Indian J. Suyarcane 9 249-25 1 
Bindra O S and Singh J 1973 Bionomics of the army.worm, Mythimna separata (Walker) (Lepidoptera: 

Noctuidae) at Ludhiana, Punjab; Indian J. Ayric. Set. 48 249-303 
Butani D K 1955 Control of an outbreak of the armyworm (Cirphis unipuncta Haw.) on sugarcane at 

Pusa, Bihar; Indian J. Entomol. 17 133-136 
Butter N S, Gill K S, Nanda G S and Mahal G S 1979 Fteld screening of wheat germplasm against 

armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae); Sci. Cult. 45 77-79 
Chaudhary J P and Ramzan M 1967 Armyworm; a menace to sugarcane; Prog. Farm. 4 7-8 



76 Ram Singh and J P Chaudhary 

Chaudhary J P and Singh Z 1980 Outbreak of armyworm, Mythimna separata (Walker) on paddy crop in 

Haryana; Haryana Agric. Univ. J. Res. 10 248-249 
Dhaliwal Z S and Bains S S 1978 Effect of temperature on the development, survival and the rate of 

increase of armyworm, Mythimna separata (Walker) (Noctuidae: Lepidoptera); Indian J. Ecol. 5 240- 

249 
Kalode M B, Varma A, Yadav C P and Israel P 1971 Feeding reaction of armyworm (Cirphis compta 

Moore) larvae fed on some cultivated and wild plants; Indian J. Agric. Sci. 41 568-572 
Kushwaha K S and Bhardwaj S C 1977 Forage and pasture insect pests of Rajasthan (New Delhi: Indian 

Council of Agricultural Research Publication) pp 186 
Sarup P, Peshwani K M, Singh D S, Srivastava V S and Lai R 1969 An unusual gutbreak of armyworm, 

Pseudaletia separata (Walker) at Delhi in 1968 and evaluation of pesticides against the larvae infesting 

wheat; Indian J. Entomol 31 265-272 
Sharma H C, Agarwal R A and Singh M 1982 Effect of some antibiotic compounds in cotton on post 

embryonic development of spotted bollworm (Earias vittella F.) and mechanism of resistance in 

Gossypium arboreuw, Proc. Indian Acad. Sci. (Anim. Sci.) 91 67-77 
Sharma H C and Davies J C 1983 The oriental armyworm, Mythimna separata (Wlk.) distribution, biology 

and control, A literature review. Miscellaneous report no. 59 (London: Centre for Overseas Pest 

Research) p 24 
Singh D and Manchanda S K 1981 Severe incidence of armyworm on rye in Kashmir valley; Sci. Cult. 47 

97-98 

Singh D and Rai L 1977 Bionomics of rice cutworm, Mythimna separata (Walker); Entomon 2 141-144 
Tripathi A K, Bhattacharya A K and Verma S K 1982 Developmental behaviour of Mythimna separata 

(Walker) on some monocotyledonous plants; Indian J. Entomol. 44 355-367 
Verma A N and Khuranfc. A D 1971 Incidence of armyworm Pseudaletia separata Walker in different dwarf 

wheat varieties; Haryana Agric. Univ. J. Res. I 20-23 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96. No. 1, January 1987, pp. 77-80. 
Printed in India. 



Food utilization and surfacing activity of Channa striatus fry in relation 
to quality of food 

K SAMPATH* and E VIVEKANANDAN** 

School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, India 
*Department of Zoology, V.O.C. College, Tuticorin 628 008, India 
**Central Marine Fisheries Research Institure, Madras 600 105, India 

MS received 13 October 1986; revised 7 January 1987 

Abstract. Effects of food items (mosquito larva,- pupa, chironomous larva and chopped 
pieces of the fish Lepidocephalichthyes thermalis) on food utilization and surfacing activity of 
Channa striatus fry were studied. Test individuals fed on live food organisms surfaced 94- 
117% more frequently, consumed 23-119% more rations/day and, on an average, converted 
33% more than those fed on pieces of Lepidocephalichthyes thermalis. Regardless of the 
quality of food, Channa striatus surfaced about 60 times/h before each meal and the 
frequency increased to 130 times/h 3-6 h after the meal was offered and returned to the 
prefeeding level 18-21 h after the meal. 

Keywords. Channa striatus fry; quality of food; food utilization; surfacing activity. 



1. Introduction 

Culture of air-breathing fishes is gaining much importance in tropical fisheries due to 
high demand for these fishes. Though sturdy as adults, the fry of the obligatory air- 
breathing fish Channa striatus have low survival rate and hence, pose considerable 
problems to fish culturists. Non-availability of suitable food organisms and lack of 
favourable conditions during the development of the air-breathing organ are the two 
major reasons for high mortality rate of fry of air-breathing fishes (Pandian 1980). As 
an attempt- to select suitable food, fry of C. striatus were offered with 4 live food 
organisms, chopped pieces of the fish Lepidocephalichthyes thermalis and the pattern 
of food utilization and surfacing activity was studied. 

2. Materials and methods 

Active and well acclimated C. striatus fry weighing 308-5 25-91 mg were randomly 
chosen and divided into 5 series. Four replicates were maintained for each series. The 
first 3 series were fed on mosquito larva or pupa, the IV series on chironomous larva 
and the V series on chopped pieces of the fish L. thermalis. Chironomous larvae were 
collected from the field every day, starved for 24 h and then served as food. Culex 
mosquito egg raft was collected from the field regularly, hatched and reared in the 
laboratory up to 4th instar or pupa (Ponniah and Pandian 1977). Third and fourth 
instar larvae of Anopheles were collected directly from the field and offered as food. 
L. thermalis was maintained separately in aquaria and were starved for 24 h before 
being offered as food. Food was offered twice a day at 0900 and 1900 h for a period 
of 1 h each; unconsumed food was collected by a pipette without disturbing the test 
animals. 

77 



78 



K Sampath and E Virekanandan 



Experiments were conducted in cylindrical aquaria (5 cm dia) containing constant 
depth of water (15 cm). Faecal matter was collected by filtering the aquarium water 
daily before changing water. Sacrifice method was followed to estimate the initial 
water and energy contents of the test fish. Energy content was estimated in a Parr 
1412 semi-micro bomb calorimeter. The scheme of energy budget followed in the 
present study is that of IBP formula (Petrusewicz and Macfadyen 1970) represented 
as C = P + R+U + F where C is the energy consumed, P the growth, jR the energy 
lost as heat due to metabolism, F the faeces and U the nitrogenous waste. Estimation 
of various energy components has been detailed elsewhere (Sampath and Pandian 
1980). 

Number of surfacing by each test individual was observed everyday for a known 
period of time (15 min) during the following timings: 3, 6, 9, 12, 15, 18, 21 and 24 h 
after feeding. The distance travelled per individual was estimated by multiplying the 
mean number of visits per unit observations with twice the depth of water. Experi- 
ments were conducted for a period of 21 days at a room temperature of 282C. 



3. Results 

Of the 5 test series, the one fed on Anopheles larva exhibited the highest rates of 
feeding, absorption, metabolism and conversion (table 1); these rates were statisti- 
cally significant compared to the corresponding rates of fry fed on other test series. 
The series fed on pieces of L. thermalis exhibited the lowest rates of feeding, 
absorption and metabolism. Test individuals fed on live food organisms surfaced 94- 
117% more frequently, consumed 23-119% more ration and, on an average, 
converted 33% faster than those fed on pieces of L. thermalis. Contrary to this., 
absorption efficiency did not vary much among the test series and averaged to 90-4%. 



Table 1. Effects of different food materials on food utilization and surfacing activity in C striatus. 



Parameter 


Anopheles 
larva 


Culex 
pupa 


Culex 
larva 


Chironomous 
larva 


Pieces of 
L. thermalis 


Feeding rate 


1895 134-9 


1382 176-7 


1069 97-7 


1122 58-7 


868 172-9 


Absorption rate 


1706 141-1 


1227 165-4 


963 105-8 


1054 59-2 


817213-5 


Conversion rate 


666 75-3 


444-1 64-5 


291 35-9 


288 36-0 


31868-9 


Metabolic rate 


936 59-9 


703 108-7 


605 69-3 


690 4-4 


432 128-5 


Absorption 












efficiency 


89-5 1-30 


88-7 0-75 


90-1 1-05 


94-0 0-41 


90-0 2-75 


Conversion 












efficiency (K 2 ) 


38-9 1-27 


36-4 2-70 


30-5 6-85 


27-3 1-84 


37-25-10 


Surfacing 












frequency/day 


2196315-0 


2052 286-2 


1962 244-8 


2106 201 -6 


1010 114-0 


Hanging 












duration 


3-0 0-25 


3-3 0-26 


3-0 0-1 3 


2-9 0-38 





Distance 












covered 


539 74-90 


508 63-50 


577 82-60 


549 51-75 


222 25- 10 



Each value'represents the average ( SD) performance of 4 individuals at 28 2C. Rates are expressed in 
J/g live fish/day, efficiencies in percentage; hanging duration and distance covered are expressed as h/day 
and m/day, respectively. 



Food utilization in relation to food quality 



79 



Conversion efficiency ranged from 27-3% (series fed on Chironomous larva) to 38*9% 
(series fed on Anopheles larva) and averaged to 34-1%. 

The series fed on live food organisms surfaced twice (average: 2079 times/day) as 
frequently as the series fed on L. thermalis pieces (1010 times/day); correspondingly, 
the distance covered to surface increased by nearly 2-5 times. After undertaking a few 
surfacings, C. striatus resorts to a short resting period in water surface called 
hanging. In the present experiment, the duration of hanging did not change in any of 
the test series and remained around 3 h/day (table 1). 

Hourly fluctuation in surfacing frequency following a meal was observed in the 
test series fed on live organisms. This study was conducted during the last 3 days of 
the experiment over a 24 h cycle. The test individuals surfaced about 60 times/h 
before feeding and the frequency increased to about 130 times/h, 3-6 h after the meal 
in the series fed on Anopheles larva and decreased thereafter to the prefeeding level 
21 h after each meal (figure 1). Vivekanandan et al (1977) have reported similar trend 
in the surfacing frequency following meal in another obligatory air-breathing fish 
4 nab as scandens. 



4. Discussion 

The test series which were offered 4 different live food organisms exhibited statisti- 
cally significant difference in food utilization parameters, viz rates of feeding, absor- 
ption, conversion and metabolism as compared to those fed on pieces of L. thermalis. 



140 - 



120 - 




9 12 15 

Time after meal (hr) 



18. 



21 



24 



Figure 1. Hourly fluctuation in the surfacing frequency/h as a function of time after meal 
in C. striatus fed on Different live food organisms (), Anopheles larva; (A), Culex pupa; 
(O), Culex larva; (A), Chironomous larva. 



80 



K Sampath and E Vivekanandan 

Table 2. Water content and caloric value of different food materials and experi- 
mental fish. 



Food 



Fish 



Food 



Water content Caloric value Water content Caloric value 
(%) (kJ/gdrywt) (%) (kJ/g dry wt) 



Anopheles larva 


83-4 8-20 


21-70-51 


76-7 0-69 


19-7 0-57 


Culex pupa 


88-7 7-50 


21-0 0-90 


77-5 1-01 


19-0 0-85 


Cu/ex larva 


88-0 7-90 


22-1 0-77 


77-0 0-87 


19-4 0-61 


Chironomous 










larva 


84-6 7-50 


23-1 0-90 


76-7 0-17 


19-7 0-54 


L. thermalis 










pieces 


80-0 6-70 


19-5 0-28 


79-2 0-69 


19-5 0-57 



This result provides clues on the possible strategies adapted by C. striatus when 
exposed to different food items: (i) when exposed to low energy food L. thermalis 
(19-5 kJ/g dry wt; table 2), C. striatus reduces the feeding rate and compensates by 
decreasing the surfacing frequency and alloting less energy for swimming activity and 
metabolic rate, (ii) the high feeding rate in the series fed on Anopheles larva might 
have resulted from a behavioural synchronisation of the properties of the predator 
and prey. Anopheles larva stays at the water surface most of the time and, while 
surfacing, C. striatus encounters Anopheles larva more frequently than other food 
organisms. This synchronisation might have resulted not only in higher feeding and 
conversion rates but also in the least allocation of ingested energy towards meta- 
bolism, and (iii) there is no evidence of changes in feeding, absorption and conver- 
sion rates and surfacing activity among the series receiving live food organisms in 
relation to the minor variation in the energy content. However, the series receiving 
the lowest food energy (L. thermalis) consumed the least ration. As the conversion 
rate of the series fed on Anopheles larva (666 J/g/day) was substantially faster than 
the rates exhibited by the other series (288-444 J/g/day), it may be advantageous to 
feed C. striatus fry on live food organisms which remain at the water surface. 

Acknowledgement 

We are grateful to Prof. T J Pandian, School of Biological Sciences, Madurai 
Kamaraj University, Madurai for valuable suggestions. 



References * 

Pandian T J 1980 Contributions to food production in India through aquacultural research; Biology 1 1-4 

Petrusewicz K and Macfadyen A 1970 Productivity of terrestrial animals: Principles and methods; in IBP 
handbook (Oxford: Black well Scientific) Vol. 13, p 190 

Ponniah A G and Pandian T J 1977 Surfacing activity and food utilization in the air-breathing fish Poly- 
acanthus cupanus exposed to constant pO 2 ; Hydrobiologia 53 221-227 

Sampath K and Pandian T J 1980 Effects of density on food utilization and surfacing behaviour in the 
obligatory air-breathing fish Channa striatus; Hydrobiologia 68 113-117 

Vivekanandan E, Pandian T J and Visalam C N 1977 Effects of algal and animal food combinations on 
surfacing activity and food utilization in the climbing perch Anabas scandens; Pol. Arch. Hydrohiol. 24 
555-562 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 2, March 1987, pp. 81-115. 
Printed in India. 



On some plant galls from the Fiji Islands 

M S MANI and P JAYARAMAN* 

Zoological Survey of India, Madras 600 028, India 

*Department of Botany, Presidency College, Madras 600 005, India 

MS received 5 October 1983; revised 27 January 1986 

Abstract. This paper describes 34 new galls and records one widely distributed gall, 
collected by the first author in the course of a two-month visit to the Fiji Islands. The midge 
galls include those on Flacourtia sp., Calophyllum sp., Heritiera ornithocephala, Canarium 
vitiense, Syzygium rubescens, Astronidium sp., Plerandra vitiense, Tapeinosperma sp., 
Pagiantha thurstonii, Cy^tandra sp., Glochidion ramnifiorum, Gironniera celtidifolia, Gnetum 
gnemon, etc. The Eriophyid galls are on Hibiscus schizopetalus, Hibiscus tiliaceus, Brucea 
javanica, Vitis vitifolia, Pometia pinnata, Ipomaea gracilis, Ipomaea sp., Macaranya rnagna 
and Nephrolepis biserrata. The Psyllid galls occur on Canarium sp., Haplolobus jloribundus, 
Maesa tabacifolia, Myristica micrantha, Cryptocarya sp. The dominance of leaf galls and 
midge galls is considered as strong evidence of the relatively recent origin of the flora of 
these islands. 

Keywords. Zoocecidea; Fiji. 



1. Introduction 

This paper deals with the plant galls, collected by the first author, during a two- 
month visit in 1978 to the Fiji Islands. The collections were made in the course of a 
series of field trips in the Viti Levu and in the Taveuni Islands. Of the 35 galls 
described here, 34 are new to science. Except one gall on the shoot axis, all the others 
occur on leaf. The majority of the galls are caused by Cecidomyiidae, 9 galls are 
caused by Eriophyidae and only 6 by Psyllidae. Except for one gall each on a 
Pteridophyte and Gymnosperm, all the other galls occur on Dicots. 

The Fiji Islands in southwest Pacific Ocean, about 2600 km to the northeast of 
Australia, comprise nearly 500 islands, of which 97 are inhabited, mostly in the 
coastal areas and in the river valleys. Of volcanic origin, the islands are generally 
rugged and mostly below 1250 m above mean sea-level and are characterized by mild 
climate. In the larger islands there is a wet zone on the windward coast and a dry 
zone on the leeward coast, with characteristic vegetation. The flora is remarkable for 
the presence, besides many introduced species, of outliers of the Oriental elements, 
which have bypassed Australia over Borneo-Sulawesi and New Guinea group of 
islands to reach their extreme eastern limits. The dominance of leaf galls and of the 
galls by Cecidomyiidae over Eriophyid galls and the high endemism of the galls are 
strong evidence of a relatively recent origin of flora of these islands. 

2. Family Flacourtiaceae 

Flacourtia sp. (figures 1-3) 
Gall No. 840 Cecidomyiidae * 

Foliar gall; hypophyllous, erect, flask-shaped, hollow, densely bristled, closed, 

81 



82 



M S Mani and P Jayaraman 




\ 



Figures 1-3. Flacourtia sp. 1. A leaf showing the distribution of galls. 2. Three galls 
enlarged. 3. L.S. of the gall. (La, Lamina; LC, larval chamber; Pa, parenchyma; SZ, 
sclerenchyma zone; VS, vascular strands). 

covering-growth sessile pouch-gall, crowded together in two submarginal rows of the 
leaf; about 4 mm long and 2 mm thick. 

The gall appears like a miniature sea-urchin due to the occurrence of dense, stiff, 
straight unicellular unbranched and heavily thick-walled acute bristles on the sur- 
face; gall cavity axial, elongate, heavily sclerotized. A zone of collapsed cells 
surrounds the gall cavity, followed by 4-5 layers of brachysclereids, sclereids radially 
elongated, progressively larger, with well developed ramiform canals in walls and 
wide cell lumen; external to the sclerotic zone are compact, vertically elongated and 
thin walled parenchyma cells, delimited by a distinct epidermis, with elongated 
cylindrical cells; trichomes deep-seated in the outer layers of the parenchyma zone. 

Locality: Taveuni: Soqulu Estate: Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 



3. Family Guttiferae 

Calophyllum sp. (figures 4-9) 

Gall No. 841 Cecidomyiidae 

Foliar gall; hypophyllous unilateral or bilateral fleshy, lobed, short or also extensive, 
glabrous, indehiscent, multilocular swellings of the midrib, with the lamina depressed 



Plant galls from Fiji Islands 



83 




Figures 4-9. Calophyllum sp. 4 Adaxial view of a gall bearing leaf. 5 and 6. Adaxial 
view of the galls on two leaf segments. 7. T.S. of midrib. 8. T.S. of the midrib gall. 
9. T.S. of the laminar gall. (BV, Bicollateral bundle; LC, larval chamber; NZ, nutritive 
zone; SC, secretory canal; SZ, sclerotic zone; TC, tanniniferous cells; VS, vascular 
strands). 



on either side of the galled vein; about 0-5-0-7 cm thick; lateral fleshy outgrowths of 
gall are 0-3 cm thick; also on leaf margin as circular hypophyllous discs, with a 
central depression, about 1 cm in diameter; minute exit holes on the abaxial side of 
gall 

When the gall develops unilaterally on the midrib, the vascular tissues of the vein 
remain unaffected. The parenchyma of cortex of the vein and lamina contribute to 
the formation of the gall, and undergo extensive hyperplasia and hypertrophy, 
resulting in the compact homogenous mass of gall cells; when the gall develops 
bilaterally, the gall is larger than when unilateral and the vascular tissues become 
broken up into distorted strands by proliferation of medullary and cortical cells of 
the midrib. 

Larval cavities in a horizontal row both in the unilateral and bilateral galls, 
irregular, surrounded by well developed nutritive and sclerotic zones; cells immedia- 
tely lining the larval cavities collapsed; cells inner to the band of crushed cells intact 
and in radial rows, with dark granular contents; sclerotic zone consists of compact 
brachysclereids, with large lumens, filled with tannin. 

The galls on leaf margin are basically similar to those of veins, hypophyllous, with 
two or more larval cavities, surrounded by the nutritive and the sclerotic zones, 
outside of which is a broad zone of tanniniferous cells, absent in the vein galls; 
mesophyll tissues have lost their identity, and have proliferated in the vertical plane 
at right angles to the surface, resulting in homogenous mass of cell layers. 

Locality: Taveuni: Soqulu Estate: Coll. M S Mani and M K Ramath, 19 
December 1978. 



84 MS Mani and P Jayaraman 

4. Family Malvaceae 

4.1 Hibiscus schizopetalus Hook. f. (figures 10-13) 

Gall No. 873 Eriophyes sp. 

Gall occurs on leaf, petiole and stem. The foliar gall irregular, epiphyllous fleshly 
agglomerate mass of pouch, rather clustered towards the major veins, with a wide 
opening on the opposite side; galls on stem and petiole irregular covering growths 
with wide apical ostiole; foliar gall 2 mm high and 3 mm in diameter and the petiole 
and stem galls slightly smaller; both yellowish-green, soft, glabrous. 

The foliar gall epiphyllous, shallow, fleshy pouch, with wide hypophyllous opening 
and irregular fleshy emergences from the inner surface of the cavity; stellate hairs 
persist unmodified on the fleshy emergences in the cavity; mucilage cavities larger 
and more numerous than in normal leaf; epidermis bordering the gall cavity with 
hypertrophied cells, enclosing prominent nuclei and dense cytoplasm; gall paren- 
chyma consists of compact, thin-walled, irregular parenchymatous cells. 

The petiole and stem galls essentially similar, arise as covering growths from 
proliferation of cortical cells, without affecting the vascular tissues; considerably 
hypertrophied and homogenous; cells around the gall cavity possess dense cytoplasm 



10 




Figures 10-13. H. schizopetalous Hook. 10. A leaf with galls. 11. Stem with galls. 
12. T.S. of foliar gall. 13. T.S. of stem gall. (GC, Gall chamber; La, lamina; MC, mucilage 
canals; St, stem). 



Plant galls from Fiji Islands 



85 



and giant nuclei; cavity smooth and wide; ostioie narrow; no hairs on the lining layer 
of the cavity; mucilage cavities of the gall larger than those in the normal portion. 

Locality: Taveuni: Waiyevo: Coll. M S Mani, M K Kamath and D S Singh, 20 
December 1978. 



4.2 Hibiscus tiliaceus Linn, (figures 14 and 15) 

Gall No. 832 Eriophyes hibiscitileus Nalepa 

Leaf gall; ephiphyllous or hypophyllous, rugose, pustuloid, pale yellow, unilocular 
pouch-gall; gall cavity large, filled with emergences from the wall of the gall; ostioie 
minute on the minute hypophyllous covering growth; on stem and petiole the gall 
arises as small covering growth, with the ostioie at the tip of the gall; size 8-14 mm in 
diameter. 

Proliferation of the ground tissues gives rise to homogenous compact layers of 
cells; several fleshy, lobed emergences grow from the wall of the gall cavity; outer 
surface of gall densely pubescent like on normal leaf; surface layer of the gall cavity 
has the same type of stellate hairs as the normal leaf; gall trichomes have a distinct 
pedicel, from the top of which the arms of the trichome spread horizontally; larger 
than those of the leaf; ostioie is narrow and straight; galls on stem and petiole flask- 




Figures 14-15. H. tiliaceus Linn. 14. A leaf with galls. 15. T.S. of leaf through a gall. 
(GC, Gall chamber with fleshy outgrowths and trichomes; La, lamina; Os, ostioie). 



86 



M S Mani and P Jayaraman 



shaped with narrow, outwardly bent neck; basically similar to the leaf gall except 
that they are covering-outgrowths formed by the proliferation of the outer cortical 
parenchyma. 

Locality: Viti Levu: Deuba beach side: Coll. M S Mani, Sundaresan and M K 
Kamath, 12 December 1978. 

4.2a Remark: This gall is reported by Mani (1973) as having wide distribution in 
India, Hongkong, Celebes, Malaya, Bismarck Archipelago, Samoa and Java 
(Houard C; 1922, 1923, Les Zoocecidies des Plantes d'Afrique, d'Asie et d'Oceanic, 1: 
1-498; 2: 499-1058; Houard, C; 1933, Les Zoocecidies des Plantes de 1'Amerique du 
sud et FAmerique Centrale, pp. 1-519; Mani M S; 1948, Zoocecidia and Cecidozoa 
from India, J. R. Asiatic Soc. Bengal, (Sci.) 14 (2): 4348; Mani M S; 1973, Plant galls 
of India, Macmillan India, pp. 1-354; Drs Van Leeuwen-Rajnvaan W M; 1926, The 
Zoocecidia of the Netherlands, East-Indies; RAibsaamen E H; Beitrage zur Kenntnis 
aussereuropaischer Zoocecidien, Marcellia 4: 65-138; 6: 110-173; 7: 15-79). 

5. Family Sterculiaceae 

Heritiera ornithocephala Kosterm. (figures 16-18) 

Gall No. 860 Cecidomyiidae 

Foliar twin-gall; short hour-glass shaped gall of two semi-circular, subsessile halves, 
one on each side of blade; the halves joined together at the blade by a small neck-like 
interconnecting portion; the two halves equal or rarely one may be smaller; smooth, 




Figures 16-18. H. ornithocephala Kosterm. 16. A leaf with galls. 17. A gall enlarged. 
18. Vertical section of a gall. (B E, Broken epidermis; I Z, inner zone of parenchyma; La, 
lamina; L C, larval chamber; M Z, middle zone of parenchyma; Pe, periderm). 



Plant galls from Fiji Islands 



87 



dark brown, nut-like and indehiscent; about 3-5 mm in diameter and 3- mm in height. 

This is a remarkable eruption gall, of two structurally identical, dome-shaped 
bodies, above and below the blade, exactly opposite to each other; two plano-convex 
halves originate from a common point on the lamina and have a short, stout, inter- 
connecting neck; each has thus the appearance of an umbrella-shaped body. The 
larval cavity small, circular, in the middle of the neck, equidistant from the two 
halves, with a single minute larva. The parenchyma cells divide periclinally and later 
elongate vertically; consequently erupting out by rupturing the leaf epidermis on 
both surfaces, the broken epidermis pushed aside as a circular membrane around the 
neck of the gall on both the surfaces of the blade; each half of the gall consisting of 3 
zones: the outermost of a narrow band of radial series of laterally stretched cells, 
which resemble the periderm tissue; the outermost layers of the zone are collapsed 
and remain as a dark line; the next inner zone of large, vertically elongated tannini- 
ferous cells with dense aggregation of starch grains; third zone extends down into the 
neck, and consists of smaller, compact irregular cells; these cells also contain starch 
grains, but no tannin; in the region of the leaf where the galls are attached, the air- 
chambers are larger than those of the normal region and are also irregular in outline. 
The tracheary elements are scattered in horizontal plane in this region. The larval 
chamber is surrounded by smaller densely cytoplasmic cells. 

Locality: Taveuni: Soqulu Estate: Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 

6. Family Simaroubaceae 

Brucea javanica (Linn.) Merrill (figures 19-21) 

Gall No. 838 Eriophyes sp. 

Foliar gall; epiphyllous, sometimes hypophyllous, hemispherical, sessile, hairy, 
multilocular, brownish pouch gall; sparsely scattered, about 3-4 mm in height and 
5 mm in width. 



.S.C 




Os 



Figures 19-21. B. javanica (Linn.) Merrill. 19. A leaf with gall. 20. Vertical section of a 
gall. 21. A gall enlarged. (G C, Gall chamber; Os, ostiole; S C, secretory cavity). 



88 



MS Mani and P Jayaraman 



The gall consists of an epiphyllous. hemispherical pouch and equally large 
projecting hypophyllous covering growth, with fleshy emergences projecting from 
the inner surface of the gall cavity, dividing the cavity into a multilocular chamber; 
straight, thick-walled hairs line the ostiole and the outer surface of the gall; similar 
to, but denser than those on the normal lamina; gall tissue consists of much hyper- 
trophied, compact, polygonal cells, without much differentiation into the usual 
components of the mesophyll tissues; tanniniferous cells abundant in the gall tissue; 
cells lining gall cavity cytologically distinct from the remaining layers, radially 
elongated, with prominent nuclei and dense cytoplasm. 

Locality: Taveuni: Soqulu Estate: Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 

7. Family Burseraceae 

7. 1 Canarium vitiense A Gray (figures 22 - 24) 

Gall No. 853 Cecidomyiidae (?) 

Hypophyllous, globose or elliptical, large glabrous, thin-walled, indehiscent, reddish- 
brown, sessile or shortly necked pouch gall, upto 10 galls per leaf; with a small 
conical epiphyllous, ostiolate, covering growth, 1 mm long, gall about 7 mm high 
and 5 mm wide. 

The gall cavity large, with smooth inner and outer surfaces; there are 4-5 layers of 
laterally stretched cells in the inner part of the gall, followed by 2-3 layers of scleren- 
chyma zone. The outer part of the gall consists of a few layers of large thin-walled 
cells, which are collapsed in the periphery. The sclerenchyma cells are large with wide 
lumen. 

Locality: Viti Levu: Colo-i-Suva: Coll. M S Mani, M K Kamath and Sundaresan, 
25 January 1978. 




Figures 22-24. Canarium vitiense A. Gray. 22. A leaf with galls. 23. A single gall 
enlarged.' 24. Vertical section of a gall. (L, Larval chamber; Os, ostiole; PZ, parenchyma 
zone; SZ, sclerotic zone). 



Plant galls from Fiji Islands 
7.2 Canarium sp. (figures 25-27) 



89 



Gall No. 862 Psyllidae 

Epiphyllous, globose or hemispherical solitary or more commonly agglomerate 
masses, mostly in the vicinity of the veins, with minute hypophyllous ostiole, and a 
mature hypophyllous covering growth about 3 mm high and 2-3 mm thick; larval 
cavity wide and shallow. In the young gall, the larval cavity is surrounded by radial 
cells from a meristematic zone; outer to this meristematic zone are compact paren- 
chyma irregular cells with undulate walls. In the mature galls, the cells bordering the 
larval cavity and those at the periphery of the dome are collapsed to form a dense 
dark layer. The cells in between the disintegrated zones are compact, thin-walled 
and stretched in the plane parallel to the cavity. 

Locality: Taveuni: Soqulu Estate: Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 



7.3 Haplolobus floribundus (Schum) H J Lam. (figures 28-31) 

Gall No. 844 Psyllidae (?) 

Epiphyllous, slightly raised, irregular, yellowish-green pouches, with circular, wide- 
open hypophyllous ostiole on hypophyllous short covering growth; solitary or 
irregular agglomerate mass, especially near the lateral veins. The solitary gall 
measures 1 mm in diameter and the agglomerate mass measures 1 cm. 

Ostiole and the larval cavities are wide; the lamina gradually merges with the gall 
and the- different tissues of the former lose their identity in the gall due to extensive 
hypertrophy and hyperplasia. In the transition from the normal lamina into the gall, 
the number of cell layers remain unaltered, but the cells are greatly hypertrophied 
and their differentiation into the spongy and palisade tissues are suppressed. In the 




Figures 25-27. Canarium sp. 25. A leaf with galls. 26. A group of galls enlarged. 
27. Vertical section of a gall. (CT, Crushed tissues; LC, Larval chamber; Os, ostiole; VB, 
vascular bundle). 



90 



M S Mani and P Jayaraman 




Figures 2&-31. H. floribundus (Schum.) H J Lam. 28. Adaxial view of a leaf with galls. 
29. Adaxial view of the gall with ostioles. 30 and 31. Vertical sections of a solitary and 
compound galls respectively. (La, Lamina; L C, larval chamber; S C, secretory canal; V B, 
vascular bundle). 



gall proper, the cells have undergone repeated transverse and vertical divisions and 
are stretched in vertical plane, without differentiating into the usual ground tissues. 
The cells that would have normally differentiated as palisade and spongy paren- 
chyma have contributed equally to the growth of the gall. Large, circular, schizoge- 
nous cavities found in the gall are smaller in the normal lamina. In the roof of the 
gall cavity, the cells have divided transversely and stretched laterally to form 
compact rectangular cells. This region of the gall is slightly thicker than the normal 
lamina. Here also the differentiation of the ground tissues is suppressed. Neither 
sclerotic nor special nutritive zones are found in the gall. 

Locality: Taveuni Queleni; Coll. M S Mani, Sundaresan and M K Kamath, 20 
December 1978. 

8. Family Vitaceae 

Vitis vitifolia Lamarck (figures 32 and 33) 



Gall No. 870 Eriophyes sp. 

Hypophyllous, minute, dot-like, scattered, densely hairy, pouch gall, with epiphyllous 
ostiole, about 1-2 mm high and 1 mm wide. 



Plant galls from Fiji Islands 



91 




Figures 32 and 33. Vitis vitifolia Lamarck. 32. Adaxial view of a leaf with galls. 
33. Vertical section of a gall (GC, Gall chamber; La, lamina; Os, ostiole). 

The gall consists of a hemispherical hypophyllous pouch, with dense trichomes on 
the surface similar to... those of the normal leaf; ostiole at the tip of a minute 
epiphyllous conical covering growth, the inner surface of the gall with short, 
irregular, solid, stout tuberculate outgrowths, no hairs on the lining layer of the .gall 
cavity. The gall consists of about 12 layers of compact, more or less homogenous 
polygonal cells. The cells of the lining layer of the gall cavity are distinct from the 
remaining cells in being radially elongated with dense protoplasmic contents. 

Locality: Taveuni: Vurevure; Coll. M S Mani, M K Kamath and D S Singh, 20 
December 1978. 

9. Family Sapindaceae 

Pometia pinnata J R and G Forst (figures 34 and 35) 

Gall No. 835 Eriophyes sp. 

Epiphyllous, blister-like, rugulose, rusty-brown, irregular swellings of the lamina 
about 2 cm large; gall cavity shallow and wide, clothed with dense brownish 
erineum; gall cells exhibit limited hyperplasia followed by hypertrophy, parenchyma 
compact, without intercellular air spaces; anticlinal divisions of both dermal and 
parenchyma and subsequent enlargement of the derivatives cause the invagination of 
the lamina into a shallow pouch; dense erineum lining the gall cavity consists of 
unicellular, unbranched hairs, longer and broader than those of the normal leaf. 

Locality: Viti Levu: Deuba; Coll. M S Mani, Sundaresan and M K Kamath, 12 
December 1978. 



10. Family Myrtaceae 

1 0.1 Metrosideros sp. (figures 36-38) 
Gall No. 875 Cecidomyiidae midge 

Stem gall; extensive fusiform or localised globose hard, smooth, indehiscent swellings 
of the internodes, with minute exit holes; numerous minute larval cavities as dark, 



92 



M S Mani and P Jayaraman 




Figures 34 and 35. Pometia pinnata J R and G Forst. 34. Leaf with galls. 35. T.S. of 
leaf passing through the gall (G, Galled portion of the leal). 




Figures 36-38. Metrosideros sp. 36. A galled twig with exit holes. 37. T.S. of a young 
gall. 38. T.S. of a mature gall. (Ca, Cambium; Co, cortex; G T, gall tissue; L C, larval 
chamber surrounded by dark crushed tissues; Pe, periderm; S P, secondary phloem; S X, 
secondary xylem). 



Plant galls from Fiji Islands 93 

irregular spots within the secondary xylem cylinder; gall measures 3-5 cm long and 
1 cm in diameter. 

In a young gall, the larval cavity is external to the vascular cambium and is 
surrounded by a dark band of collapsed tissue, which is again followed by radiating 
rows of thin-walled, unlignified tissue. The minute larval cavity and its surrounding 
cylinder of radiating cells appear as if embedded in the furrow of the vascular 
cylinder of the stem. The gall tissue cylinder originates by the centripetal activity of a 
circular meristem. As the girth of the gall tissue increases, it inhibits the activity of a 
strip of cambium adjacent to it, while the remaining portion functions normally. This 
causes the furrowing of the vascular cylinder and thrusting of the gall tissue cylinder 
into the furrow. The normal vascular tissues and the gall tissues grow as coaxial 
cylinders; however, the activity of the vascular cambium supersedes the growth of the 
gall tissue and the secondary xylem produced by the cambium grows over and 
around the gall tissue cylinder, thus engulfing it. At a later stage, the cambium forms 
a complete cylinder, so that its derivatives push the larval cavity and its surrounding 
reaction tissues deep into the xylem cylinder. Thus, in a mature gall, there is a broad, 
complete cylinder of secondary xylem with the central pith, and one or more intraxy- 
lary larval cavities, each surrounded by its own reaction tissues. 

The cells around the larval cavity resemble the secondary xylem, but are isodia- 
metric and lack the rays, fibers and vessels. These cells and those in the vicinity of the 
gall tissue are larger in size, thin walled and lack lignification. The vascular elements 
of the galled stem are shorter, broader and sometimes curiously shaped. Proliferation 
of the cortical tissue also adds to the growth of the gall. 

Locality: Viti Levu: Suva; Coll M S Mani, Sundaresan and M K Kamath, 12 
December 1978. 

Syzygium ruhescens (A Gray) C Muell. 

Gall No. 852 Cecidomyiidae midge 

The gall occurs on the stem, petiole and veins. On the stem, the gall is a diffuse 
cylindrical and often a moniliform swelling, extending from node to node; on leaves 
the gall develops along the entire length of the petiole, as fusiform, much elongated 
largely hypophyllous swelling on the midrib; both the stem and foliar galls indehi- 
scent, dark brown, rugose and covered with membranous scales; stem gall 7 mm in 
diameter and the vein gall 4 mm thick and 9 mm broad. 

The stem gall is roughly circular in cross section (figures 43-44). There are one or 
two narrow larval cavities in the region of secondary phloem. The central pith cavity 
is absent. Due to reduction in the activity of the cambium, the secondary xylem 
cylinder appears thinner and especially in the vicinity of the larval cavities this 
tissue is much reduced. The ground tissues have proliferated considerably to 
contribute to the increase in the thickness of the gall. The secondary xylem cylinder 
also increased in perimeter concomitant with the increasing girth of the gall and this 
circumferential increase is by proliferation of the medullary rays which conse- 
quently broke the xylem cylinder into narrow radial bands. The ray cells are 
stretched laterally and the bands of ray are wedge shaped in transection. The 
secondary phloem also increased and the cortical fibres are better developed forming 



94 



M S M-ani and P Jayaraman 



a distinct continuous zone. The larval cavity is bound by peculiar type of sclereids, 
with secondary thickenings only on the inner tangential and part of the radial walls. 
The periderm of the gall is thicker than that of the normal stem. Tanniniferous 
cells and druses are abundant in the ground tissues of the gall. 

10. 2a Vein gall (figures 39-42): The gall is confined to the midrib. Any part of the 
midrib such as basal, middle and terminal, may be galled. The gall projects more on 
the lower side than on the upper side which is flat or even somewhat concave. Along 
with the vein a part of the lamina on either side is also involved in galling, so that the 
gall is pronouncedly wide. One or two larval cavities are found on the lower or lateral 




Figures 39-42. S. rubescem (A Gray) C Muell. 39. A shoot showing stem and vein 
galls. 40. T.S. of mid-vein. 41. T.S. of gall at the terminal part of the vein. 42. T.S. of 
gall at the middle part of the vein. (LC, Larval chamber; Pe, periderm; SC, secretory canal; 
SG, stem gall; VA, vascular arch; VG, vein gall). 



43 




Figures 43 and 44. 5. rubescens (A Gray) C. Muell. Stem gall; 43. T.S. of stem. 44. T.S. of 
stem gall. (Ca, Cambium; Co, cortex; LC, larval chamber; PC, pith cavity; Pe, periderm; Pi, 
pith; PS, proliferated secondary xylem; SC, secretory canal; SP, secondary phloem; SZ, 
sclerotic zone; XR, xylem rays). 



Plant galls from Fiji Islands 



95 



part of the vascular arch. The vascular strand expands laterally due to proliferation 
of the ray cells and consequently the xylem tissue occurs in the form of narrow 
bands, alternating with the proliferated rays. The ground tissues, especially those 
bordering the larval cavities, proliferate by periclinal divisions increasing the bulk of 
the gall. The surface layers of cells also divide periclinally giving rise to a zone of 
periderm like cells. The periderm like zone may occur on both adaxial and abaxial 
sides x>r only on the abaxial side. The sclereids with thickened inner tangential and 
lateral walls occur a few layers away from the larval cavities. The cells around the 
larval cavity exhibit features of the nutritive tissues. 

Locality: Viti Levu: Colo-i-Suva; Coll. M S Mani, M K Kamath and Sundaresan, 
25 January 1979. 

11. Family Melastomaceae 

11. 1 Astronidium sp. (figures 45-48) 

Gall No. 837 Cecidomyiidae 

Large, fusiform or ovoid smooth, hard indehiscent nodal or internodal swellings, 




Figures 45-48. Astronidium sp. 45. A flowering shoot with galls on different parts of the 
axis. 46. T.S. of stem. 47. T.S. of a gall with a single central larval chamber. 48. T.S. of a gall 
with many larval chambers. (LC, Larval chamber; MB, medullary bundles; NZ, nutritive 
zone; SP. secondary phloem; SX, secondary xylem; SZ, sclerotic zone). 



96 



M S Mani and P Jayaraman 



measuring about 2-3 cm long and 2 cm in diameter on the stem and primary axes of 
the paniculate inflorescence. 

The gall may have either a single axial larval cavity or many scattered cavities, 
with a single larval cavity having the same tissue systems as in the normal stem, but 
showing extensive hyperplasia and hypertrophy. The cortical cells of the gall have 
increased only to a limited extent, while the vascular tissues have increased both in 
the number of cell layers and in size of the cells. The sieve tubes of the internal 
phloem are well developed. The pith cells are much hypertrophied and the medullary 
bundles are dilated. The striking feature of the medullary bundles of the gall is that 
the differentiation of their xylem elements is completely suppressed and the sieve 
tubes are well developed. In the gall with many larval cavities the medullary bundles 
are absent. A functional cambium exists in the gall, and its inner derivatives are 
found in radial sedations but they do not develop secondary wall. They remain as 
unlignified parenchymatous tissue. The larval cavities are wider and irregular in 
outline. Each cavity is surrounded by nutritive cells and sclerotic zones. The nutritive 
cells have dense granular contents and the sclerotic cells are of brachysclereid type. 
In certain parts of the gall a distinct periderm is seen which is absent in a normal 
stem. The effect of the formation of periderm tissue in the gall is to increase 
circumference of the stem in order to accommodate the increased girth of the gall. 

Locality: Taveuni: Soqulu Estate; Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 

11.2 Astronidium sp. (figures 49 and 50) 
Gall No. 867 Cecidomyiidae midge 

Vein gall; spindle-shaped, moniliform, smooth, solid, soft, fleshy, indehiscent swell- 
ings of the midrib and lateral veins. The gall projects more on the abaxial side and 
measures 7 mm long and 3 mm in diameter. 




Figures 49-50. Astronidium sp. 49. Abaxial view of the vein gall. 50. T.S. of vein. 
50A. T.S. of vein gall (BV, Bicollateral vascular bundle; CT. gall tissue; VS, vascular 
strands -distorted). 



Plant galls from Fiji Islands 



97 



The gall has a narrow axial larval cavity, opening to the outside by a minute exit 
hole in the mature gall. The growth of the gall is due to hypertrophy and hyperplasia 
of parenchyma, as well as the perivascular and vascular parenchyma. Due to inten- 
sive proliferation of the inter and intra-vascular parenchyma, the original vascular 
bundles are broken into numerous islands, arranged in a wide ring. The procambium 
of each strand is stimulated to divide periclinally to form radial files of cells. 
However, the derivatives are not lignified; there are only a few lignified tracheary 
elements in each strand which are widely spaced. In the medullary portion of the gall 
are seen a few islands of smaller cells surrounded by larger cells. The epidermis of the 
gall expands in surface by anticlinal divisions and lateral expansion and keeps pace 
with the growing size of the gall. The annular sclereids are absent in the gall. 

Locality: Taveuni: Soqulu Estate; Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 

13. Family Araliaceae 

Plerandra vitiensis (Seem) Baillon (figures 51-54) 
Gall No. 849 Cecidomyiidae midge 

Hypophyllous, ovoid, shortly pedicellate, fleshy, soft, yellowish-green, with epiphyl- 
lous ostiole; either solitary or crowded, erect or lopsided; about 1 cm long and 0-5 cm 




Figures 51-54. Plerandra vitiensis (Seem) Bailon. 51. Abaxial view of a leaf with galls. 
52. A single gall enlarged. 53. Vertical section of a gall. 54. T.S of gall. (LC, Larval 
chamber; NZ,. nutritive zone; Os, ostiole; VS, vascular strands). 



98 



M S Mani and P Jayaraman 



thick; sometimes the midrib is also galled, forming fusiform hypophyllous swellings. 

The gall consists of fleshy, ovoid hypophyllous pouch and a minute conical epi- 
phyllous covering growth. The ostiole at the tip of the covering growth leads to a 
canal like larval cavity, which is irregular in outline in transection. The cells 
immediately surrounding the larval cavity, especially in the upper part, show 
centripetal meristematic activity, as a result of which some interior cells are crushed. 
The gall parenchyma consists of thin-walled highly hypertrophied undifferentiated 
compact parenchyma polygonal cells outside, and radially elongated in the interior; 
cells generally diminish in size outwards; cells lining larval cavity collapsed and the 
cells of the meristematic zone do not contain any special inclusions. The vascular 
bundles at the periphery dilated due to the hyperplasia and hypertrophy of the 
parenchyma cells. However, the different components of the vascular bundles are 
distinct and the tracheary elements remain normal The gall is 6 times thick as the 
normal leaf and the bulk of the gall tissue is derived from the spongy mesophyll 
tissue. 

Locality: Viti Levu: Colo-i-Suva; Coll. M S Mani, M K Kamath and Sundaresan, 
25 January 1979. 

14. Family Myrsinaceae 

14.1 Maesa tabacifolia Mez. (figures 55-57) 
Gall No. 833 Psyllidae 

Epiphyllous, scattered, surrounded by a chlorotic halo; with large epiphyllous 
hemispherical pouch and a small hypophyllous conical covering growth; the cover- 




Os 



Figures 55-57. M. tabacifolia Mez. 55. Abaxiai view of the leaf with galls. 56. An entire 
gall enlarged. 57. Vertical section of gall. (EP, Epiphyllous pouch; HC, hypophyllous 
covering growth; LC, larval chamber with larva; Os, ostiole). 



Plant galls from Fiji Islands 99 

ing growth ends with a recurved bilobed lip, enclosing a slit-like ostiole, leading into 
a large smooth larval cavity with a single larva inside. 

The gall tissues show marked deviation from those of the normal leaf. The 
parenchyma has undergone hyperplasia and hypertrophy in specific planes in addi- 
tion to hypoplasia at certain regions. In the mature gall, the following 3 distinct 
regions may be observed. 

(i) Transitional region represents the portion of the gall where the lamina merges 
into the gall proper, where the palisade and spongy tissues are recognisable. The 
palisade cells, which are normally 2-layered, have divided periclinally resulting in 4-6 
layers of small compact cells. The spongy cells are similar to those of the normal leaf; 
but air cavities and the surrounding cells are much larger. Towards the larval cavity, 
the spongy rnesophyll cells have divided both periclinally and anticlinally and the 
derivatives have undergone extensive vertical stretching, forming compact elongated 
cells, lying parallel to the long axis of the covering growth. However, the palisade 
cells do not show such a stretching; they remain short and compact. The vascular 
bundle is dilated due to proliferation of parenchyma cells within the bundle. 

(ii) The roof of the gall cavity exhibits suppression of usual tissue differentiation; 
the number of cell layers in this part is doubled as compared to normal leaf. 
However, the cells are small, compact and uniform; the thickness remains normal. 

(iii) The region of covering growth develops entirely from the spongy mesophyll 
cells by the downward elongation of cells. The cells in this region are thin, compact, 
elongated parallel to the long axis of the cover cone. The, cells lining the gall cavity 
are small without any cytological features of special interest. 

Locality: Viti Levu: Cuvu, Navouvoa Road; Coll. D S Singh, 12 January 1979. 

14.2 Tapeinosperma sp. (figures 58 and 59) 

Gall No. 847 Cecidomyiidae midge 

Scattered, solitary, lenticular gall, with hypophyllous ostiole dark green circular spot; 
upto 50 galls per leaf; size 2-3 mm in diameter. 

The gall is more than 4 times thicker than the leaf and the gall tissues are formed 
by proliferation of all ground tissues in varying degrees. The upper epidermal cells 
divided periclinally at certain regions to form 2-3 layers of cells. The single 
hypodermal layer of cells is also proliferated to form 4 layers of much hypertrophied 
cells. The palisade cells are divided in periclinal plane to form 4 layers of cells which 
are recognisable by the presence of chloroplasts. The spongy parenchyma is divided 
profusely in all planes and the derivatives are enlarged, circular and more or less 
compact. The bundle sheath cells are not distinguishable. The larval cavity is wide 
and is surrounded by 2-3 layers of small empty cells. Following this is the sclerotic 
zone, which is made up of brachy sclereids. 

Locality: Viti Levu: Colo-i-Suva; Coll. M S Mani, M K Kamath and Sundaresan, 
25 January 1979. 

14.3 Tapeinosperma sp. (figures 60-62) 

Gall No. 854 Cecidomyiidae midge 

Hypophyllous, small inconspicuous, fusiform or cylindrical, rusty-brown swellings 



M S Mani and P Jayaraman 




Figures 58 and 59. Tapeinosperma sp. 58. Abaxial view of the leaf with galls. 59. Vertical 
section of a gall (HD, Hypodermal derivatives; Os, ostiole; PD, palisade derivatives; SZ, 
sclerotic zone). 




Figures 60-62. Tapeinosperma sp. 60. Abaxial view of the leaf with vein galls. 61. T.S. of 
vein. 62. T.S. of vein gall, (LC, Larval chamber; Ph, phloem; SB, sclerenchymatous 
bundle sheath; SZ, sclerotic zone; VB, vascular bundle; X, xylem). 



mostly of the lateral veins; covered by membranous scales; fully mature gall measures 
upto 8 mm long and 1-1-5 mm broad. 

In a section of the gall larval cavity is 'Y' shaped, spaced in the centre, which splits 
the vascular arch into two segments. The parenchyma shows diffuse proliferation 
and the outermost layers form a suberised periderm of 3-4 layers. The sclerenchyma 
sheath of the vascular bundle persists around the vascular tissues. A few layers of 
cells around the cavity are lignified and no nutritive tissue is evident in the mature 
gall. 



Plant galls from Fiji Islands 



101 



Locality: Viti Levu: Colo-i-Suva; Coll. M S Mani, M K Kamath and Sundaresan, 
25 January 1979. 

15. Family Apocynaceae 

Pagiantha thurstonii Seemann (figures 63 and 64) 
Gall No. 864 Cecidomyiidae 

Lenticular, pale green gall, scattered on the lamina, larger hypophyllous and thinner 
epiphyllous swellings, measuring about 2 mm in diameter and 1 mm thick. 

There is a single, laterally stretched disc-shaped larval cavity, with one larva. In 
the region where the normal leaf merges with the gall, the cells show pronounced 
hypertrophy obliterating the air spaces. In the region of the gall proper, parenchyma 
undergone extensive periclinal divisions, giving rise to vertical tiers of cells; these cells 
smaller with dense contents around the larval cavity, and highly vacuolated and 
vertically stretched away from it, especially so in the abaxial part of the gall. Thus, 
the gall consists of several layers of compact cells, without histological distinction; 
lower epidermis ruptured due to stretching in surface plane. 

Locality: Taveuni: Qeleni Road; Coll. M S Mani, M K Kamath and D S Singh, 20 
December 1978. 

16. Family Convolvulaceae 

16.1 Ipomaea gracilis R. Br. (figures 65-68) 
Gall No. 872 Eriophyes sp. 

Epiphyllous or hypophyllous globose pouch gall on leaf and covering-growth gall on 
the petiole and stem; scattered and solitary, measuring about 1-2 mm high and 1 mm 



63 




12 



Figures 63 and 64. Pagiantha thurstonii Seemann. 63. Abaxial view of the leaf with galls. 
64. Vertical section of a gall. (LC, Larval chamber with larva; TZ, tiered zone of cells). 



102 



M S Mani and P Jayaraman 




Figures 65-68. I. gracilis R. Br. 65. Abaxial view of a leaf with galls. 66. Stem 
galls. 67. Vertical section of a leaf gall. 68. T.S. of stem gall. (CG, Covering-growth gall; 
GC, gall chamber with mites; Os, ostiole; PG, pouch gall; St, stem portion). 



in diameter; covering-growth galls are slightly larger than the foliar galls; both 
yellowish-green glabrous and fleshy. 

The foliar gall has a hemispherical pouch portion and an equally projecting 
covering-growth portion, with the ostiole at its summit; the ostiole leads into a 
narrow and curved passage, ending in the irregular gall cavity with fleshy emergen- 
ces. The gall consists of compact layers of homogenous parenchyma cells; one or two 
layers of cells bordering the gall cavity destroyed and form a dark lining layer all 
around the cavity. 

The stem and petiole galls develop by proliferation of the cortical tissue, the 
vascular tissues not affected; from the cortex develops a small bowl-shaped structure, 
which later grows into a spherical body with the ostiole at the summit. The gall 
cavity irregular due to development of the fleshy emergences. The gall tissue consists 
of large compact irregular and variable parenchyma cells. The cells around the gall 
cavity are smaller and densely cytoplasmic. The bordering cells are destroyed, 
forming a dark lining around the cavity as in the leaf gall. The laticiferous ducts of 
the gall are larger in size and more in number than those of the normal organ. 

Locality: Taveuni: Lavena; Coll. M S Mani, M K Kamath and D S Singh, 20 
December 1978. 



16.2 Ipomaeasp. (figures 69-71) 



Gall No. 830 Eriophyes sp. 

Epiphyllous or hypophyllous, small, hemispherical galls about 2 mm large on leaf, 
stem and petiole, scattered and numerous, more than hundred per leaf; each gall has 
a hemispherical pouch on one side and a minute conical covering growth on the 



Plant galls from Fiji Islands 



103 




Figures 69-71. Ipomaea sp. 69. Abaxial view of the leaf with foliar and petiole 
galls. 70. T.S. of petiole gall. 71. Vertical section of foliar gall. (G, Gall part; GC, gall 
chamber; Os, ostiole; P, petiole). 

opposite side, with narrow ostiole at the tip, leading to the gall cavity, in which 
several fleshy emergences project from the surface. On the stem and petiole, the galls 
are small covering growth with the ostiole at the summit of the outgrowth. 

16.2a Foliar gall: The tissues of the foliar gall differ strikingly from those of the 
normal leaf. The outer epidermis of the gall is continuation of the upper epidermis of 
lamina; cells are slightly laterally expanded, but retain the tannin contents. The 
palisade cells have undergone periclinal divisions, increasing the number of layers 
upto seven of irregular cells, not arranged in rows as in the normal portion. These 
cells form a narrow outer zone of the domed part of the gall. The spongy cells form 
the major part of the gall and show two distinct zones. About 4 layers of cells around 
the gall cavity show pronounced anticlinal divisions and their derivatives allign 
themselves in regular semi-circular rows. Outer to this zone are larger, irregular, less 
compact cells without regular allignment. These cells are largely hypertrophied. The 
cells in the covering growth are uniformly elongated parallel to the long axis of the 
gall. The lower epidermis, which is the seat of attack by the mites, consists of densely 
cytoplasmic cells with prominent nuclei. In the older galls, this layer is destroyed by 
the excessive feeding activity of the mites and the subepidermal cells resemble the 
changed epidermal cells. 



16.2b Petiole and stem galls: In these cases, only the cortical cells take part in the 
formation of the galls and the vascular tissues are least involved. The parenchyma 
cells of the cortex proliferate around the point of infection to produce a globular 
covering growth. The ostiole at the summit of the covering growth leads to the gall 



104 



M S Mani and P Jayaraman 



cavity. The gall tissue consists of compact much dilated parenchyma cells. The lining 
layer of the gall cavity consists of distinct cells with dense cytoplasm and prominent 
nuclei, as those of the foliar gall. 

Locality: Viti Levu: Deuba Beach; Coll. M S Mani, Sundaresan and M K Kamath, 
12 December 1978. 

17. Family Gesneriaceae 

Cyrtandra sp. (figures 72-76) 

Gall No. 839 Cecidomyiidae Midge 

Hypophyllous, fusiform or irregular exclusive fleshy, soft, solid, indehiscent and 
yellowish-green swellings of the midrib, lateral veins and veinlets; sometimes, 
unilateral bulging of the midrib; the galls do not project above; measurement 6 mm 
thick and 1-2 cm long. 

The midrib gall has a single centrally situated larval cavity. The vascular bundles 
are scattered among the much dilated, irregular parenchyma cells, containing sphae- 
roraphides; collenchyma tissue found in the outer region of the normal vein is not 
differentiated in the gall. In the adaxial portion of the gall, the cells of the multiple 
epidermis and palisade tissue have undergone periclinal divisions, increasing in the 




Figures 72 - 76. Cyrtandra sp. 72. Abaxial view of the leaf with vein galls. 73. T.S. of a 
lateral vein. 74. T.S. of lateral vein gall. 75. T.S. of a midvein. 76. T.S. of midvein gall. 
(Co, Cortex; DM, derivatives of multiple epidermis; DP, derivatives of palisade tissue; DV, 
distorted vascular strands; LC, larval chamber; NT, nutritive tissue; VB, vascular bundle)! 



Plant galls from Fiji Islands 



105 



number of layers. The gall on the lateral vein also has a single layer larval cavity. 
Vascular bundles collapsed and consist of xylem elements in the middle of disinte- 
grated phloem tissue. The multiple epidermis and the palisade tissue divided in 
periclinal plane and the ground parenchyma has also divided, giving rise to irregular 
compact cells. In the lateral vein gall the place of the vascular bundle is occupied by 
the larval cavity. The hypodermal as well as the palisade cells have increased in 
radial plane and the parenchyma cells of the abaxial part have multiplied forming 
large irregular cells. 

Locality: Taveuni: Soqulu Estate; Coll. M S Mani, M K Kamath and D S Singh, 
20 December 1978. 

18. Family Myristicaceae 

18.1 Myristica macrantha A C Smith (figures 77-79) 

Gall No. 843 Psyllidae 

A large hypophyllous dome-shaped, beaked covering-growth about 4mm and a 
smaller epiphyllous conical, curved pouch about 3 mm long; hard, brittle, smooth, 
bright reddish-brown; mature gall dehisces by a circular line along the subbasal part 
and falls off as a lid, leaving behind the epiphyllous portion as a cup; median part of 
the gall 5 mm wide. 

Each gall consists of two distinct parts: a larger hypophyllous dome-shaped part 
with a small beaked tip; the ostiole occurs at the summit of the beak. The epiphyllous 




Figures 77 - 79. Myristica macrantha A. C. Smith. 77. Abaxial view of the leaf with some 
intact and some dehisced galls. 78. One entire gall enlarged. 79. Vertical section of a gall. 
(Ep, Epiphyllous portion of the gall; Hy, hypophyllous covering growth portion; La, 
lamina; LC, larval chamber; NZ, nutritive zone; SZ, sclerotic zone). 



106 



M S Mani and P Jayaraman 



part is hemispherical with a short sharply curved beak. The ostiole leads to the 
elliptical axial smooth wide larval cavity, surrounded by several layers of small 
rectangular nutritive cells filled with dark contents; followed by a broad zone of 
sclereids, which encircles the entire gall cavity and extends upto the path of the 
ostiole. External to the sclerotic zone, the gall tissue consists of homogenous layers of 
irregular thin-walled cells, stretched tangentially to the plane of the blade and 
represent apparently derivatives of the exaggerated palisade tissue; vascular strands 
do not extend into the hypophyllous part of the gall, but occur as small islands in the 
epibasal portion only. All gall cells, including the sclereids, contain spherical globules 
of tannin. The passage of the ostiole is clothed with minute trichomes. 

Locality: Taveuni: Soqulu Estate; Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 



1 8.2 Myristica sp. (figures 80-82) 

Gall No. 861 Psyllidae 

Hypophyllous, cylindrical and pyramidal, scattered, sparse, dark brown, closed, 
indehiscent pouch gall; marked by a circular epiphyllous depression and a central 
bluntly conical spot; each gall measures about 3 mm long and 1 mm broad. 

In vertical section the gall appears as a pyramidal structure, with flat apex and 
wavy surface, with large, smooth, obovoid axial larval cavity; gall tissue consists of 
homogenous layers of compact, thin-walled highly tanniniferous cells of varying size 
and shape. A few layers of cells around the larval chamber possess small spherical 




DP 



Figures 80-82. Myristica sp. 80. Abaxial view of the leaf with galls. 81. One gall 
enlarged. 82. Vertical section of the gall (DP, Derivatives of palisade tissue; LC, larval 
chamber; SC, secretory canal). 



Plant galls from Fiji Islands 



107 



starch grains. The secretory cavities similar to those of the normal leaf are found in 
the peripheral region of the gall. The vascular traces do not extend beyond the 
middle of the gall. The outer boundary of the gall is formed by a distinct, cuticulari- 
sed epidermis. 

Locality: Taveuni: Soqulu Estate; Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 



19. Family Lauraceae 

Cryptocarya sp. (figures 83-84) 

Gall No. 842 Psyllidae 

Hypophyllous, conical or subtruncate, pyramidal, sessile, indehiscent, smooth, 
reddish-brown, solitary and scattered; 3-5 mm long and 2 mm thick; ovoid gall cavity 
is basal, the upper part being solid; a minute truncated projection occurs on the 
opposite side of the gall. The gall tissue consists of homogenous, compact, thin- 
walled, polygonal, tannin-filled, parenchyma cells. A few layers of cells around the 
gall cavity are smaller and darker. Some of the cells are much elongated and canal- 
like, with granular contents. The lining layer of the gall cavity consists of small 
rectangular cells. The outer epidermis differs from that of leaf in being less papillate. 
The vascular tissues of the leaf extend into the gall upto the upper solid part. 

Locality: Taveuni: Qeleni Road; Coll. M S Mani, Sundaresan and M K Kamath, 
20 December 1978. 




Figures 83-84. Cryptocarya sp. 83. Abaxial view of the leaf with galls. 84. Vertical 
section of a gall. (L C, Larval Chamber; N Z, nutritive zone; V S, vascular strand). 



108 M S Mani and P Jayaraman 

20. Family Euphorbiaceae 

20.1 Glochidion ramiflorum JRandGForst. (figures 85-87) 

Gall No. 836 Cecidomyiidae 

Hypophyllous, solitary, scattered, hairy, indehiscent, two-chambered, conical 
covering growth gall, 3 mm high and 3 mm wide, with the ostiole on the summit of 
the minute hypophyllous covering growth; the gall is marked above by a smooth, 
circular ridge. 

Erect or curved, conical covering growth, with an apical ostiole; surface studded 
with long, uniseriate, unbranched, multicellular hairs, which almost mask the gall. 
The epiphyllous part is a glabrous hemispherical pouch. Ostiolar canal with straight, 
stiff outwardly directed hairs; spaceous gall cavity is divided into 2 incomplete, 
laterally disposed chambers by short, thick, pillar-like outgrowths from the floor and 
by pendulous tissue from the roof of the gall cavity, meeting each other, but do not 
fuse, so that the incomplete central partition divides the gall cavity into lateral, 
spherical empty atrium and a larval chamber proper. 

The gall cavity is surrounded by 7 or 8 layers of thin-walled and polygonal 
nutritive cells; followed by 4 or 5 layers of brachysclereids, extending upto the ostiole 
through the ostiolar canal. External to the sclerotic zone are several layers of 
parenchyma cells. In the region of the adaxial pouch, these parenchyma cells, are 
compact and polygonal. In the basal region of the conical portion, they are less 




" Figures 85-87. G. ramiflorum J.R. and G. Forst. 85. Abaxial view of the leaf with 
galls. 86. One gall enlarged. 87. Vertical section of gall (Ep, Epiphyllous part of the gall; 
Hy, hypophyllous conical part of the gall; L, larva; Os, ostiole; NZ, nutritive zone; SZ, 
sclerotic zone; Tr, trichome). 



Plant galls from Fiji Islands 



109 



compact, vertically stretched and gradually merge into the different tissues of the leaf. 
The surface cells of the central pillar and of the pendent mass elongate into straight, 
stiff, unbranched unicellular hairs. The annular sclereids of the mesophyll are 
considerably hypertrophied and much elongated along with other cells of the gall. 

Locality: Viti Levu: Cuvu, Novouvou Road; Coll. M S Mani, M K Kamath and 
Sundaresan, 12 January 1979. 

20.2 Macaranga magna Turrill (figures 88 and 89) 

Gall No. 831 Eriophyes sp. 

Epiphyllous, shallow, wide open, blister-like pouches, about 7 mm large; often very 
much crowded; young galls circular and pale green; mature ones irregular and 
brown, with soft and velvety dense tomentum. 

The gall consists of considerably hypertrophied epidermal cells. The upper 
epidermal cells divided in anticlinal plane and stretched laterally, thus accommodat- 
ing the increased surface area of the gall. The palisade cells widened several times, 
accompanied by reduction in the height of the cells and they remain single layered. 
The spongy mesophyll cells have also undergone considerable hypertrophy thus 
obliterating the intercellular space. The hypertrophy of different tissues leads to the 
arching over the lamina to form the shallow pouch. Dense unicellular, unbranched 
epidermal hairs occur on both outer and inner surfaces of the gall curled, with 
granular inclusions. 

Locality: Viti Levu: Deuba beach side; Coll. M S Mani, Sundaresan and M K 
Kamath, 12 December 1978. 

21. Family Ulmaceae 

21.1 Gironniera celtidifolia Gaud, (figures 90-92) 
Gall No. 857 Cecidomyiidae midge 

Hypophyllous, solitary, scattered or agglomerate, indehiscent, glabrous, reddish- 
brown pouch gall, with a circular raised spot above and is a minute curved beak-like 



89 




Figures 88 and 89. Macaranga magna Turrill. 88. A portion of the leaf with galls. 89. T.S. 
of leaf through gall (G, Galled portion; La, lamina). 



110 



M S Mani and P Jayaraman 




Figures 90-92. G. celtidifolia Gaud. 90. Adaxial view of the leaf with galls. 91. Abaxial 
view of the galls. 92. Vertical section of the gall (L, Larva; Os, ostiole; SZ, sclerotic zone; 
Tr, trichome). 



hypophyllous covering growth, about 4 mm in diameter and 2 mm high in the 
median part. 

The gall is more or less fusiform in cross section; larval cavity horizontally 
elongated in the middle with a single larva; ostiolar canal horizontal for a short 
distance then bent down to ostiole, situated at the tip of the small pointed, excentric 
covering growth. The passage in the region of the covering growth is filled by dense, 
brownish, straight hairs, directed downwards, and thicker and sturdier than those on 
the leaf. The mass of gall is of compact thin-walled homogenous polygonal cells; in 
the roof of the larval cavity, the upper epidermis is immediately followed by 5-7 
layers of brachysclereids, which form a horseshoe-shaped zone around the larval 
chamber; inner to the sclerotic zone are several layers of laterally stretched 
parenchyma cells. The gall tissue contains large and dense starch grains and most 
cells are tanniniferous. 

Locality: Taveuni: Soqulu Estate; Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 



21.2 Gnetum gnemon Linn, (figures 93-95) 
Gall No. 848 Cecidomyiidae 

Foliar gall; blister-like, yellowish-green, smooth indehiscent, solitary or agglomerate, 
closed gall, more hypophyllous than epiphyllous; lamina depressed around the gall, 



Plant galls from Fiji Islands 



111 




Figures 93-95. G. gnemon Linn. 93. Abaxial view of the leaf with gall type 
851. 94. Vertical section of gall No. 851. 95. Vertical section of gall No. 848 (L C, Larval 
chamber; M Z, meristematic zone; N Z, nutritive zone; T Z, thick walled cell zone). 



so that the gall appears as if it arises out of a pit. The gall tissue consists of highly 
proliferated and hypertrophied cells. One or more large, irregular larval cavities 
occur either at the centre or peripherally in the gall; surrounded by a thick layer of 
collapsed cells; followed by a meristematic zone; asterosclereids scattered in the gall 
tissue; they are more numerous and larger than in normal leaf; gall stomata 
epiphyllous; hypophyllous galls display certain modifications as compared to the 
normal leaf stomata; subsidiary cells of the gall stomata proliferated, losing their 
identity and the epidermal cells have also highly hypertrophied. 

Locality: Viti Levu: Colo-i-Suva; Coll. M S Mani, M K Kamath and Sundaresan, 
25 January 1979. 



Gall No. 851 (figures 93 and 94) Cecidomyiidae midge 

Foliar gall; irregular, blistej-like, solitary or agglomerate, closed, indehiscent, 
smooth, yellowish-green, pocken-gall, projecting more or less equally on both 
surfaces of the blade; 7 mm wide and 4 mm thick; more or less elliptical or circular in 
transection with 1 or 2 larval cavities in the middle; cavities small spherical, 
surrounded by wide concentric zone of thin walled, compact, polygonal nutritive 
tissue, with prominent nuclei and numerous starch grains; outer of this zone is the 
parenchyma zone with similar, but slightly thick walled, empty cells; delimited by a 
distinct epidermis, cells of which have undergone anticlinal divisions and lateral 
expansion; asterosclereids are scattered in the periphery of the gall and they resemble 
those of the normal leaf except for a slight increase in size. 

Locality: Viti Levu: Colo-i-Suva; Coll. M S Mani, M K Kamath and Sundaresan, 
25 January 1979. 



112 M S Mani and P Jayaraman 

22. Family Filicales 

Nephrolepis biserrata (Sewartz) Schott. (figures 96-99) 
Gall No. 863 Eriophyes sp. 

Hypophyllous, cup-shaped, sessile or subsessile, covering-growth of irregular size 
and shape occurring distally on the margins, or on the midrib of the pinnule; bowl- 
shaped gall has a wide opening, the rim of the cup being fringed and incurved. The 
outer surface of the gall is smooth and the interior is brownish. Measurement 2 mm 
thick and 4 mm broad. 

The gall develops as a covering growth from the lower epidermis and a few layers 
of the subepidermal ground tissue to assume a fleshy hollow cup-like structure. The 
cells of inner lining layer of the cup grow into many irregularly branched filaments or 
thin lobed plates, one cell thick; plate-like emergences several cells thick at base and 
ultimately become uniseriate at the tip; plates and filaments fill up the entire interior, 
of the gall and mites crowded in the narrow spaces of these outgrowths; cells of the 
emergences filled with tannin, the characteristic cytological features of the lining 
layer of cells, usually observed in most of the mite galls, are not evident here. The 
cells constituting the gall are much hypertrophied, irregular in shape, loosely 
arranged and possess thin wavy walls. 

Locality: Taveuni: Qeleni; Coll. M S Mani, M K Kamath .and D S Singh, 20 
December 1978. 




Figures 96-99. Nephrolepis biserrata (Sewartz) Schott. 96. Pinnule with terminal galls. 
97. Pinnule with cup shaped gall on the midrib. 98. Vertical section of the gall in the middle 
portion of lamina. 99. Vertical section of gall at the apex of lamina. (La, Lamina; PO, plate- 
like outgrowths). 



Plant galls from Fiji Islands 
23. Species Incertae sedis 



113 



Galls on unidentified plants (figures 100 and 101) 
Gall No. 845 Cecidomyiidae midge 

Hypophyllous, flask-shaped, fleshy, solitary and sessile; erect or curved; with a 
dilated basal part measuring about 3 mm in diameter and an apical part produced 
into narrow cylindrical slender, beaked straight or somewhat curved horn-like 
process, 1 mm diameter; gall measures 7 mm high from base to the tip; on the upper 
surface of leaf the basal part of gall forms a hemispherical raised spot, ostiole occurs 
on the summit of the beaked horn-like process; exit hole at the dilated basal part of 
the gall. 

The gall develops as a small conical hypophyllous covering growth, the gall cavity 
in the initial stage is filled with club-shaped, glandular outgrowths from the surface 
of the cavity; a few hairs are seen at the ostiolar region, in the mature gall glands and 
trichomes absent in ostiole; gall cavity axial and it runs upto the tip of the gall, is 
wide at base and narrow and canal-like inside the horn; the basal wider part of the 
larval cavity is surrounded by a dark layer of collapsed cells, inside which follow a 
zone of wound healing tissue and a broad zone of sclereids; gall fully vascularised, 
vascular strands extend upto the extreme tip of the gall; path of the exit hole is also 
surrounded by the wound healing tissue; cells bordering the narrow passage of the 
larval cavity, leading to the ostiole are intact, thin-walled and elongated parallel to 
the canal. Outer to the sclerotic zone occurs the vascular strand followed by several 
layers of homogenous compact polygonal cells, which become less compact and ir- 
regular towards the periphery; epiphyllous hemispherical projection also consists of 
compact homogenous mass of cells. 

Locality: Viti Levu: Novouvou Road; Coll. D S Singh, 12 January 1979. 




Figures 100 and 101. Gall No. 845. An unidentified plant. 100. Leaf with 
galls. 101. Vertical section of gall. (LC, Larval chamber; Os, ostiole; SZ, sclerotic zone; 
VS, vascular strand). 



114 



M S Mani and P Jayaraman 



Gall No. 846 (figures 102-105) Cecidoniyiidae midge 

Hypophyllous, cylindrical, straight or lopsided, scattered covering-growth gall, 
measuring about 1 cm long and 2 mm in diameter; with a hemispherical epiphyllous 
pouch; the entire outer surface of the gall covered by straight, rough, bristle-like, 
unicellular, unbranched trichomes; a circular ostiole occurs at the tip of the cylinder. 
The gall develops as a small conical hypophyllous covering growth with a minute 
epiphyllous projection. The covering growth is derived entirely from the spongy 
tissue and consists of small compact transversely dividing cells, arranged in regular 
rows parallel to the vertical axis of the young gall; ostiole very narrow, outer surface 
covered by trichomes; a few layers of cells in the outer zone of the young gall do not 
show much divisions and the cells are somewhat irregular; epiphyllous projection is 
formed by periclinal divisions of the adaxial epidermis and the palisade cells. In the 
mature gall, the cells have extensively elongated in a vertical plane at right angles to 
the leaf surface, forming the cylindrical body of the gall. As the elongation stops, a 
few layers of cells inner to third or fourth layers from the larval cavity differentiate 
into sclerotic zone, which lines the entire larval cavity from the base upto the tip of 
the gall. Outer to the sclerotic zone occurs the vascular strands which extend to the 
extreme end of the cylinder and these strands are extensions of the vascular system of 
the lamina. A few layers of cells, inner to the outer epidermis, which are derived from 
the cells of less meristematic activity at the initial stage of the gall, are filled with 
tannin. The epidermal hair has a bulbous base, which is deeply embedded below the 
epidermal layer. In the mature gall, the ostiole becomes wider and circular through 
which the insect escapes. 



102 




Figures 102-105. Gall No. 846. An unidentified plant. 102. Abaxial view of leaf with 
galls. 103. A single gall enlarged. 104. Vertical section of gall. 105. T.S. of gall. (LC, Larval 
chamber, Os, ostiole; PZ, parenchyma zones; SZ, sclerotic zone; V S, vascular strands). 



Plant galls from Fiji Islands 



115 



Locality: Viti Levu: Colo-i-Suva; Coll. M S Mani, M K Kamath and Sundaresan, 
25 January 1979. 

Gall No. 871 (figures 106-108) Cecidomyiidae midge 

Hypophyllous covering growth gall, occurring mostly near the veins, thick, quite 
conspicuous, cylindrical, sessile, greyish-green, softly hairy, indehiscent, straight or 
slightly curved; ostiole minute and terminal, 1-5 cm long and 0-5 cm thick; the gall 
develops as a small covering growth on the abaxial side by proliferation of 
mesophyll; cells of the palisade and spongy mesophyll tissues divide repeatedly by 
periclinal walls and give rise to a hemispherical mass of small cells filled with dense 
cytoplasmic contents. These cells stretch in vertical plane at right angles to the 
surface of the leaf and develop into a long cylindrical gall with the ostiole at the tip. 
The ostiole leads to the narrow straight canal-like axial larval cavity which extends 
down to the base of the cylinder. 

The mature gall has 4 distinct zones of tissues. The gall cavity is surrounded by a 
zone of radial files of regular rectangular cells, the cells bordering the cavity are 
collapsed. Outside of this lies a sclerotic zone of either angular or circular cells with 
secondary wall thickness gradually diminishing outwards; outside of this is a wide 
zone of large thin-walled parenchyma, containing large concentric starch grains; 
vascular tissues occur in this zone as narrow as anastomosing strands. The outermost 
zone is made up of somewhat thick-walled, smaller cells, the lateral walls of which 
possess circular primary pits. A distinct epidermis delimits the fourth zone. Simple 
and stellate hairs occur on the surface of the gall. 

Locality: Taveuni: Soqulu Estate; Coll. M S Mani, M K Kamath and D S Singh, 
19 December 1978. 




Figures 106-108. Gall No. 871. An unidentified plant. 106. A portion of lamina with a 
gall. 107. Vertical section of a gall. 108. T.S. of gall. (La, Lamina; LC, larval chamber; 
NZ, nutritive zone; PZ, parenchyma zone; SZ, sclerotic zone; TCZ, thick walled cell zone; 
V S, vascular strands). 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 2, March 1987, pp. 117-127. 
Printed in India. 



Biochemical correlates in Rhipiphorothrips cruentatus Terminalia 
catappa interactions with special reference to leaf infestation patterns 

G SURESH and T N ANANTHAKRISHNAN 

Entomology Research Institute, Loyola College, Madras 600 034, India 

MS received 10 December 1986 

Abstract. Leaf age correlated infestation patterns of Rhipiphorothrips cruentatus on 
Terminalia catappa revealed specificity in stage and site selection. An indepth analysis of the 
quantitative as well as qualitative profiles of proteins, amino acids and fatty acids in age 
specific leaves exemplified a correctable influence on the infestation patterns. 

Keywords. Rhipiphorothrips cruentatus Terminalia catappa; biochemical correlates; site 
selections; host selections. 

1. Introduction 

Phytophagous thrips species tend to selectively exploit certain areas of leaves of 
different ages (Fennah 1963) and also show vertical stratification restricted to diffe- 
rent nodes (Ananthakrishnan 1984; Ananthakrishnan et al 1982), in order to locate 
nutrient rich solutes at specific sites. Infestation is known to decline with the age of 
the leaves following a reduction in the availability of soluble nitrogen, an aspect 
exemplified by Selenothrips rubrocinctus on Anacardium occidentale (Fennah 1963). 
The amino acid concentration occurring within the leaves of varying age also offers a 
clue to the ability of the insect to establish themselves on the host. In this connection, 
the role of free amino acids in host preference has been discussed in relation to 
Retithrips syriacus infesting Ricinus communis (Ananthakrishnan and Muraleedharan 
1972), as also the allied panchaetothripine species Rhipiphorothrips cruentatus, a 
highly polyphagous species (Ananthakrishnan and Muraleedharan 1974). A reduc- 
tion in the quantity/quality of food of adult females tends to result very often in the 
delay and decline in the rate of egg production (Scriber and Slansky 1981). Besides, 
feeding is strongly influenced by olfactory and gustatory responses resulting from 
both nutritional and non-nutritional components in the food so that such behavi- 
oural responses as change in feeding rates could result from changes in food quality. 
Though a broad-based idea of the intake of major nutrients like total nitrogen, 
carbohydrates, proteins and lipids is generally assessed, an indepth analysis of the 
nature of lipids and amino acids in feeding, growth and development appear essential 
for a better understanding of the utilization of these substances. An attempt has 
therefore been made in this paper to analyse the above aspects with reference to 
Rhipiphorothrips cruentatus infesting the myrtaceous tree host Terminalia catappa, 
which shows marked preferences to leaf age. 

2. Materials and methods 

Based on their position on the branch in relation to the shoot apex, the following 

,J:XT 4. i~ A f ~ /r \T\ ~r nr -.,*. ~ 11 *~JL *u^ ,,.,:ii > ~: i i i~ 



118 G Suresh and T N Ananthakrishnan 

with the I leaves unopened; the II leaves from the terminal bud, partially opened; 
completely opened and green leaves III from the terminal bud; dark green comp- 
letely opened IV leaves; and mature V leaves respectively. 

Ethanolic extracts of the leaf material were prepared (Harborne 1973), an aliquot 
of which was acidified with 2 M hydrochloric acid and incubated for 0-5 h, the 
resultant solution cooled and filtered. The phenols taken into ether from the extract 
was washed, evaporated to dryness, and taken into ethanol for the estimation of total 
phenols (Bray and Thorpe 1954) and o-dihydric phenols (Johnson and Schall 1952). 
The ethanol extract devoid of phenols was used for the quantitative estimation of 
amino acids (Moore and Stein 1948) and carbohydrates (Dubois et al 1956). Total 
chlorophyll was estimated by spectrophotometry of the acetone (80% v/v) extract of 
the fresh leaves (Yoshida et al 1976). Buffer soluble proteins were extracted from the 
fresh leaves in phosphate buffer saline (pH 7-2, 0-1 M with 0-85% sodium chloride) 
and estimated (Lowry et al 1951). Total lipids were extracted from fresh leaves in 
CHC1 3 :CH 3 OH (2:1) by the method of Folch et al (1957) and estimated by 
gravimetry. 

Five g of fresh leaves were ground in ice cold acetone using a prechilled mortar and 
pestle, and later using an ultrasonic disintegrator. The homogenate was centrifuged 
at 12500 g for 30 min, at 5C using a Hitachi refrigerated centrifuge. The residue free 
of pigments was used for enzyme analysis. 200 mg of the acetone powder was 
suspended in 4 ml of buffer [phosphate buffer, pH 7-0, 0-3 M for peroxidases; borate 
buffer, pH 8-8, 0-1 M for phenylalanine ammonia lyase (PAL) and tyrosine ammonia 
lyase (TAL)] and extracted for an hour at 4C. The extract was centrifuged at 
1 1500 g for 30 min and the clear supernatant was used for enzyme assay. Peroxidase 
was assayed (Loebenstein and Linsay 1961) using 2-5 ml of pyrogallol (0-1%), 0*1 ml 
of 0-1% hydrogen peroxide and 0-1 ml of the enzyme extract and the absorbance at 
420 nm was taken at an interval of 30s for 10 min, using a Hitachi UV-Vis 
Spectrophotometer. Increase in absorbance by 0-01/min/ml of enzyme used/mg pro- 
tein was expressed as a specific unit. For the estimation of PAL and TAL (Higuchi 
1966), 2 ml of the substrate (0-1% L. phenylalanine and L. tyrosine respectively) was 
incubated with 1 ml of the enzyme in borate buffer (pH 8-8, 0-1 M) and 1 ml of 
distilled water for 1 h. 0-4 ml of 3 N HC1 was added to stop the reaction and the 
reaction mixture was twice extracted with ether and dried. The residue was redissol- 
ved in 2-5 ml of 0-05 N NaOH and read at 268 nm for PAL and 333 nm for TAL 
respectively. Specific units were expressed as /*g trans. cinnamic acid released/ml 
enzyme used/mg protein for PAL activity and as increase in absorbance by 0-1/ml 
snzyme used/mg protein for TAL activity. For the analysis of the qualitative profiles 
sf amino acids in different leaf stages, Hewlett Packard High Performance Liquid 
:hromatography was used. o-Phthalaldehyde derivatives of the amino acids in the 
alcoholic extract was chromatographed (Lindroth and Mopper 1979) using Hypersil 
ODS 5 /^m column. 0- 1 M phosphate buffer pH 7-7 and methanol were used as 
mobile phase and fluorescent derivatives were detected at 340 nm. The amino acids 
were identified based on standard chromatograms of individual amino acids and the 
area per cent of individual amino acid peaks were calculated. 

To the total lipids extracted from the leaf tissue, 10 ml of ethanol, 3 ml of 28% 
ammonium hydroxide, 25 ml of petroleum ether and 25 ml of diethyl ether were 
added to a separating funnel, shaken for 5 min and allowed to stand for 20 min. The 
bottom phase was drained off, the ether phase was dried to which 3 ml of 0-5 N 



Biochemical correlates in R. cruentatus 119 

NaOH in methanol was added and heated in a steam bath for 15 min. To this, 5 ml 
of water was added and 2 N HC1 were added slowly until the pH was approximately 

2. The fatty acid methyl esters were then extracted into 5 ml of petroleum ether and 
5 ml of diethyl ether from the acidified methylated lipid extract. Fatty acids were 
analysed by a Hewlett Packard high performance liquid chromatograph (HPLC) 
system at two detection wavelengths, i.e. 210 nm and 230 nm using an Hypersil ODS 
5 ^m column with water and acetonitrile as mobile phase at a flow rate of 0-45 ml 
according to the gradient programme as per Schuster (1985). Retention times and 
area per cent of fatty acid methyl esters were recorded. 

Polyacrylamide gel electrophoresis (PAGE) was carried out (Davis 1964) using 
7-5% gel on a slab gel electrophoretic apparatus in an alkaline buffer system (Tris- 
glycine buffer, pH 8-6). Acetone powder was suspended in borate buffer and the 
resultant supernatant was used for qualitative profiles of proteins. 0*02% Coomassie 
brilliant blue G250 in methanol, acetic acid and water (25:7:68) was used for 
staining the gels. Zymograms were drawn from the protein stained gels. 

3. Results 

R, cruentatus, a panchaetothripine species, infests the abaxial side of the leaves of 
T. catappa during the months of March-September. Infestation in terms of number 
of leaves attacked in several tree branches examined during the above period showed 
the maximum incidence to be around 56% and the feeding index (which is the area 
damaged in cm 2 /total laminar area in cm 2 x number of individual thrips) to be 
34-4 8-24. The infestation ratio, which is the area of damage of laminar surface/total 
laminar area in cm 2 was found to be 0-240*21 for March-April, 0-23 0-14 for 
May, 0-23 0-1 3 for July and 0*25 0-1 3 for August-September during the period of 
observation. Analysis of the infestation pattern in different leaf stages indicated the 
infestation ratio and the percentage ratio to be 0*21 0-31 and 53% for the leaf 
stage III and 0*20 0-31 and 58% for the leaf stages IV and V, while the leaf stages I 
and II as well as the senescing yellow leaves were not infested by this thrips species. 

Observations on the life cycle of the thrips species on T. catappa leaves revealed 
the number of eggs laid by a single female to be varying from 15-50 and the 
oviposition rate to be 2-6 per day, the total duration of life cycle ranging from 14-28 
days inclusive of the preoviposition period, with the individual stages having the 
following duration in days: preoviposition period 5-7 0*4; I instar 2*36 0*39; II 
instar 3*38 0-41; prepupa 1 -day; and pupa 2*20 0*45 making the total duration to 
be around 19 days. However, the total duration and fecundity were 21-06 and 25 
2-94, 14-11 0*08 and 36*41 2*94, and 11 -86 0*09 and 45*53 0-09 at 25C and 
84% RH, 30C and 81% RH and 35C and 74% RH respectively. 

Since infestation by R. cruentatus was on the flush leaves (stage III) and on 
subsequent leaf stages, an analysis of the basic biochemical components in relation to 
the distribution pattern of this species, appeared relevant. Quantitative analysis of 
chlorophyll of different leaf stages revealed an increasing trend in terms of concen- 
tration, the maximum being 2*188 mg/g fresh weight in completely mature leaves. 
The quantity of carbohydrates which indirectly indicates the functional efficiency of 
the quantum of chlorophyll present did not reveal much of variation among different 
leaf stages, though considerably high quantity (6*5 mg/g fresh weight) was noticed in 
leaves of stages I and III (figure la). 



10 



G Suresh and T N Ananthakrishnan 



3l 



i 



(-9000 (A) 

_ Carbohydrates 

2 " / A 

i-^x: 



2 



- jf^Ct\\orotf\y\\ ^ 



180 



Protein 





Figure 1. Quantitative biochemical profile in relation to leaf stages of differing age. 



Since life-history patterns of herbivores and the associated plants tend to be 
3sely correlated in relation to the availability of nitrogen, investigations on the 
oteins as well as the free amino acids in the host plants had to be attempted, 
terestingly enough, quantitative analysis of proteins revealed again a pattern simi- 
r to that of carbohydrates in that the maximum quantity was present in leaves of 
age III (153 mg/g fresh weight) (figure Ib), while the qualitative profile based on 
\GE revealed maximum number of bands in the extracts of the leaf stage I (figure 
. Minimum number of bands were found in the III stage leaves indicating the 
esence of higher concentrations of a few proteins present in the completely open 
een leaves. Examination of the free amino acid distribution in the cell sap of the 
aves of T. catappa which might form one of the major components of food for this 
sect species, revealed a trend of decreasing concentrations in relation to increasing 
af age (figure Id). Valjue as high as 1-525 mg/g fresh weight was recorded for I 
aves, while the leaf stages III, IV and V did not show much of variation in their 
lantitative profile, the values being 0-200, 0-125 and 0-225 mg/g fresh weight of 
isue analysed. 
The qualitative profile of free amirio acids and their individual concentrations 



Biochemical correlates in R. cruentatus 



121 



0-n 



S1 S2 S3 S4 55 



20 - 1 
cm 



IkT 



. 

xl 
c 



-6 



Figure 2. Zymogram of the slab gel electrophoresis of proteins from different leaf stages of 
T. cat&ppa. S1-S5, Indicates leaf stages; B, bromophenol blue; O, origin. 



sased on the area per cent of the peaks) were examined using a HPLC (figure 3 and 
able 1). Based on the retention time of standard amino acids, individual ammo acids 
/ere detected. In the leaf stage I, the notable amino acids that could not be detected 
/ere threonine, argiriine, alanine and valine, and the highest concentrations of total 
mino acids in this appear to be due to the 10 free amino acids as indicated in table 
. From stage II onwards, the availability of free amino acids increased in terms of 
[uality, with a minimum of around 14 amino acids inclusive of the known essential 
.mino acids for insect feeding like threonine, arginine, phenylalanine, methionine etc. 

While increasing total lipid content was typical with increasing leaf age (figure 4b), 
IPLC analysis of the fatty acid methyl esters both at 210 nm as well as 230 nm 
ndicated that decreasing number of fatty acids to be present during increasing leaf 
.ge (figure 4a, table 2). Among the CIS fatty acids, stages I, III and V had linolenic, 
>leic and stearic acids, while stage II had oleic and stearic acids, and stage IV had 
nly linolenic acid. The fatty acids of differing retention time in the 5 leaf stages are 
>rovided in table 2. 

Phenolics which form the product of secondary metabolism of plants, is of interest 
ti insect-plant interactions since they could either be feeding attractants forming part 
>f the food for insects along with other components like proteins, amino acids, 
arbohydrates etc or as feeding deterrants. Hence, a quantitative study of the total 
>henols, o-dihydric phenols and the important enzymes involved in secondary 
metabolism such as peroxidase, phenylalanine ammonia lyase and tyrosine ammonia 
yase were also attempted. Decreasing trends in the quantitative profiles of the total 
ihenols and the o-dihydric phenols (figure Ic) as well as of activities of peroxidase 
figure le), phenylalanine ammonia lyase and tyrosine ammonia lyase (figure If) were 
vident from the leaf stages II-V correlating well with increasing leaf age. However, 
he terminal buds with I leaves recorded low level of phenolics and comparatively 
dgher levels of activities of all the 3 enzymes studied. 



122 



G Suresh and T N Ananthakrishnan 



(V) 




Retention time (min) 



Figure 3. HPLC chromatograms of amino acids in 'different leaf stages of T. catappa. 



4. Discussion 

Natural infestation of leaves of T. catappa by R. cruentatus indicated a specific 
pattern in relation to both time and space, infestation occurring during a specific 
period i.e. March-September with the peak population of adults during the months 
of August/September. Infestation being specific to the abaxial side of the compara- 
tively young, completely opened, fresh maturing. leaves (stage III and continuing to 
stages IV and V), selective exploitation for feeding site and subsequent reproduction 
were very evident. The patterns of infestation, feeding and reproduction as well as the 
population build-up appear to be in close correlation with the initial leaf age and its 
subsequent stages indicating the influence of chemical changes in the host leaves. The 
availability of a substrate with an optimal mixture of suitable nutrients as well as 
feeding stimulants offered by any part of the leaf at any time cause such specific 



Biochemical correlates in R. cruentatus 



123 



Table 1. Amino acid profile in different stages of T. catappa leaves. 





Amino acid 




Area percentage* 




Retention 


Leaf stages 




Lime 
(min) I 


II III 


IV V 


Asparagine 
Glutamine 


3-50 02-99 
7-77 12-55 


00-346 13-849 02-240 00-847 
4-704 14-742 3-619 06-944 




Serine 


8-24 ND 


ND ND 


ND ND 




Histidine 


10-30 07-57 


03-357 03-458 00-392 05-729 




Glycine 
Threonine 


11-65 ND 
12-20 ND 


05-654 04-840 05-175 05-706 
04-875 01-486 ND 02-377 




Arginine 
Alanine 


13-35 ND 
14-50 ND 


10-795 26-466 30-462 09-205 
04-091 00-376 08-736 03-854 




Tyrosine 
Cysteine 
Tryptophan 
Methionine 


15-25 05-50 
16-75 03-056 
17-40 02-715 
17-75 02-977 


03-611 08-130 09-216 11-610 
06-174 03-387 06-728 06-306 
03-875 01-465 02-667 05-920 
03-132 01-703 03-588 01-544 




Valine 


18-20 ND 


23-596 11-946 16-984 18-585 




Phenylalanine 
Isoleucine 


19-10 03-799 
19-70 01-778 


ND 02-559 
10-246 ND 


ND 03-614 
ND 11-261 




Leucine 


20-45 00-293 


11-013 00-889 03-038 04-339 




Lysine 


21-55 03-867 


04-801 00-378 


ND ND 


*Based on amino acid peak areas only. 
ND, Not detectable. 


2 

X* 
U Ki 

I 


O co 

S>23 %< 
s * % I 

* i ~ | 
3 

I li s jr 


(1) 210 (2 

/ps 
J < 

CM <\j <u 
fV <\i r\j 
16 N d> g 

UL;_ ^ 

J (3) 210 

/ 


) 230 a 

S 

j *-. \J 


2)210 

LL. 

(4) 2IO 


(3) 230 II 

I 


1 1 "* i 


J 


LJ1 

, (5) 230 1 


JL v. 


Jl -Hi 


\1 


(5) 210 '0 


2O'0 


2O 




J 


J400r 


Total lipids (B) 8Q w 


ater content (C) 


* 


F J 


I 


(%) 




k^s/ 


L llN- / 


1 


^ 


n 


i 


1 


o> 
E 


+-+r^f C 


"V\ 


i 
o 




200"- 


TO'- 


\ 


1 1 

20 O 


-j p- 

20 


II III IV V 


II III IV V 



Retention time (min) 



Leaf stages 



Figure 4. A. HPLC chromatograms of fatty acid methyl esters of different leaf stages of 
T. catappa. Numbers in circles indicate leaf stages. 210 and 230 indicate detection wave- 
lengths. S, Start position. B. Total lipids in different leaf stages. C. Water content in 
different leaf stages. 



124 



G Suresh and T N Ananthakrishnan 

Table 2. Qualitative profile of fatty acid methyl esters in different leaf stages of T. catappa. 



Area percentage* 


Retention 
time 
(min) 






Leaf stages 




I 


II 


III 


IV V 


0-76 











1-448* 


1-20 





9-803* 





0-114* 


2-50 










1-204" 




0-589 C 






96-184 f 


2-75 


0-438* 












0-503 C 


99-44 c 






3.00 


76-206" 


0-540* 


1-396* 







4-96 l c 




1-435 C 




3-25 







2-124* 


4-823* 




~65-685 c 




0-789' 


71-020'' 


4-00 








0-490* 


39-892* 








89-620 f 




4-15 


0-854* 





76-071* 


1-760* 


5-5 





44-922* 





16-450* 


6-0 


5-184* 


2-347* 








6-25 





2-430* 








6-65 





1-069* 








8-50 Lino!enic 


8-606* 





6-691* 


33-351* 11-737* 


C18:3 






H32 f 




16-25 Oleic 


0-606* 


15-527* 


0-393* 


28-930* 


C18:l 










17-25 Stearic 


2-709* 


15-385* 


11-118* 


3-607* 




25-253' 




5-097 c 


28-930 c 


18-90 


5-154* 













8-944 c ' 









"Based on fatty acid methyl esters peak areas only 
*At 210 nm. 
c At 230 nm. 



infestation patterns. Alternatively it may also be due to the optimal substrate 
availability 'within a unit of actual feeding time' at specific circumstances sufficient to 
let the thrips live, grow in size and to lay eggs (Fennah 1963). The latter postulate 
may explain the infestation of the III, IV and V stage leaves due to the fact that 
the optimal concentrations of different nutrients are not available in all stages of leaf 
development. 

Differential feeding requirements of insects and the diversified proportional com- 
position of food chemicals among different plant species makes it a finely balanced 
problem for the insect to obtain an adequate intake of proteins and amino acids. 
This gets more complicated due to the variation in the leaf nitrogen content as well 
as water content in relation to seasonal changes. A comparative analysis of leaves of 
differing age of T. catappa in relation to infestation revealed an increasing protein 
concentration upto the III and IV stages, while the quantitative amino acid profiles 
showed a diametrically opposite pattern. However, the qualitative analysis and 
quantitation of individual amino acids showed that higher values were recorded 
either in stage III or IV, which coincides with the infestation and population build- 
up of R. cruentatus. A similar result was obtained, in the case of the sap sucking 



Biochemical correlates in R. cruentatus 125 

insects, where the bulk of the population becoming adults coincided with the higher 
levels of amino acids in the infested leaves, and higher methionine levels correlated 
well with alate production. This could be true in the case of R. cruentatus since 
higher methionine levels were recorded in stage IV. In the senescing yellow leaves the 
infestation is absent, and that could be correlated with low nitrogen levels due to 
remobilization of unspent nitrogen by the plant. 

Varying concentrations of nutrients in different feeding sites of a plant result in 
many insects feeding on different parts of their host plants, an aspect correlated with 
the sites of maximal nitrogen availability (Feeny 1970; Parry 1974; McNeill 1973). 
This could explain the infestation by R. cruentatus near the venal regions in the III 
leaves and on the laminar sides in the IV and V stages. In seasonal habitats synchro- 
nous flushing is a fundamental defence mechanism telescoping the essential transport 
of nitrogen into the shortest possible period (McNeill and South wood 1978), and this 
high degree of synchrony may be disadvantageous to T. catappa since this thrips 
species seems to have been able to synchronise with the timing of the leaf flush and 
nitrogen content. 

Qualitative profiles of fatty acid methyl esters in different stages of leaf 
development clearly revealed higher numbers in the early stages and from stage III 
onwards where infestation was initiated, as many as 7 fatty acid methyl esters were 
recorded^ the important ones being the polyunsaturated CIS: 3 linolenic acid, the 
monoenoic CIS: 1 oleic acid and C18:0 stearic acid. Linolenic acid was present in 
higher concentrations in stage IV, while high stearic and oleic acid contents were 
noted in stage V, well correlating with the development of larvae, eclosion and at 
finality the egg deposition by females. It is well known that insects depend on dietary 
fatty acids in higher concentrations for the aforesaid and there is unequivocal 
evidence that some fatty acids might be more than a facultatively utilizable energy 
source since the di- and trienoic polyunsaturated fatty acids appear necessary for 
larval growth, pupal eclosion and wing expansion in insects. Among several satu- 
rated monoenoic fatty acids, oleic (CIS : 1) was most effective for silkworms, while in 
the majority of the cases, mixtures of fatty acids were superior to any one alone 
(Dadd 1973). Hence, it is not surprising that R. cruentatus had a preference for the 
leaf stages III-V since they not only provide higher fat contents than the rest of the 
stages, but also a mixture of essential fatty acids including linolenic, oleic and stearic 
acids, for the larval growth, pupal eclosion and subsequent reproduction within the 
specified time of the duration of different instars. 

Available information on the influence of secondary plant substances, especially 
phenolics, largely gives the impression that they are the main defence arsenals of the 
plants and once the insect succeeds in dealing with these compounds, it could 
successfully colonise and utilise the host, though it extends far beyond the host selec- 
tion phenomenon. Quantitative estimations of the key enzymes of secondary meta- 
bolism namely the phenylalanine ammonia lyase, tyrosine ammonia lyase and pero- 
xidase showed a correctable influence on the levels of total phenols as well as o- 
dihydric phenols that are of importance in host selection and feeding as well as the 
selection of the feeding stages and site. Of considerable relevance is the selection by 
R. cruentatus of the leaf stages of T. catappa where decreasing levels of total and o- 
dihydric phenols were recorded. If it were a rule that plant phenolics restrict insect 
colonization, then it may not be surprising to note that JR. cruentatus chose a stage of 
decreasing phenolic contents with decreasing enzyme levels thereby having limited 



126 G Suresh and T N Ananthakrishnan 

profiles of repellent/resistant compounds including quinones in the leaf tissue. A 
further analysis of terpenoids and tannins on one hand and the alkaloids and 
glucosinolates on the other, the former providing barriers and the latter being 
utilised by the insects, would reveal reasons for the host selection and site selection 
phenomena of R. cruentatus on differing leaves in terms of age. 

Total carbohydrate levels showed lesser variation among different leaf stages 
indicating that total carbohydrates may not be a limiting factor for specificity and 
feeding site selection, though in combination with other nutrients it would have a 
limiting influence. 

Variation in leaf water content was not marked among different leaf stages of 
T. catappa (figure 4c) although it is known to decline with increasing age (Scriber and 
Slansky 1981). Rapid decline in nitrogen and generally lower leaf water in trees alone 
as influencing factors for feeding by insects is more difficult to interpret due to other 
accompanying chemical changes. Hence, within plant variability in nutritional and 
allelochemic composition such as phenolics would form important factors influ- 
encing site selection, synchronized life cycle pattern in relation to leaf flush, feeding 
and reproduction of R. cruentatus. Scriber and Slansky's (1981) conclusion that it is 
apparent to identify the specific proximate factors associated with the food quality 
and quantity of plant nutrients and allelochemics change through time and in res- 
ponse to environment, the behavioural responses resulting from change in food 
quality, as well as the utilization of food by arthropods, involves dealing with a 
multitude of covarying biochemical and morphological mechanisms that interact as 
food is ingested might exemplify the complexities of R. cruentatus T. catappa inter- 
actions. 



Acknowledgement 

Instrumentation facilities utilised in this investigation were made available by the 
generous funding of the University Grants Commission, New Delhi under the 
COSIST programme for which thanks are due. 

References 

Ananthakrishnan T N 1984 Bioecology ofThrips (Michigan: Indira Publishing House) 
Ananthakrishnan T N, Daniel A M and Suresh Kumar N 1982 Spatial and seasonal distribution patterns 

of some phytophagous thrips (Thysanoptera : Insecta) infesting Ricinus communis Linn. 

(Euphorbiaceae) and Achyranthes aspera Linn. (Amarantaceae); Proc. Indian Nad. ScL Acad. B48 183- 

184 
Ananthakrishnan T N and Muraleedharan N 1972 Free amino acids in relation to host plant preferences 

in the polyphagous heliothripine Rhipiphorothrips cruentatus Hood and Retithrips syriacus (Mayet); 

Curr. ScL 43 846-847 
Ananthakrishnan T N and Muraleedharan N 1974 On the incidence and effects of infestation of 

Selenothrips rubrocinctus (Giard) (Thysanoptera : Heliothripinae) on the free amino acids of some 

susceptible host plants; Curr. Sci. 43 216-218 
Bray H C and Thorpe W V 1954 Analysis of phenolic compounds of interest in metabolism; Methods 

Biochem. Anal 1 27-52 
Dadd R H 1973 Insect nutrition: Current developments and metabolic implications; Annu. Rev. EntomoL 

18 381-420 
Davis B J 1964 Disc Electrophoresis: II Method and application to human serum proteins; Ann. N.Y. 

Acad. Sci. 121 404-427 



Biochemical correlates in R. cruentatus 127 

Dubois M, Gilles K A, Hamilton J K, Rebers P A and Smith F 1956 Colorimetric determination of sugars 

and related substances; Anal, Chem. 26 351-356 
Feeny P 1970 Seasonal changes in oak leaf tannins and nutrients as a cause of spring feeding by winter 

moth caterpillars; Ecology 51 565-581 
Fennah R G 1963 Nutritional factors associated with seasonal populations increase of cacao-thrips 

Selenothrips rubrocinctus (Giard (Thysanoptera) ) on Cashew, Anacardium occidental; Bull Entomol 

Res. 53681-713 
Folch J, Lees M and Sloane-Stanley G H 1957 A simple method for the isolation and purification of total 

lipids from animal tissues; J. Bioi Chem. 226 497-506 

Harborne J B 1973 Phytochemical methods (London, New York: Chapman and Hall) 
Higuchi T 1966 Role of Phenylalanine deaminase and Tyrase in the lignification of bamboo; Agric. BioL 

Chem. 30 667-673 
Johnson G and Schall L A 1952 Relation of chlorogenic acid to scab resistance in potatoes; Science 115 

627-629 
Lindroth P and Mopper K 1979 High performance liquid chromatographic determination of subpicomole 

amounts of amino acids by precolumn fluorescence derivatization with o-phthalaldehyde; Anal. Chem. 

51 1667-1674 
Loebenstein G and Linsey N 1961 Peroxidase activity in virus infected sweet potatoes; Phytopathology 51 

533-537 
Lowry O H, Rosebrough N J, Far A L and Randall R J 1951 Protein measurement with Folin phenol 

reagent; J. BioL Chem. 193 265-275 
McNeill S 1973 The dynamics of a population of Leptoterna dolabrata in relation to its food resources; 

J. Anirn. Ecol. 42 495-507 

McNeill S and South wood T R E 1978 The role of nitrogen in the development of insect/plant relation- 
ships; in Biochemical aspects of Plant-Animal coevolution (ed) J B Harborne (London: Academic 

Press) pp 77-98 
Moore S and Stein W H 1948 Photometric ninhydrin method for use in the chromatography of amino 

acids; J. BioL Chem. 176 367-388 

Parry W H 1974 The effects of nitrogen levels in Sitka spruce needles on Elatobium abietinum in north- 
eastern Scotland; Oecologia 15 305-320 
Scriber J M and Slansky Jr F 1981 The nutritional ecology of immature insects; Annu. Rev. Entomol. 26 

183-211 
Schuster R 1985 Determination of fatty acids in margarine and butter by on-column derivatization; 

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3rd edition (International Rice Research Institute, Los Banos, Philippines). 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 2, March 1987, pp. 129-134. 
Printed in India. 



Effect of sublethal doses of dichlorvos and carbaryl on the nervous 
system of Pericallia ricini Fabr. (Lepidoptera : Arctidae) 

Y N SINGH and F SHAHEEN 

Department of Zoology, University of Allahabad, Allahabad 211 002, India 

MS received 30 December 1986; revised 9 February 1987 

Abstract. Topical application and injection of various doses of dichlorvos and carbaryl to 
the fourth instar larvae of Pericallia ricini did not alter the normal differentiation of various 
centres of the brain during metamorphosis but certain changes were produced in the 
neurons and neurites. These changes included the thickening of the neurilemma, clumping 
of chromatin, disintegration of cells and swelling of nuclei. The neurilemma of treated larvae 
became wavy, remained as such till the early adult stage and then degenerated for a very 
short period. The optic lobe anlagen cells exhibited increased mitotic division. 

Keywords. Pericallia ricini; sublethal doses of insecticides; histopathology of nervous 
system. 

1. Introduction 

One of the main factors which may account for the lack of information on the action 
of the sublethal doses of insecticides is the production of insecticide resistant 
individuals. Thus most of the earlier workers paid their attention to study mode of 
action and histopathological effects of various insecticides at lethal levels (Richards 
1941, 1943; Wigglesworth 1941; Hartzell and Scudder 1942; Salkeld 1951). Very 
recently Shaheen and Singh (1986) gave an account of the effects of sublethal doses of 
two insecticides on the nervous system of Diacrisia obliqua. They found a number of 
histopathological changes in the neurons and neurites by the application of very low 
doses of these insecticides, however, they did not find any alteration in the general 
differentiation of the brain centres during metamorphosis which was an interesting 
phenomenon. These observations fascinated us to gather some more information in 
other insects. 

2. Materials and methods 

Larvae of Pericallia ricini Fabr. were collected and reared in the laboratory. Fourth 
instar larvae were selected from the stock and all the experiments were performed at 
this stage. Four sets of 50 larvae each were placed separately in petridishes. Two 
insecticides viz dichlorvos [0, 0-dimethyl 2, 2-dichlorovinyl phosphate (DD VP)] and 
carbaryl [1-naphthyl-N-methyl carbamate (sevin)] were used for direct injection and 
topical applications. These were diluted with acetone to make 0-2, 04, 0-6, 10, 14, 1-6, 
2-0, 4*0, 6-0 and 8-0 /*g. Insects of the first set were treated with these insecticides 
topically on the dorsal region of the abdomen with a microapplicator. In the 
abdomens of the larvae of the second set, above mentioned doses of these insecti- 
cides were injected. The third set was treated with the same amount of acetone only 
and the fourth set was left untreated. Two to three insects of each set were dissected 



1 30 Y N Singh and F Shaheen 

at an interval of 2 days till the emergence of adults and the entire nervous system was 
fixed in aqueous Bourn's fixative for 18-24 h. The adults obtained from these treated 
larvae are termed here after as t-adults. The sections were cut at 6-8 tm thickness 
and staining was done with haematoxylin-eosin and Heidenhains Azaii stains. 

3. Observations 

3.1 Changes in the neurilemma 

The normal thickness of the neurilemma of the brain and ganglia of P. ricini larva 
was about 0-75-1-0 /an (figure 1). After 4-12 days of topical application of 0-2, 0-4, 1-0 
and 1-6 tig of sevin and 0-6, 14 and 1-6 fig of DDVP its thickness increased (figure 2). 
It was about 2-7-3-0 jum and 2-25 /an in the brain and nerve cord respectively. It also 
became wavy in the larval period. This type of wavy neurilemma appears during the mid 
pupal stage, prior to its degeneration in untreated insects. In the adults produced by 
the insecticide treated larvae (t-adults) the neurilemma of the thoracoabdominal 
ganglionic mass showed greater degree of wavy appearance. It also increased in 
thickness by the treatment of larvae with 1-6 /zg of sevin. This wavy nature of the 
neurilemma appeared 4 days after the treatments of the larvae and did not degene- 
rate during pupal period (figure 3). 

3.2 Changes in the cortical and neuropilar regions 

Various histopathological changes were produced by the topical applications and 
direct injection of the insecticides. These changes were more or less similar in both 
the methods except that the direct injection of insecticides produced these changes 
more rapidly. The 4th instar larvae of P. ricini treated with various doses of two 
insecticides and the two methods exhibited differentiation of all the centres of the 
brain in 5th instar larvae, pupae and adults like normal insects, however, changes 
were produced in the structure of the nerve cells and their neurites. 

The central nervous system of the t-adults produced by the treatments of 4th 
instar larvae (8-0 /<g of DDVP and sevin) showed slight changes in its histology. The 
neurilemma covering the thoracoabdominal ganglionic mass and abdominal ganglia 
became more wavy. The brain of t-adults showed swollen nuclei with an increase in 
the number of degenerating cells. The connective tissue sheath which appears during 
the absence of the neurilemma, also showed pyknosis. 

Application of 6-0 jig of DDVP to the 4th instar larvae, after 2-7 days of 
treatment, showed swelling in the neurons with slight degeneration of chromatin and 
cytoplasm in the brain. Distinct swelling of globuli cells was noted in the calyx with 
chromatin attached to the periphery of the nuclei. The neuropilar regions of the 
larval brain and adults ganglia showed disintegration and degeneration of the longi- 
tudinal fibres. The newly emerged t-adults showed degeneration of neurons and 
irregular spaces in the perineurium and in between the cortical and neuropilar region 
of the abdominal ganglia. Such histopathological changes were not met within the 
control insects Increased intercellular spaces and degeneration of longitudinal fibre 
tracts were noted in the thoracoabdominal ganglionic mass. The t-adults produ 
by the treatments with 4-0 M g of DDVP did not show any marked change in 



Effect ofsublethal doses ofdichlorvos and carbaryl on P. ricini 131 




Figures 1-6. 1. Frontal section (FS) of 5th instar larval brain of untreated P. ricini 
showing normal thickness of the neurilemma (NE). (scale bar 400 /mi). 2. FS of 5th 
instar larval brain showing an increase in thickness of the neurilemma (NE). Sevin, 0-2 /ig. 
(scale bar 200 /im). 3. FS of the thoraco abdominal ganglionic mass (TAG) of adult 
showing degeneration of the neurilemma and shrinkage of cells in the cortical region 
(arrow). Sevin, 1-6 //g. (scale bar 200 /mi). 4. FS of 5th instar larval brain showing mitic 
division (M), swollen nuclei (SW) and peripheral chromatin (ari;ow). Sevin, 1-6 ^g. (scale 
bar 100 /mi). 5. FS of 5th instar larval brain showing peripheral accumulation of chro- 
matin (arrow). Sevin, 2-0 jug. (scale bar 100 /mi). 6. FS of thoracoabdominal ganglionic 
mass of adult showing degeneration of neurons (arrow) and appearance of vacuoles (V) in 
nuclei. DDVP, 1-6 /zg (scale bar 100 /mi). 



cellular integrity of the brain. In the ganglia, like 2-0 jag of sevin, the disappearance of 
chromatin with more marked degeneration of cells and production of dark stained 
particles around the neuropile was noted. No change was noted in the differentiation 
of optic centres and central complex. However, an enormous increase of the mitotic 
division of the optic lobe anlagen cells was observed (figure 4). 



132 



Y N Singh and F Shaheen 



Treatment with 2-0 /ig of DDVP and sevin on the larvae of P. ricini showed the 
clumping of chromatin which got attached to the nuclear membrane (figure 5). 
Gradual disintegration of cytoplasm and appearance of intercellular spaces were also 
noted. No marked changes were noted in the prepupal brain with sevin. With 1-6 fj.% 
of DDVP degeneration and shrinkage of cells and partial atrophy of fibres occurred 
in the adult's central nervous system. The ganglia showed enlargement of cells with 
enormous vacuolation of the nuclei (figure 6). With 1-0 ^g of DDVP, pyknosis and 
breakage of the cell wall and thickening of the larval neurilemma was noted. 

Very little cellular disintegration and changes in the nuclei were noted with 0-2, 0-4 
and 0-6 /ig of sevin and DDVP. The abdominal ganglia of treated larvae (0-2 /xg of 
sevin) showed the appearance of clusters of small neurons placed in the peripheral 
part of the cortex very near to the thickened neurilemma (figure 7). 0-4 jug of sevin 
produced dark stained particles in the larval ganglia abundantly (figure 9) whereas 
0-2 //g caused chromatin clumping, swelling of nuclei with intracytoplasmic vacuoles 
and cell degeneration in the larval ganglia (figure 8). Adults produced by the 
treatment with 0-8 /jg of DDVP to the larvae showed degeneration of neurites and 
the peripheral accumulation of chromatin in the globuli cells of the calyces of the 
mushroom bodies (figure 10). 




Figures 7-10. 7. FS of 5th instar larval abdominal ganglion showing clusters of small cells 
(CL). Sevin, 0-2 //g. (scale bar 100 jan). 8. FS of 5th instar larval abdominal ganglion 
showing swollen nuclei (SW) and degeneration of cells (arrow). Sevin, 0-2 /ig. (scale 
bar 100 /im). 9. FS of 5th instar larval thoracic ganglion showing dark particles (arrow) 
and vacuoles (V) in the cytoplasm of motor neurons. Sevin, 0-4 /*g. (scale bar -100 //m). 
10. FS of adult's brain (t-adult) showing degeneration of fibres (F) and peripheral 
accumulation of chromatin. DDVP, 0-8 /*g. (scale bar 100 /mi). 



Effect ofsublethal doses ofdichlorvos and carbaryl on P. ricini 133 

4. Discussion 

Wilcoxon and Hartzell (1933) observed vacuolation and trigrolysis of the Nisle 
granules and tissue disintegration in the brain and ganglia of the ventral nerve cord 
and to some extent in the connectives also in the larvae of Tenebrio molitor after 
treatment with pyrethrum. Klinger (1936) in the larvae of gypsy moth, Porthetria 
dispar, observed that a high dose of pyrethrum, 24 h after application, caused 
extensive damage to the nerve cells of the abdominal ganglia, especially towards the 
periphery. On the other hand, a number of well known insecticides do not produce 
any visible histopathological changes in insects. Kruger (1931) and Hartzell (1934) 
failed to find any effect on the central nervous system by applying rotenone. Thus it 
is evident that the mode of action and effects of different insecticides is not uniform in 
all the insects. 

It is also known that the neurilemma of the insect central nervous system plays a 
role in the transport of nutrients in the nervous system (see Chandra and Singh 
1982), assists to control the movements of ions into and out of the nervous system 
(Hoyle 1953; Pichon et al 1972; Lane 1972; Schofield and Treherne 1978) and 
performs an osmoregulatory function (Lorente de No 1952; Shanes 1953). Like 
nutrients, the insecticides also might be entering through the neurilemma, perineu- 
rium and ultimately reaching to the glial cells, neurons and axons. The presence of 
bound lipid nerve sheath and their probable relation to the penetration of neurotoxic 
insecticides is of major interest in any study of mode of action of these toxins. Most 
of the earlier workers reported the destruction of this sheath by the insecticidal 
action thus facilitating the insecticides entry, but we do not find this type of 
destruction after the treatments with sublethal doses of dichlorvos and carbaryl on 
P. ricini. Thus there may be two possibilities for the entrance of these chemicals 
inside the cortex of the central nervous system of the insects (i) either the entire 
lipid sheath, covering the nervous system degenerates or (ii) the insecticides diffuse 
through the tissue barriers (connective tissue sheath, neuriiemma, perineurium and 
cortex). The former type of insecticide entry is reported by Klinger (1936), Richards 
(1941, in mosquito larva) and Wigglesworth (1941). Shaheen and Singh (1986) in 
Diacrisia obliqua and in the present study found cellular destruction in treated insects 
but did not include the destruction of the nerve sheath. This may be possible that 
either lower doses of these chemicals do not cause sheath destruction or that this 
change is insecticide specific. 

Insecticides induce the division of cells. An enormous cell death due to insecticidal 
actions is most probably compensated by this increased cell division. The increase in 
the mitotic figures in treated insects might be due to this reason. The larval neural 
lamella is retained for a maximum period. In normal development it is degenerated 
in the early pupal period but in the treated insects it is retained till the early adult 
stage. It indicates that the treated insects try to retain the neural lamella as far as 
possible to avoid the entry of the chemical (s) present in the haemolymph. Other 
morphogenetic changes and differentiation of various nervous centres are not 
affected by the insecticidal treatment because the metamorphic changes in the central 
complex, optic ganglia and deutocerebrum etc take place normally and timely. 

Treatments with dichlorvos and carbaryl produce vacuoles in the brain and gang- 
lia. Clumping of chromatin, disintegration of cellular components and neurites, 
swelling and deformities in the nerve cells have also been noted. Particle formation is 



134 '* Y N Singh and F Shaheen 

also noted (Richards and Cutkomp 1945; Shaheen and Singh 1986). Appearance of 
vacuoles in the perineurium is also noted which may be due to the neuronal death 
caused by insecticidal action. 

On the basis of the present study we have come to the following conclusions: 
(i) Treatments with dichlorvos and carbaryl do not interfere with the normal deve- 
lopment, morphogenetic changes and differentiation of various centres of the brain 
and ganglia, but cellular and fibre disintegration of various degrees occurs, (ii) that 
treated insects try to retain the larval neural lamella for a maximum period and 
(iii) that insecticides probably induce cell division. 

Acknowledgements 

We are thankful to the University Grants Commission, New Delhi for providing 
financial assistance in the form of a research scheme to YNS. We are also grateful to 
CIBA-GEIGY and Union Carbide India Ltd. for gifting the technical grades of 
DDVP and sevin respectively. 

References 

Chandra P and Singh Y N 1982 Histochemical analysis of the central nervous system of Diacrisia obliqua 

during metamorphosis with reference to the breakdown and reformation of the neural lamella; Indian 

Zool 6 37-45 
Hartzell A 1934 Histopathology of insect nerve lesions caused by insecticides; Contrib. Boyce Thompson 

Inst. 6211-223 
Hartzell A and Scudder H I 1942 Histological effects of Pyrethrum and an activator on the central 

nervous system of the housefly; J. Econ. Entomol 35 428-433 
Hoyle G 1953 Potassium ion and insect nerve muscle; J. Exp. Biol 30 121-135 
Klinger H 1936 Die Insektizid-Wirkung und ihre Abhangigkeit von dem Insektenkorper; Arb. Physiol. 

Angew. Entomol. Berlin-Dahlem 3 115-151 
Kruger F 1931 Untersuchungen uber die Giftwirkung von dalmatischem Insektpulver auf die Larven von 

Corethra plumicornis; Ztschr. Ange\v. Entomol. 18 344 
Lane N J 1972 Fine structure of a Lepidopteran nervous system and its accessibility to peroxidase and 

lanthanum; Z. Zellforsch. Mikrosk. Anat. 131 205-222 
Lorente de No R 1952 Observations on the properties of the epineuriurn of frog nerve; Cold Spring 

Harbour Symp. Quant. Biol. 17 299-315 
Pichon Y, Sattelle D B and Lane N J 1972 Conduction processes in the nerve cord of the moth Manduca 

sexta in relation to its ultrastructure and haemolymph ionic composition; J. Exp. Biol. 56 117-134 
Richards A G Jr 1941 Differentiation between toxic and suffocating effects of petroleum oils on larvae of 

the house mosquito (Culex pipiens); Trans. Am. Entomol. Soc. 67 161-196 
Richards A G Jr 1943 Lipid nerve sheaths in insects and their probable relation to insecticide action- 

J- N.Y. Entomol. Soc. 51 55-66 ' 



,nc Cutkom P L K 1945 Neuropathology in Insects; J. N.Y. Entomol. Soc. 53 313-354 
Salkeld E H 1951 A toxicological and histophysiological study of certain new insecticides as stomach 

poisons to the honey-bee Apis mellifera L.; Can. Entomol. 83 39-61 
Schofield P K and Treherne J E 1978 Kinetics of sodium and lithium movements across the blood brain- 

bacner of an insect; J. Exp. Biol. 74 239-251 
Shaheen F and Singh Y N 1986 Effect of sublethal doses of dichlorvos and carbaryl on the nervous system 

of Biacnsia obliqua (Lepidoptera); Ada Entomol. Bohemoslov 83 340-346 
Shanes A M 1953 Effect of sheath removal on bull frog nerve; J. Cell Comp. Physiol 12 305-311 



Wilcoxon F and Hartzell A 1933 Some factors affecting the efficiency of contact insecticides III. Further 
chemical and toxicological studies of pyrethrum; Contrib. Boyce Thompson Inst. 5 115-127 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 2, March 1987, pp. 135-140. 
Printed in India. 



Effect of starvation on food utilization in the freshwater snail Pila 
globosa (Swainson) 

M A HANIFFA 

Post-Graduate and Research Department of Zoology, St. Xavier's College, Palayamkottai 

627 002, India 

MS received 20 August 1986; revised 27 January 1987 

Abstract. Increase in food supply produced elevated values of several parameters in energy 
budget of both normal and starved individuals (P< 0-001). Starvation accelerated the rates 
of feeding, absorption and metabolism (3 times) and conversion (1% times; P< 0-001). Pila 
globosa starved for a month lost 79-5 mg dry weight/g live weight and the recovery periods 
to attain the original body weight were estimated as 66, 72, 88, 32, 28 and 23 days between 8 
and 20% rations. 

Keywords. Pila globosa', starvation; feeding; conversion. 



1. Introduction 

This paper is in continuation of a series on food utilization in freshwater snails 
exposed to different rations (Haniffa and Pandian 1974; Vivekanandan et al 1974; 
Haniffa 1982), food combinations (Haniffa et al 1984; Haniffa and Sethuramalingam 
1985), crowding and water level (Haniffa 1980) and aestivation (Haniffa M A, 
unpublished results). Starvation is by definition a condition in which the animal's 
ration is not sufficient to balance its basal demand for energy. Under this condition 
the reserve food energy within the body is utilized in order to make good the 
difference between ration and respiratory loss (see Bayne 1973). Studies on the effect 
of starvation on metabolism of molluscs are well documented (Martin and Goddard 
1966; Emerson 1967; Emerson and Duerr 1967; Stickle and Duerr 1970; Uma Devi et 
al 1986), but that on food utilization is meagre (Haniffa and Mullainathan 1982). The 
present work examines the effects of starvation on mortality, weight loss and food 
utilization in a freshwater snail Pila qlobosa. 

2. Material and methods 

Active individuals of P. globosa were collected from the pond Idumban (Palni, Tamil 
Nadu) and acclimated to laboratory conditions. About 150 snails of 122g live 
weight were recruited after 12 h of exposure to air (Haniffa 1978a) and introduced in 
groups of 25, into 6 glass aquaria (25 1 capacity). Water was changed once in two 
days and the snails were starved for a period of two months; every week, mortality 
rates and weight losses were recorded. Fifty snails, starved for a month were divided 
into 10 groups, each containing 5 individuals and kept in troughs of 5 1 capacity. 
Experimental snails were fed on Chora fragilis at chosen feeding levels ranging from 



136 MA Haniffa 

2-20% of their respective live weights. Everyday a control sample of 5 g food plant 
was dried at 85C and weighed to measure its water content. Fifteen such starved 
snails were dried and weighed to determine the initial dry weight. Unconsumed plant 
was removed daily, dried and weighed to estimate the amount of food consumed. 
Faecal pellets were collected daily by filtering the water from the entire trough 
through a fine sieve of 160 /zm. This experimental setup was maintained for a period 
of one month. 

The scheme of energy budget followed here is a slightly modified IBP formula 
(Petrusewicz and Macfadyen 1970). 

C (consumption) = P (growth) + R (metabolism) + F (faeces). 

The sacrifice method (Maynard and Loosli 1962) was followed to estimate the initial 
and final dry weight (inclusive of shell) of the test snails. Following the same 
procedure energy budget was estimated for normal P. globosa and a comparative 
analysis was made between starved and normal snails. 

3. Results and discussion 

The data presented in table 1 show the mortality rate and weight loss of one month 
starved P. globosa. Survival was 99*6% for one week-starved individuals and it 
decreased to 70-6% for the 7 week-starved snails. Total mortality during the tenure 
increased from 0-4% in the first week to 29-4% in the last week and mean mortality 
estimated after one month starvation was 10-5%. Following the least mean square 
method, the slope was calculated as 1-032 (Y= 1-032x4- 0-072). Analysis of correlation 
coefficient confirmed that increase in the period of starvation resulted in heavy 
mortality (P<001). As the test individuals were forced to starve, it is possible to 
suggest that the absence of food energy and the utilization of body reserve during the 
course of starvation resulted in mortality (Uma Devi et al 1986). Weight loss via 
metabolism (shell and body tissue) amounted to 2-65 mg dry weight for one month 

Table 1. Effect of starvation on survival and weight loss of the freshwater snail 
P. globosa. 



Period of 


Mortality 


Weight lo^s frna Hrv/cr live. anaiU 


starvation 


Survival 


Week 


Total 












(weeks) 


(%) 


(%) 


(%) 


Shell 


Flesh 


Total 


1 


99-6 


0-4 


0-4 


1-20 


3-01 


4-21 


2 


97-8 


1-8 


2-2 


0-74 


3-21 


2-96 


3 


93-9 


3-9 


6-1 


0-62 


1-70' 


2-32 


4 


89-2 


: 4-7 


10-8 


0-39 


0-71 


1-10 


5 


83-3 


5-9 


16-7 


0-33 


0-59 


0-92 


6 


76-9 


6-4 


23-1 


0-23 


0-54 


0-77 


7 


70-6 


6-3 


29-4 


0-32 


0-006 


0-33 



Correlation coefficient 

r- 0-987 0-845 0-987 0-899 -0-676 -0-956 
P< 0-001 <0-01 < 0-001 < 0-001 < 0-001 < 0-001 

?<0-01, Significant; P< 0-001, Highly significant. 



Food utilization in the freshwater snail, Pila globosa 



137 



starved P. globosa and this accounted for 0-74 mg loss of shell weight/g live snail/day 
i.e. those snails which survived one month starvation, exhibited 25% decrease in shell 
weight. Increase in starvation period resulted in a loss of shell weight from 1-2 mg in 
the first week to 0-32 mg in the 7th week (P< 0-001). According to Haniffa (1978b), 
shell weight of P. globosa which successfully survived 5 months of aestivation 
exhibited 5-25% decrease in total shell weight. P. globosa is capable of aestivating for 
a period of 5 years (Hyman 1967) but starvation is not possible beyond 90 days. 
Hence the weight loss via metabolism during starvation is comparatively more. 
Working on the mortality rate of Lymnaea stagnalis, Jose et al (1968) reported that 
pond snails can survive a starvation period of 6 weeks by utilizing the reserve food 
energy. Uma Devi et al (1986) reported that the tropical intertidal gastropod Morula 
granulata can be subjected to prolonged starvation of 70 days. 

Increase in food supply/day enhanced the energy budget of P. globosa. Feeding 
rate of normal P. globosa increased from 2-2 mg/g/day at 2% to 18-4 mg/g/day at 
20% ration level (figure 1). Corresponding increases in the rates of absorption and 
metabolism were from 1 -7 - 13-7 mg/g/day and 4-2 -114 mg/g/day. The gain in body 
substance increased from 0-5 mg at 10% to 2-2 mg/g/day at the maximum ration level 
of 20% . Feeding rates estimated for one month starved P. globosa at different ration 
levels ranged from 5-1 to 53-3 mg/g/day (figure 1). Starvation caused about 2 times 
increase in food consumption; it might have stimulated the nerve centres controlling 
the appetite and in response to this the snail consumed more food. 

Both normal and starved P. globosa showed weight losses at lower rations (2-8%). 
For feeding rates of 12-1 and 20-9 mg/g/day, a gain in weight of 0-5 and 1-2 mg/g/day 
was ''observed in normal and starved snails respectively. Maximum growth rate was 



O 

E 60 



40 - 



20 - 



Feeding rate 5tQrve d x A 


4 


Conversion rate -- 


^^ O 




Starved ^^ ^ A ^-* 9 


A- "& O 




. Ar-A^__^---* Q_ 








/ V 




AA / ^/ 


/ Normal ^ 




^^x-* Normal 


A / 4 -** 




m-^* 


A X ' X """"*"* 


-4 




^ P"""* | 1 1 1 




\ \ \ 




60 




Absorption rate 


Metabolic rate 


__ Starved A 


40 


~~ Starved ^-^ 


^-^ 8 




A **^ 8 


~ / A Normal ^ 


20 


A-- A ^ 6 
A X Normal ^ 


f^*"^l i i 1 




A ^.A^ " -0 ^" 

*=< i i 


4 8 12 16 20 4 8 12 16 20 



Ration level (% ) 



Figure 1. Effects of starvation on the rates of physiological energetics of P. globosa. 



138 MA Haniffa 

2-2 mg/g/day in normal snails and it increased to 3-4mg/g/day for the starved 
P. globosa (figure 1). Haniffa and Mullainathan (1982) have also reported enhance- 
ment of conversion rate in the freshwater snail Viviparus variatus as a function of 
starvation. 

Vivekanandan et al (1974) reported 14-7 gcal/g/day as the minimum energy cost of 
maintenance of P. globosa exposed to starvation and 67-4 gcal/g/day as the maxi- 
mum energy cost of living for an active P. globosa fed ad libitum on Ceratophyllum 
demersum at 28C. Considering the calorific content of P. globosa as 3682 444 g cal/g 
dry weight (Haniffa. 1978b), the energy lost through metabolism in one month 
starved P. globosa in the present investigation amounted to 9-8 gcal/g/day. When 
compared with normal snail, the metabolic level of one month starved P. globosa was 
lowered by l/7th. The metabolic level of the aestivating snail was lowered by l/18th 
of that of the normal snail (Haniffa M A, unpublished results). Similar observations 
on the capacity of the snail to lower its metabolic level have also been reported in 
P. virens (Meenakshi 1964), P. ovata (Visser 1965) andMomlagranulata (UmaDevi 
ef of 1986). 

Increase in ration level caused rapid decrease in metabolism in normal snails but 
the decrease was slow in starved P. globosa (table 2). Starved snails also showed 
higher metabolic cost of 69-79% of consumption or 92-97% of absorption at higher 
rations (12-20%) than normal snails (62-71% of consumption or 84-93% of 
absorption). Hence it is possible to suggest that higher metabolic cost may be 
responsible for poor conversion efficiency in starved snails. 

Increase in food supply enhanced the conversion efficiency from 5-6-1 2-1% in 
normal and 5-7-8-4% in starved P. globosa. Starvation elevated the values of 
conversion rate though the conversion efficiency actually showed decreases (table 2). 
Net conversion efficiency in the present investigation ranged from 5-6-13-0 and from 
3-0-8-4% for normal and starved snails respectively. Increase in ration level caused a 
decrease in absorption efficiency from 83-74% and from 93-76% in normal and 
starved P. globosa (table 2) respectively (Vivekanandan et al 1974; Haniffa 1982). 
Absorption efficiency values reported for gastropods range from 73-88% for 
P. globosa (Haniffa 1982), 57-89% for Littorina littorea (Grahame 1973), 73-88% for 



Table 2. Effect of ration level on efficiencies of absorption and conversion and recovery period 
to attain original weight by starved P. globosa. 



Ration 
level 

/o/\ 
(/o) 


Absorption 
efficiency (%) 


Conversion 
efficiency (%) 


Total loss of 
body weight 
(mg dry/g 
live snail) 


Recovery 
period (days) 


Normal 


Starved 


Normal 


Starved 


2 


83-4 


92-7 








79-5 





4 


82-6 


84-6 








79-5 





6 


84-4 


80-7 








79-5 





8 


80-6 


82-6 





5-7 


79-5 


66 


10 


78-8 


78-7 





4-5 


79-5 


7 3 


12 


76-7 


81-7 


5-6 


3-7 


79-5 


72 


14 


75-2 


79-8 


74 


3-0 


79-5 


88 


16 


73-6 


78-4 


7-7 


7-5 


79-5 


32 


18 


74-4 


11 -I 


13-0 


7-5 


79-5 


28 


20 


74-2 


75-6 


12-1 


84 


79-5 


23 



Food utilization in the freshwater snail, Pila globosa 1 39 

Gyraulus convexiusculus (Vivekanandan and Pandian 1976) and 64-80% for 
V. variatus (Haniffa and Mullainathan 1982). 

Elevations in the rates of feeding, absorption, metabolism and conversion as a 
function of ration level in normal and starved P. globosa were statistically significant 
(P< 0-001). Starvation accelerated the energy budget of P. globosa and Student's t 
test confirmed that this elevation was statistically significant (P< 0-001; figure 1). 
Regression equations were fitted by the method of least squares and the coefficients 
were used in analysis of variance (Zar 1974). It was confirmed that feeding and ration 
level significantly influenced the absorption efficiency (P<O05 and <0-01) and 
conversion (P<0-05) but the difference in metabolic rate (as percentage of 
consumption or absorption) was not significant (P > 0-05) as a function of feeding or 
ration level (table 1). 

Acknowledgements 

This research work was carried out at Arulmigu Palaniandavar Arts College, Palni. 
Grateful acknowledgements are due to Council of Scientific and Industrial Research 
and University Grants Commission, New Delhi for financial assistance. 



References 

Bayne B 1973 Aspects of the metabolism of Mytilus edulis during starvation; Neth. J. Sea Res, 7 399-410 
Emerson D N 1967 Carbohydrate oriented metabolism of Planorbis corneus (Mollusca, Planorbidae) 

during starvation; Comp. Biochem. Physiol. 22 571-579 
Emerson D N and Duerr F 1967 Some physiological effects of starvation in the intertidal prosobranch 

Littorina planaxis (Philippi 1847); Comp. Biochem. Physiol. 20 45-53 

Grahame S D 1973 Assimilation efficiency of Littorina littorea (L); J. Anim. Ecol 42 383-389 
Haniffa M A and Pandian T J 1974 Effects of body weight on feeding rate and radula size in the 

freshwater snail Pila globosa; Veliger 16 415-418 

Haniffa M A 1978a Secondary productivity and energy flow in a tropical pond; H ydrobiologia 59 49-65 
Haniffa M A 1978b Energy loss in an aestivating population of the tropical snail Pila globosa; 

H ydrobiologia 61 169-182 
Haniffa M A 1980 Influence of crowding and water level on food utilization in the freshwater snail Pila 

globosa (Swainson); Indian J. Exp. Biol. 18 71-73 
Haniffa M A 1982 Effects of feeding level and body weight on food utilization of the freshwater snail Pila 

globosa; H ydrobiologia 97 141-149 
Haniffa M A and Mullainathan P 1982 Studies on energy transformation in the freshwater snail, Viviparus 

variatus: Influence of feeding and starvation; Int. J. Ecol. Environ. Sci. 8 187-194 
Haniffa M A, Amaladoss A J, Murugesan A G and Isai Arasu L 1984 Influence of plant and animal food 

on food utilization of a herbivorous snail Pila globosa (Swainson); Indian J. Exp. Biol. 22 482-483 
Haniffa M A and Sethuramalingam T A S 1985 Food utilization and mineral absorption efficiency in the 

freshwater snail Pila globosa; Indian J. Com. Anim. Physiol. 3 56-64 
Hyman L H 1967 The Invertebrates (New York: McGraw Hill) Vol. VI Part I 

Jose J, Boer M A and Cornelisse C J 1968 Physiology and ecology of molluscs (New York: Academic Press) 
Martin A and Goddard C 1966 Carbohydrate metabolism; in Physiology of Mollusca (eds) K M Wilbur 

and C M Yonge (New York: Academic Press) pp 275-308 
Maynard A L and Loosli K J 1962 Animal Nutrition (New York: McGraw Hill) 
Meenakshi V R 1964 Aestivation in Pila; Comp. Biochem. Physiol. 11 365-368 
Petrusewicz K and Macfadyen A 1970 Productivity of terrestrial animals. Principles and Methods (Oxford: 

Blackwell Scientific Publications) 
Stickle W B and Duerr F G 1970 Effects of starvation on the respiration and major nutrient stores of 

Thais lamellosa; Comp. Biochem. Physiol. 33 689-695 



140 M A Haniffa 

Uma Devi V, Prabhakara Rao Y and Prasada Rao D G V 1986 Starvation as a stress factor influenting the 

metabolism of a tropical intertidal gastropod Morula granulata (Duclos); Proc. Indian Acad. Sci. (Anim. 

Scl) 95 539-547 
Visser S A 1965 A study of the metabolism during aestivation of the amphibious snail Pila ovata; West 

AfrL J. Biol. AppL Chem. 8 41-47 
Vivekanandan E, Haniffa M A, Pandian T J and Raghuraman R 1974 Studies on energy transformation 

in the freshwater snail Pila globosa. 1. Influence of feeding rate; Freshwater Biol. 4 275-280 
Vivekanandan E and Pandian T J 1976 Food utilization and mineral absorption efficiency in the snail 

Gyraulus Convexiusculus\ J. Madurai Univ. 5 66-72 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 2, March 1987, pp. 141-149. 
Printed in India. 



Limnology of river Cooum with special reference to sewage and heavy 
metal pollution 

BERNICE ANANTHARAJ, V BAGYALAKSHMI and R LAKSHMI 

Department of Zoology, Presidency College, Madras 600 005, India 

MS received 26 June 1986; revised 8 January 1987 

Abstract. The physical, chemical and biological variables and pollution of river Cooum 
with sewage and heavy metal were studied in 4 stations within Madras city for a period of 
one year from August 1982 to July 1983. The physical, chemical and biological parameters 
presented complex pattern of variations indicating that the biotic life of Cooum river is 
severely affected by the excessive discharge of sewage effluents within the city limits which 
excessively eutrophicate the river water. The number of species and density of zooplankton 
are very low. Heavy metals such as iron, nickel, lead and zinc are recorded to be above 
permissible levels. 

Keywords. Lotic limnology; sewage pollution; eutrophication; heavy metals. 



1. Introduction 

Information on lotic limnology is mainly restricted to the pioneer works of 
Ganapathi and Alikunhi (1950), Chacko and Ganapathi (1949, 1952). lyengar and 
Venkataraman (1951), Sreenivasan and Sounderaj (1967) and Govindan and 
Sundaresan (1979) in South India. Relatively little is known about the sewage and 
heavy metal pollution of South Indian rivers except the study of physical, chemical, 
biological and microbiological aspects of the river Cooum made by Narayanan 
(1980); the report of the effects of pollution on the algal flora of the Cauvery river by 
Paramasivam and Sreenivasan (1981) and the detailed work done by Ravichandran 
(1985) in the Buckingham Canal with special reference to sewage pollution. In the 
present study a survey of the sewage and heavy metal pollution of Cooum river was 
made to know the effects of eutrophication and heavy metal pollution on zooplank- 
ton population. 

2. Materials and methods 

2.1 Study area and study design 

River Cooum which starts from Sattarai village in Chingleput district flows just 
65 km, passes through the city of Madras (Lat 13 N; long 80 15' E) and opens into 
the Bay of Bengal near Chepauk (figure 1). Bangaru Channel taking off from 
Kesavaram Anicut across Koratalyar joins this river and overflow from a number of 
tanks fall into the river before it reaches Madras. 

Four stations were chosen for the collection of water samples in the stretch of the 
river within the city. Station 1 was located at Arumbakkam; station 2 at Chetpet; 
station 3 was at Chepauk while station 4 was the estuary. 



142 Bernice Anantharaj, V Bagyalakshmi and R Lakshmi 



Cooum river" 
Study area 



Arumbakkom 
Chetpet 
Chepauk 
Estuary 




Figure 1. River Cooum - Sampling stations within Madras City. 

2.2 Sampling programme 

The 4 stations were sampled for a period of one year from August 1982 to July 1983. 
Once in a month samples were collected before 1000 h. A Vondorn sampler was used 
for the purpose. Two litres of water were collected in clean polythene bottles and 
transported immediately to the laboratory for analysis. 



2.3 Analytical methods 

The various methods used for the estimation of the variables are listed below: 



(i) Physico-chemical variables: 

Temperature 

pH - 

Dissolved oxygen (fixed 

at the site itself) 

Free carbon dioxide (fixed 

at the site itself) 

Alkalinity 



Recorded by a sensitive 0-50C mercury thermo- 
meter. 

Recorded by Elico Model Li- 10 pH meter. 
Azide modification of Winkler method (APHA 
1980). 

Titrating against 0-045 N sodium carbonate with 
phenolphthalein as indicator (APHA 1980). 
Phenolphthalein and methyl orange alkalinity was 
estimated by titrating 100 ml samples against stan- 
dard acid (Welch 1948). 



Limnology, sewage and heavy metal pollution of river Cooum 



143 



Phosphate 



Silicate 
Nitrate 

Sulphate 

Iron 

Lead 

Nickel 

Zinc 

(ii) Biological variable: 
Zooplankton 



Aqueous stannous chloride method after digestion 
with sulphuric acid-nitric acid reagent (APHA 
1980). 

- Silicomolybdic acid method (APHA 1980). 
Phenol-di-sulphonic acid method (Mackereth 
1957). 

- Turbidimetric method (APHA 1980) 

- Phenonthroline method (APHA 1980) 

- Dithizone method (APHA 1980) 

- Dimethylglyoxime method (APHA 1980) 

- Dithizone method (APHA 1980) 

-Zooplankton were sampled by filtering 100 1 of water 
through a plankton net of 32 ^m mesh size, concen- 
trating to 100 ml and preserving in 4% formal saline 
solution. Identification was done with standard keys 
(Ward and Whipple 1958) and plankters were enu- 
merated with a sedgewick rafter counting cell. 



3. Results and discussion 

3.1 Temperature 

The atmospheric and surface water temperature showed seasonal variations in all the 
4 stations having an unimodal peak during summer. The surface water temperature 
closely followed the air temperature in all the 4 stations. This might be due to the 
shallowness and slow flow of the river; the temperature pattern of the surface waters 
following the atmospheric temperature is not an uncommon phenomenon and in 
most of the lotic systems this has been observed (Ganapathi 1956; Rai 1974a; 
Narayanan 1980; Ravichandran 1985). There was not much spatial difference 
between different stations. 

3.2 pH 

The pH value slightly increased during summer. The seasonal variations in pH of 
surface waters exhibited same pattern in different stations. The pH values fluctuated 
between 6-5-8-0. This narrow range of fluctuation in pH value throughout the year 
reflects the v; ill buffered nature of the water. The range of pH values of this tropical 
river is identical with other than Indian rivers recorded by Subramanian (1979). 



3.3 Carbonate alkalinity 

The phenolphthalein alkalinity was observed only during summer months and not 
detected during most of the study period. The absence of carbonates might be due to 
the time of sampling (0900-1000 h). Similarly Sinada and Karim (1984a) and 
Ravichandran (1985) were not able to detect phenolphthalein alkalinity during these 
hours in blue and white Nile and Buckingham canal respectively. 



144 Bernice Anantharaj, V Bagyalakshmi and R Lakshmi 

3.4 Bicarbonate alkalinity 

Slight spatial and annual variations were observed in bicarbonate alkalinity. Higher 
values in bicarbonate alkalinity were always recorded in station 4, which was grossly 
affected by sewage. Pollution by sewage and its subsequent decomposition has been 
quoted as possible causes for the increase in bicarbonate alkalinity of the waters of 
river Sabarmati (Venkateshwarlu and Jayanthi 1968) and the river Yamuna (Rai 
1974a). The bicarbonate alkalinity values recorded in Cooum river were higher than 
that of Buckingham canal (Ravichandran 1985) showing the heavy impact of sewage 
pollution. 

3.5 Dissolved oxygen 

Dissolved oxygen content in the surface waters were very low fluctuating between nil 
values to 8 mg/1. Depletion of oxygen in all stations was mainly due to eutrophi- 
cation and degradation of organic matter present in the sewage by the heterotrophic 
bacteria which completely take up all available dissolved oxygen (DO) (Hynes 1970; 
Welch 1980). Similar observations were made by Ravichandran (1985) in Bucking- 
ham canal which was also highly polluted by sewage. 

3.6 Free carbondioxide 

Fluctuation in the concentrations of free CO 2 in Cooum river was slightly linked 
with seasons, showing higher values during monsoon and lower values during 
summer. Occasional higher values of free CO 2 might be due to the release from the 
sediments during breakdown of organic matter. Similar sudden shoot up of free CO 2 
was observed by Paramasivam and Sreenivasan (1981) in river Cauvery and 
Ravichandran (1985) in Buckingham canal. 

3.7 Phosphate 

Phosphate contents seemed to be high at stations 1 and 2. Phosphate varied from 
0-1-7-94 mg/1. Maximum phosphate content was observed at station 2. The high 
values of total phosphate at stations 1 and 2 might be due to the sewage input and 
the excessive eutrophication of waters of the Cooum. High values of total phosphate 
were usually recorded in rivers/streams affected by domestic sewage (Shane et al 
1971; Ravichandran 1985). 

3.8 Silicate 

Silicate values were high in all the stations except station 1. It ranges from minimum 
value of 0-2 mg/1 to maximum value of 24-8 mg/1. Higher silica content was also 
recorded in various rivers of India by Subramanian (1979). The high silica content 
might be due to the decomposition of the diatoms as well as storm discharge 
(Sreenivasan 1970). 



Limnology] sewage and heavy metal pollution of river Cooum 145 

3.9 Nitrate 

Nitrate nitrogen level varied between 0-77-11-76 mg/1. Higher values were recorded 
at stations 3 and 4. Concentration of nitrate nitrogen indicated clearly the impact of 
sewage discharge and eutrophication in Cooum. Similar higher values were also 
observed in Buckingham canal (Ravichandran 1985). 

3.10 Sulphate 

Sulphate in the surface layers of Cooum river showed a slight increase during 
monsoon in all the 4 stations. Sulphate values ranged from 80-408 mg/1. The values 
recorded were greater than those encountered in other Indian rivers (Subramanian 
1979). Sulphate becomes the electron acceptor when the dissolved oxygen is 
completely depleted and used for the organic matter break down by bacteria and 
results in the production of H 2 S which forms as bubbles at the surface (Welch 1980). 
High sulphate levels were also associated with sewage pollution. Similar observa- 
tions were recorded by Govindan and Sundaresan (1979) and Ravichandran (1985) 
in tropical polluted lotic environments, Adyar and Buckingham canal respectively. 

3.11 Heavy metals 

Heavy metals when in normal concentration are essential components of biochemi- 
cal functions but toxic when present in higher concentrations. 

3.1 la Iron: The iron content was found to be high in the first 2 stations. Maximum 
value of 900 mg/1 was observed at station 2. The source of excess iron might be 
sewage. The concentration of iron was at the maximum during rainy season and it 
was below detection limits during January. Similar observations were recorded in 
Blue Nile (Sinada and Karim 1984a). 

3.1 Ib Lead and nickel: Maximum amount of lead (275 mg/1) was observed in station 
1. Maximum nickel concentration of 1312-5 mg/1 was also observed at station 1. The 
concentrations of lead and nickel were higher during rainy season. Irregular 
fluctuation of lead content in all the stations, especially at station 4, could be due to 
the fluctuations in the speed of river water (Ellaway et al 1980). The drastic 
fluctuation of nickel content was found to be greater in first 2 stations. Similar fluc- 
tuations in nickel content were made by Welch (1952) in polluted aquatic environ- 
ments. The high values of nickel in this river might be due to the electroplating 
industries which are located on the banks of the river between stations 2 and 3. 
Further, the high values of nickel might be due to the failure of monsoon which 
resulted in evaporation of water and concentration of contents (Welch 1952). The 
high values might also be due to high pollution caused by sewage and organic matter 
(Lichtfuss and Gerbard 1981). It could also be due to the mixing of small amounts of 
enriched bottom waters with outflowing surface waters (Ellaway et al 1980). 

3.1 Ic Zinc: Maximum value of zinc (32-5 mg/1) was observed at station 1. Totally 
there was drastic fluctuations in the levels of zinc in all the 4 stations. The drastic 



146 



Bernice Anantharqj, V Bayyakikshmi and R Lakshmi 



fluctuation of zinc content might be due to the slow speed of the Cooum river (Welch 
1952). The content of the metal varies which could be due to high pollution caused 
by sewage entry and monsoon failure (Lichtfuss and Gerbard 1981). 

3.12 Zooplankton (figures 2-5) 

In the present study 14 species of zooplankton composed of protozoans, rotifers, 



IJL 



6000 ~ 
O - 

6000 - 
- 

16000 - 
O - 

16000 ~ 

26000 ~ 

- 

26000 ~ 

100OOO ~ 

50.000 - 



50.000 - 

10O,OOO - 
54,000 - 
2 7.OOO 



27000 - 
54,OOO - 



Station 1 



Protozoans 



Rotifers 




Crustacean larvae 



Aug Sep Oct Nov Dec Jan Feb Mar Apr May June July 
1982 1983 



Figure 2. Zooplankton in river Cooum. 



8000 


8000 
72OOO 



72000 
76000 
380OO 

, 
380OO 
760OO 
20000 
1 0000 

1 0000 

20000 
600O 


6000] 



Station 2 



Protozoans 



Rotifers 



Cladocerans 





Copepods 
Crustacean larvae 



P Oct Nov Dec Jan R& Ma7 
1982 , 1983 



May June July 



Limnology, sewage and heavy metal pollution of river Cooum 



147 



ll 



400OO 

o 

40000 

26000 

O 

2600O 

12000 
O 

12000 
3OOOO 



30000 

8000 

O 

800O 



Station 3 



Protozoans 



Rotifers 





Copepods 
Crustacean larvae 



Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul 
1982 1983 



Figure 4. Zooplankton in river Cooum. 



7600O 
38000 

o 

38000 

76000 
60000 

o 

60000 

10000 

o 

10000 

24000 

12000 



12000 

24000 

4000 



4000 




Station 4 



i 



I 




Protozoans 



Rotifers 



Clodocerans 



Cope pods 



Crustacean larvae 



Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul 
1982 1983 



Figure 5. Zooplankton in river Cooum. 



copepods, crustacean larvae and cladocerans were observed. Cladocerans and 
copepods were present in large numbers. Rotifers occupy numerically the last place 
in order of abundance. Rotifers might have been replaced by copepods and clado- 
cerans (Hynes 1970). The occurrence of crustacean larvae and copepods were patchy 
in different seasons in almost all the stations. Similar sporadic occurrence of cope- 



148 Bernice Anantharaj, V Bagyalakshmi and R Lakshmi 

pods have been recorded by Klimowicz (1973) who suggested that these organisms 
are present only when physical and chemical conditions were highly favourable. 
Ciadocerans were recorded in all the stations. Off all the stations zooplanktons were 
abundant in station 1. A decrease in zooplankton was noticed in all the stations 
during rainy season. Similar observations of decrease in zooplankton during 
monsoon was seen in Buckingham canal (Ravichandran 1985). The impact of sewage 
pollution in Cooum was clearly evident as the number of species recorded and 
density of zooplankton were very low. Identical recordings were made by Narayanan 
(1980) and Ravichandran (1985) in Cooum and Buckingham canal respectively. The 
low levels of dissolved oxygen, higher levels of free CO 2 nutrients such as phosphate, 
sulphate and nitrate due to eu'trophication and the toxic levels of heavy metal 
pollutants in Cooum river waters might be the cause for its inability to support 
greater number and density of zooplankton populations. 

References 

APHA 1980 Standard methods for the examination of water and waste water 5th edition (New York: 

American Public Health Association) 
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hydrobio logical conditions; Indian Geogr. J. 3 1-15 
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India to determine its suitability for the introduction of the rainbow trout; Arch. Hydrobiol. 46 128 - 

141 
Ellaway Mark, Ronald Bedt and Barryt Hart 1980 Behaviour of iron and manganese in the yana estuary, 

Australia; Aust. J. Mar. Freshwater Res. 31 597-610 
Ganapathi S V 1956 Studies on the source of the Madras city water supply and on other waters of Madras 

State; D.Sc. Thesis, University 'of Madras, Madras 

Ganapathi S V and Alikunhi K H 1950 Factory effluents from Mettur chemical and industrial Corpo- 
ration Ltd., Mettur Dam and their pollutional effects on the fisheries of Cauvery; Proc. Natl. Inst. Sci. 

India 16 189-200 
Govindan V S and Sundaresan B B 1979 Seasonal succession of the algal flora in polluted region of Adyar 

river; Indian J. Environ. Health 21 131-142 

Hynes H B N 1970 The Ecology of running waters (England: Liverpool Univ. Press) p 555 
lyenger MOP and Venkataraman G 1951 The ecology and seasonal succession of the river Cooum at 

Madras with special reference to the Diatomaceae; J. Madras Univ. 21 140-192 
Klimowicz 1973 Microfauna of activated sludge part III. The effect of physicochemical factors on the 

occurrence of microfauna in the annual cycle; A eta Hydrobiol. 15 167 - 188 

Lichtfuss Rudolf and Gerbard Bruemmer 1981 Distribution of organic matter, heavy metals and phos- 
phorus among special gravity fractions of aluvial sediments; Geoderma 25 245-256 
Mackereth F J H 1957 Water analysis for limnologists (Revised) (Fresh water Biological Association, 

Ambleside, England) 
Narayanan K 1980 Hydrobiological studies on river Cooum (Madras, S. India) with special reference to 

aquaculture, Ph.D. Thesis, University of Madras, Madras 
Paramasivam M and Sreenivasan A 1981 Changes in algal flora due to pollution in Cauvery river; Indian 

J. Environ. Health 23 222-238 
Rai H 1974a Limnological studies on the river Yamuna at Delhi, India. Part I. Relation between the 

chemistry and state of pollution in the river Yamuna; Arch. Hydrobiol. 73 369-393 
Ravichandran S 1985 Limnological studies on Buckingham Canal (Madras, India) with special reference to 

sewage pollution, Ph.D. Thesis, University of Madras, Madras 
Shane M S, De Michele E and Cannon R 1971 Water quality and plankton Ecology. The Christiana river, 

Delaware; Environ. Pollut. 2 81-95 
Sinada F and Karim A G 1984a Physical and chemical characteristics of the Blue Nile and White Nile at 

Khartoum; Hydrobiologia 110 21-32 
Sreenivasan A 1970 Limnology of tropical impoundments - a comparative study of major reservoirs 

in Madras State; Hydrobiologia 36 443 - 469 



Limnology, sewage and heavy metal pollution of river Cooum 149 

Sreenivasan A and Sounderaj R 1967 Effects of certain wastes on the water quality and fisheries of rivers 

Cauvery and Bhavani; Environ. Health 9 13-21 
Subramanian V 1979 Chemical and suspended sediment characteristics of river of India; J. Hydrohioi 

(Amst) 44 37-56 
Venkateshwarlu T and Jayanthi T V 1968 Hydrobiological studies on river Sabarmati to evaluate water 

quality; Hydrobioloyia 31 442-448 

Ward H B and Whipple G C 1958 Fresh water Biology (New York: McGraw Hill and Company) p 826 
Welch PS 1948 Limnological methods (Philadelphia: The Blakiston Company) p 381 
Welch P S 1952 Limnology 2nd edition (New York: Me Graw-Hill) p 538 
.Welch E B 1980 Ecological effects of waste water (London: Cambridge Univ. Press) p 337 



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Dated 1st March 1987 Signature of Publisher 



FOREWORD 

Nutrition and reproduction are interrelated processes occurring in conjunction with 
behavioural and chemosensory facets of the biology of the insects and hence form 
essential parameters in our knowledge of host relationships, whether it be insect- 
plant interactions or parasite/predator-host interactions. While it is well known that 
insects do not differ considerably in their fundamental nutritional requirements, 
quantitative aspects combined with biochemical composition of the food in terms of 
the nutritional quality a A pears to have some bearing on the developmental biology 
of insects. Differences m oie rates of larval development, survival, adult emergence, 
longevity and fecundity depend on the quality of the host tissues. As such an under- 
standing of the nutritional indices in relation to the rates of ingestion, digestion, 
assimilation and conversion into insect tissues plays a useful role. Essential pre- 
requisites for identification of specific factors influencing the efficiency of utilization 
relate to precise estimation of the biochemical factors involved. While many com- 
mon nutrients are known to function as phagostimulants, gustatory chemical stimuli 
are crucial to a normal rate of feeding, especially in phytophagous and blood sucking 
insects. 

Recent researches in insect nutrition studies involve an increased understanding of 
the metabolism of nutrients subsequent to their assimilation and ultimately directed 
towards their physiological functions. In other words, nutritional data are inter- 
preted in terms of physiological functions using microanalytical procedures. Current 
interest is centred on lipidnutrient needs and it is well known that insects require diet- 
ary sterols for proper growth and utilization of sterols available in the plants which 
influence egg production. Essential fatty acids needed for metabolism combined 
with the identification of prostaglandins have opened up new vistas in insect nutri- 
tional studies. The role of sterols in moulting hormone physiology, in particular the 
newer patterns of phytosterol metabolism, has enabled a better understanding of 
insect-plant interrelationships integrating nutrition and reproduction. In 
haematophagous insects nutritional adequacy of the blood meal js closely associated 
with fecundity. 

Bio-energetic studies have shown that protein-rich food is among the principal fac- 
tors influencing fecundity. Feeding regimes also seem to influence the pattern of 
energy allocation for reproduction. The rate at which food is consumed appears also 
to be a basic factor on which the energy of the terminal larva, pupa or adult depends 
so that feeding rate tends to determine energy allocation for egg production. The 
degree of host preference also has a definite correlation with the age specific changes 
in the foliage of host plants coupled with the nutritive value of the food substances, 
which in turn influences the reproductive potential of the concerned species. 

It is therefore of significance that the papers presented in this issue of the Proceed- 
ings, Animal Sciences in such areas as nutritional modulation of reproduction in 
phytophagous, haematophagous, predatory and parasitic insects, neuroendocrine 
control of insect nutrition and reproduction, as well as energy components involved 
will go a long way in furthering our knowledge in this very important field of 
entomological research. 

T N Ananthakrishnan 



151 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 153-169. 
Printed in India. 



Nutritional modulation of reproduction in two phytophagous insect pests 

S S KRISHNA 

Department of Zoology, University of Gorakhpur, Gorakhpur 273 009, India 

Abstract. In Earias vittella (F.) (a major noctuid pest of malvaceous crops) changes in the 
larval food quality (developing seeds, mesocarp, epicarp of okra fruit or the entire fruit; 
ovary of shoeflower) in either or both sexes led to pronounced disparities in the repro- 
ductive performance of the emerging moths. Highest breeding potential was observed when 
males and females obtained their nourishment, during postembryonic period, from 
developing seeds of okra of 0-8 days old and such nutrition must be made available for the 
caterpillars at least for the first 3 days of their lives to attain enhanced level of reproductive 
efficiency later as moths. During adult life, a carbohydrate nutrient was mandatory for these 
mated females to realize full fecundity which got tremendously boosted when the sugar was 
raffmose. 

Appreciable increase in ovarian weight with marked improvement in oviposition occurred in 
Triholium castaneum (Herbst) (a serious tencbrionid pest of cereals and other plant-derived 
stored commodities) when both larvae and adults ate whole flour instead of semolina, both 
enriched with yeast. This became further augmented when the flour ingested by these beetles 
during 'adult life was previously extracted in 100% ethanol and then reinforced with yeast. 
However, whole wheat flour or yeast alone given as food to such reared adults caused a 
sharp fall in the egg deposition of females. Number of eggs laid by mated females also 
differed considerably when their imaginal diet consisted of one of certain selected nutrients, 
oilseeds or spices all supplemented with yeast. Egg hatchability was always 100% 

Keywords. Earias iittclla; Triholium castaneum; larval; adult nutrition; reproductive 
potential. 



1. Introduction 

It is well known that reproduction in insects is influenced by a variety of external and 
internal factors and, amongst them, nutrition seems to be the most crucial single 
factor in affecting the total egg output in many ways in a majority of the species 
(Engelmann 1970). Its profound impact on the breeding potential of the adult can be 
a reflection of differences in the nourishment acquired by the concerned individual 
during its immature (larval/nymphal) and/or imaginal stage (Wigglesworth 1960; 
Johansson 1964; Adiyodi and Adiyodi 1974; Nayar 1977). Study of such effects of 
nutrition on the reproductive performance of the spotted bollworm, Earias vittella 
(F.) (a notorious noctuid pest of malvaceous crops like cotton and okra in the 
tropics) (Butani and Jotwani 1984) and the red flour beetle, Tribolium castaneum 
(Herbst) (a major tenebrionid cereal and other plant-derived stored product pest of 
cosmopolitan distribution (Sokoloff 1974; King and Dawson 1977) have been 
reported in diverse periodicals, during the past little over a decade by this author and 
his co-workers (Viswapremi and Krishna 1974b, 1975; Krishna et al 1977; Singh and 
Krishna 1980, 1983; Narayan Singh and Krishna 1980, 1981, 1983; Mani et al 1986). 
Findings from all these contributions are incorporated in this communication so as 
to constitute one comprehensive body of information. 



154 S S Krishna 

2. Materials and methods 

Laboratory stock cultures of E. vittella raised on tender seeds separated from 0-8 days 
old fruits of okra (Abelmoschus esculentus Moench) and of T. castaneum reared on 
whole wheat flour containing 5% (w/w) powdered yeast (BPC product, Alembic 
Chemical Works Co. Ltd., Baroda) supplied the required numbers of newborn larvae 
( < 24 h old) of these insects which served as the starting material for inclusion in the 
different investigations. Cultures of E. vittella were maintained at temperatures 
ranging between 25 and 28C and relative humidity (RH) varying from 80-100% 
while those of T. castaneum were held within a thermal range spanning between 30 
and 32C and RH values fluctuating between 70% and 80%. All experiments 
described in this study and related to each of these two species were conducted at the 
same temperature and humidity ranges as were chosen for maintaining their cultures. 
These tests were adequately replicated and, wherever desirable, the data were 
subjected to appropriate statistical analysis (Paterson 1939; Snedecor 1961). 

For the sake of convenience, details of the technical procedures involved in every 
experiment are separately given below for the two selected pest species. 

2.1 . vittella 

In all the trials set up here, single-reared, freshly emerged adult moths of either sex 
were employed. Pairing, mating and individual oviposition tests were performed in 
muslin-covered glass containers (70 mm diameter; 90 mm height) each provided with 
a hanging glass capillary filled with 15% sucrose solution (unless otherwise stated) 
that formed the food of the adults. A small section of the epicarp portion of okra fruit 
serving as oviposition substrate was placed at the bottom of the container. In most 
cases, the daily monitoring of egg laying was limited to the first 4 days of oviposition 
(the period when mated females in their life time deposited most of their eggs) 
although in a few experiments it was continued further even up to the death of the 
females. The hatchability of the eggs was also ascertained. 

2. la Effect of variation in larval nutrition: Three types of experiments were 
designed to examine this issue. In the first type, the caterpillars were nourished right 
from the day of their birth on 0-8 days old whole fruit of okra or on its epicarp, 
mesocarp or seeds removed from it to yield male and female moths which were 
subsequently single-paired (both sexes belonging to the same food regimen) and the 
reproductive potential of the females assessed in relation to such nutritional 
differences. In the second type, the reproductive efficiency of moths associated with 
larvae allowed to feed only during their first 24 or 72 h of their lives on normal and 
fresh tender seeds taken out of okra fruit and then shifted to a diet consisting of 
normal and ripened seeds excised from 9-11 days old fruits was compared with that 
of counterpart adults when both sexes of reproducing pairs were reared all through 
on the latter victual or on petroleum ether-extracted tender seeds 72 h after they 
derived nourishment from normal and tender seeds. 

Extraction of the seeds in petroleum ether was performed in a soxhlet extraction 
assembly. For this purpose, 100 tender seeds collected from fresh okra fruits (age as 
mentioned above) were first properly ground using a mortar and pestle in 25 ml of 
the solvent. Later, this crude seed extract was wholly transferred to the soxhlet 
assembly containing the same solvent and subjected to repetitive extractions within 



Earias and Tribolium reproduction 155 

the apparatus to ensure removal of all ether-soluble constituents. Such extracted 
seeds were subsequently taken out of the assembly and air-dried to eliminate 
completely the odour of ether present in them before offering them as diets to the 
caterpillars. 

In the third type, okra seed diet-reared newly eclosed males or females were 
coupled with emerging members of the opposite sex grown on epicarp diet and the 
breeding capacity of females in these pairs determined. Pairs of individuals reared on 
the ovary component of shoeflower (Hibiscus rosasinensis L.) or on tender seeds or 
epicarp of okra fruit were also arranged to serve identical purposes. 

2.1b Effect of variation in adult nutrition: Here male and female moths, developed 
on okra seed diet and paired as before for determination of the reproductive 
potential of this pest, were during their adult lives, allowed to imbibe only distilled 
water or solutions of one of 5 selected carbohydrates (raffinose, sucrose, fructose, 
galactose and glucose) provided through the hanging glass capillary and replenished 
daily. Although the concentration of all these sugars was maintained at 15%, the 
trisaccharide raffinose was tested at 5 different lower strengths (0-1, 0-25, 0-5. 1-0 and 
5-0%) as well. 

2.1c Effect of variation in larval and adult nutrition: Consideration of this aspect 
led to a comparison of reproductive competency of mated females included in pairs 
fed daily on fresh 15% solution of glucose and whose larval and pupal lives were 
raised on okra seeds with those associated with couples ingesting raffinose of similar 
concentration and developed from individuals reared on epicarp diet. 

2.2 T. castaneum 

This part of the investigation mainly related to determination of (i) ovarian weight 
and reproductive potential of the female reared and maintained on whole wheat flour 
or semolina supplemented with 5% yeast (w/w) and (ii) the influence of adult diet on 
the reproductive potential of the female. 

For studying the first aspect, 10 larvae procured from the laboratory culture were 
collectively permitted to continue their postembryonic development up to pupal 
stage on whole wheat flour or on semolina enriched with yeast provided in sufficient 
amounts within glass containers (30 mm diameter; 105 mm height) covered at the top 
with muslin cloth. A male and a female pupa, distinguished on the basis of sexual 
dimorphic characters akin to those described for T. confusum (Chapman 1918), 
formed from such group-reared larvae were transferred as a single pair to a small 
muslin-capped glass vial (10 mm diameter; 50 mm height) consisting of adequate 
quantity of larval food for their eventual emergence into adults. The eclosed females 
held on yeast-added whole wheat flour or semolina were sacrificed as virgins in 
preoviposition state within 24 h of their emergence or as egg laying mateds on the 
5th day of their adult life to obtain their ovaries (of one side only) made available by 
dissections performed on a clean microslide containing minute amounts of distilled 
water under a stereoscopic binocular microscope. Fresh weight of these ovaries 
removed from each of such 5 females in the different age groups associated with 
whole wheat flour or semolina were quickly determined separately on a single pan 
semi-micro electrical balance and the mean fresh weight of this reproductive tissue in 
respect of each experimental condition was calculated. 



156 S S Krishna 

For evaluating the reproductive capacity of females reared on these two diets, 
male and a female beetle, both associated with the same food and freshly emerge 
from single-paired pupae were coupled to facilitate mating and subseque] 
oviposition by the females. These tests were performed in glass vials similar to tho; 
described already for accommodation of pupae for their metamorphosis into adult 
The diets, given in sufficient amounts and replenished only at the end of 20 days i 
the paired beetles inside the vials were identical to those fed by them during the 
larval lives. Oviposition was monitored daily for a period of 40 days since emergen< 
of these insects into adults. The decision to terminate the experiments at the end < 
an arbitrarily selected 40-day duration, evidently for practical convenience, wj 
mainly based on the already known facts about the extended longevity of thej 
beetles (Feakin 1976; King and Dawson 1977). The hatchability of the eggs laid t 
the females was also noted. 

Investigations into the second aspect entailed utilization of single pairs of newbor 
male and female insects raised in the laboratory, as in previously described tests, o 
whole wheat flour supplemented with yeast and provision of one of the followin 
food items fortified with a specified amount of yeast (w/w) during their imaginal live 
(i) normal whole wheat flour with or without 5% yeast or only yeast, (ii) whoi 
wheat flour extracted in distilled water, chloroform, diethyl ether or 100% etham 
(extraction procedure using solvent extraction assembly same as already describe 
. earlier with the difference that for each extraction 10 g of flour and 200 ml of 
solvent were used) and then supplemented with 5% yeast, (iii) certai 
carbohydrates [starch, sucrose, glucose (BDH, England), fructose (E. Merc! 
Germany)] or a protein [vitamin-free casein (ICN Pharmaceuticals Inc., USA)] or 
lipid [cholesterol (Sisco Research Laboratories Pvt. Ltd., India)], all enriched wit 
5% yeast, (iv) a mixture of some vitamins of the B group [Glaxo Laboratory 
(India) Ltd.] consisting of 80 fig of thiamin, 30 //g of riboflavin, 230 /ig of niacin, 2 ju 
of calcium pantothenate and 0-63 ^g or 63 /^g of folic acid per gram of yea; 
and (v) certain oilseeds, viz cotton seed (Gossypium arboreum), ground nut (Arach, 
hypogea), linseed (Linum usittatisisnum), sesamum (Sesamum indicum), mustar 
(Brassica campestris) or toria (Brassica napus) or some spices such as cardamor 
(Ammonum subulatum), cinnamon (Cinnamomum zeylanicum\ clove (Syzygiut 
aromaticum), coriander (Coriandrum sativum), black pepper (Peper nigrum) or cumir 
seed (Cuminum cyminum) each material mixed with 5% yeast. 

Every diet, before offering it to these tenebrionids for feeding, was powdered an 
sieved properly so as to maintain uniformity in its particle size. With the exception c 
fructose which, on account of its hygroscopic nature, was replenished daily, all othe 
foods were renewed only once (on the 10th day) during the experiment. The tenure c 
these oviposition trials, whose general layout was similar to that described in th 
previous set of tests, was, however, limited to 20 days following emergence of th 
adults. As before, egg viability was also recorded. 

3. Observations 

3.1 E. vittella 

A break-up of the oviposition data indicating summarized information about certai] 
specific features of this reproductive activity in this moth in relation to larval rearm; 



Earias and Tribolium reproduction 157 

Table 1. Data relating to length of oviposition period (LOP), peak oviposition day (POD), 
mean oviposition period (MOP) and mean egg number (MEN)/mated female in E. vittella in 
pairs reared on whole fruit of okra or on its components. 





Number of 


LOP* 


Rearing medium 
okra fruit 


individuals 
tested 


Min 


Max 


POD** 


MOP 


MEN 


Seeds (separated from fruit) 


12 


12(2) 


1.6(3) 


1(83) 


14-4 


882 


Whole fruit 


10 


3(1) 


14(3) 


1(77) 


10-1 


625 


Mesocarp 


12 


6(1) 


12(2) 


1(56) 


10-1 


375 


Epicarp 


15 


9(1) 


15(3) 


6(18) 


12-8 


151 



* Figures in parentheses relate to number of females showing the corresponding minimum (min) and 
maximum (max) lengths of oviposition period for each rearing medium. 

**Figures in parentheses relate to the highest mean number of eggs (adjusted to the nearest integer) laid 
on the corresponding oviposition day by females for each rearing medium. 



on whole fruit of okra or on its components is given in table 1. A greater proportion 
of females in pairs reared on seeds or on epicarp have a longer oviposition period 
than counterparts associated with couples grown on other two diets. The mean daily 
oviposition reached its peak value on the first day of egg laying itself in all the 
females except those in pairs raised on epicarp diet where it occurred only on the 6th 
day of oviposition. The overall mean fecundity was highest in females belonging to 
seed-reared couples and this was nearly 6 times more than the lowest mean fecundity 
score recorded for individuals reared on epicarp diet. 

From the results presented above, one can assume that differences observed in the 
total length of oviposition period of moths in relation to their rearing media can 
themselves be responsible as an indirect effect of the dietary variations in the rearing 
media to produce differences in the total number of eggs laid by the females reared 
variously. Hence a correct evaluation of the direct effect of the dietary differences in 
the rearing media on the reproductive potential of these moths will be possible only 
on the basis of comparison of the total number of eggs laid by the females during the 
same length of oviposition period. Accordingly, this evaluation was based on the 
total eggs laid by the females during a period of 10 days when one-half to all of the 
total number of moths that emerged from each rearing medium completed laying 
more than 65% of the aggregate number of eggs deposited by them during their 
oviposition period. The observations (table 2) again clearly indicated the competency 
of females in seed-reared pairs to lay maximum number of eggs during this stipulated 
oviposition period. Interestingly, this was significantly different even from the total 
egg output recorded for individuals raised on whole fruit which also contained its 
seeds. 

Having realized that nutrition obtained during larval life from developing seeds is 
unquestionably superior to that provided by other parts of okra fruit for E. vittella to 
achieve highest reproductive capability, it was of interest to ascertain the time-bound 
compulsory requirement for the various seed-resident nutritive constituents for the 
caterpillar to complete its development and to subsequently function as a sexually 
mature and efficiently productive adult. Table 3 summarizes the data relating to this 
aspect of the investigation. Mated females in pairs whose nutrition during the first 3 
days of their larval lives was provided by developing seeds and then by ripened seeds 
of okra were only fit enough to lay significantly greater number of eggs than their 



58 



S Krishna 



Table 2. Number of eggs laid during the first 10 days of oviposition 
by mated females of E. vittella reared on whole fruit of okra or on its 
components*. 



Rearing medium okra 
fruit/components 


Number of 
individuals, 
tested 


Mean number 
of eggs laid 


Seeds (separated from fruit) 


12 


675-1 


Whole fruit 


5 


545-0 


Mesocarp 


9 


355-9 


Epicarp 


14 


119-4 


Mean square: 






(i) Treatment 




710367-3 


(ii) Error 




4507-2 


F(l%) 




157-6 



*Data subjected to analysis of variance for samples of unequal size 
(Snedecor 1961). 



Table 3. Egg deposition during the first 4 days of oviposition and hatchability of eggs laid 
by mated females of E. vittella in pairs reared on various okra seed-related dietary regimens 
(data pooled from 5 females per test)*. 


Mean number of 


Mean number of 
)ietary regimen eggs laid 


viable eggs 
deposited 


'airs reared during the first 72 h of their larval lives on normal 
and tender seeds and later on normal and ripened seeds 267-00 a 


227-20 a 


>airs reared during the first 24 h of their larval lives on normal 
and tender seeds and later on normal and ripened seeds 174-40 b 


1 53-40 ab 


'airs reared during the first 72 h of their larval lives on normal 
and tender seeds and later on petroleum ether-extracted seeds 170-40 b 


1 02-80 b 


'airs reared all through on normal and ripened seeds 160-00 b 


1 39-40 b 


Mean 192-95 


155-70 


LSD(1%) 105-19 


114-74 


(5%) 76-34 


83-27 


Means in the same vertical column followed by the same letter do not differ 
te 1% or 5% level by the least significant difference test (LSD) (Paterson 1939). 


significantly at 



ounterparts reared on other dietary combinations (P<001 or 0-05). Further, 
tatchability of eggs deposited by these highly fecund females was markedly higher 
P<0-01 or 0-05) than that recorded for eggs released by mateds in couples 
leveloped from larvae and pupae maintained on remaining dietary regimens tested 
lere excepting the one where the caterpillars during the first 24 h of their lives were 
leriving nourishment from developing seeds and later from ripened seeds of okra 
P>0-05). 

An examination into how far larval dietary regimen consisting of (i) tender seeds 
>f okra fruit for one sex and epicarp of this fruit for the other sex or (ii) ovary of 
hoeflower would affect egg deposition and egg viability in this noctuid species 
ielded additional interesting results. The findings (table 4) compared with such data 



Earias and Tribolium reproduction 



159 



Table 4. Estimates of egg output and egg viability in E. vittella in tests where males and 
females of copulating pairs were held during rearing, on varied or on similar dietary 
regimens (data pooled from 5 females per test)*. 





Mean number 


Mean number 




of total 


of viable 


Dietary regimen 


eggs laid 


eggs laid 


Okra fruit developing seeds for both sexes 


243-80 a 


176-40 a 


Okra fruit epicarp for both sexes 


1 58-00 b 


70-60 b 


Okra fruit epicarp for males and developing 






seeds for females 


1 54-80 b 


1 07-60 b 


Okra fruit developing seeds for males and 






epicarp for females 


145-20 b 


75-60 b . 


Ovary of shoeflower for both sexes 


103-00 b 


80-60 b 


Mean 


160-96 


102-16 


LSD(1%) 


86-00 


80-49 


(5%) 


63-06 


59-01 



*Same notations as in table 3. 

Table 5. Number of eggs laid by mated females of E. vittella 
in pairs fed on different carbohydrate diets or distilled water 
during their adult lives (data pooled from 5 females per 
test)*. 



Adult food 


Mean number of 
eggs laid 


Raffinose 


604-6 a 


Galactose 


509-8 ab 


Sucrose 


445-4 b 


Fructose 


420-4 be 


Glucose 


317-4 c 


Distilled water 


164-8 d 


Mean 


410-4 


LSD(1%) 


155-1 


(5%) 


114-5 



*Same notations as in table 3. 

rocured from moths where both sexes were raised on developing seeds or on 
picarp clearly revealed statistically significant increments in mean egg output and 
gg viability in females following mating between the sexes reared exclusively on 
kra seed diet. Nourishment obtained by pairs from other food combinations tested 
ere gave rise to mateds having pronouncedly low and statistically similar 
eproductive capacity. 

The influence of adult diet (one of various selected sugars or plain distilled water) 
n the reproductive potential of this insect was also assessed. Notwithstanding 
bsence of any major difference in longevity or in length of oviposition period 
etween moths ingesting a carbohydrate diet and those feeding on water, a 
ignificantly greater number of eggs were laid by the females fed on any one of the 
sst sugars than on water (table 5). Amongst the various carbohydrates, raffmose at 
5% strength helped the individuals to deposit maximum number of eggs and 
viposition by the moths was equally at a significantly higher level when they 
igested this trisaccharide even when maintained at as low a concentration as 1% 
P< 0-01) (table 6). 



160 S S Krishna 



Table 6. Number of eggs laid by mated females of 
E. vittella in pairs fed on different concentrations of 
raffmose solutions during their adult lives (data pooled 
from 5 females per test)*. 



Concentration 


Mean number of 
eggs laid 


15-00 


604-6 a 


5-00 


569-6 a 


1-00 


534-6 a 


0-50 


316-2 b 


0-25 


293-2 b 


0-10 


254-8 b 


Mean 


428-8 


LSD(1%) 


147-7 



*Any two means followed by the same letter do not 
differ significantly at the 1% level by the least signifi- 
cant difference (LSD) test. 

Table 7. Number of eggs laid by-mated females of E. vittella in pairs 
raised on epicarp or developing seeds during pre-imaginal stages and 
adults given raffinose or glucose solution respectively (data pooled 
from 5 females per test). 

Mean number of eggs 
Experimental condition laid ( SE) 

*Epicarp-reared and raffinose given 

in adult stage 427-2 18-66 a 

*Seed-reared and glucose given in 

adult stage 322-0 20- 50 

*Okra fruit component. 

"Significantly different at 1% level from the value just below in the 

column (t test) (Paterson 1939). 

SE, Standard error. 



In the light of informations presented above, egg yield values in this moth was 
compared when raffinose was provided as adult food for pairs whose immature 
stages were reared on epicarp diet with couples fed on glucose during their imaginal 
lives and whose immature stages were raised on developing okra seeds. Number of 
eggs deposited by moths associated with the former dietary regimens was decidedly 
greater (?<O01) (table 7) than that laid by those related with the latter set of foods. 

3.2 T. castaneum 

Mean fresh weight of ovaries of females reared and fed on whole wheat flour, whether 
unmated and remaining in preoviposition state (<24h old beetles) or mated and 
ovipositing (5-day old beetles), was comparatively higher than that determined for 
counterpart individuals that ingested semolina following their development on the 
same diet (table 8). Also, egg output in these beetles was more if they were held, right 
from their birth, on whole wheat flour instead of on semolina (figure 1). 
Females whose adult food, like those of males which mated with them, consisted of 



Earias and TriboUum reproduction 



161 



Table 8. Weight (//g) of ovaries (left side only) of unmated or 
mated females of T. castaneum reared and maintained on whole 
wheat flour or semolina supplemented with yeast (data pooled 
from 5 females per test). 

Mean fresh weight of ovaries ( SE) 



Diet +.5% yeast 


Unmated 


Mated" 


Whole wheat flour 
Semolina 


180 37-51 
140 24-56 


600 70-90 
540 51-12 



fl <24 h old females 'in preoviposition state. 

*5day old ovipositing females which mated with males 

belonging to the same dietary regimen. 

SE, Standard error. 



100 



80 



D 
CL 

-4 

D 
O 

O> 

cn 

0} 

c 
o 
o> 



60 



40 



20 







W 



Figure 1. Histogrammic representation of mean egg output by a mated female of 
T. castaneum in a pair reared on whole wheat flour (W) or semolina (S) enriched with yeast 
and adults maintained during a 40-day experimental period on the same diet. as their larvae 
(data pooled from 5 females per test). 



whole wheat flour extracted in 100% ethanol instead of in water, ethyl ether or in 
chloroform and then reinforced with yeast showed, subsequent to mating, maximal 
egg deposition possessing high statistical significance (table 9), Curiously enough, 
this egg yield value was significantly higher (P<001) than that obtained from 
mateds allowed to eat normal whole wheat flour fortified with yeast. 



162 S S Krishna 

Table 9. Number of eggs laid during a 20-day period, 
commencing from the day following emergence, by mated 
females of 71 castaneum in pairs fed on normal or differently 
extracted whole wheat flour during their adult lives (data 
pooled from 5 females per test)*. 

Mean number of 
Diet (enriched with 5% yeast) total eggs laid 

100% ethanol-extracted whole 

wheat flour 131 -20 a 

Normal whole wheat flour 57-20 b 

Ether-extracted whole wheat flour 13-80 c 

Water-extracted whole wheat flour 10-60 c 
Chloroform-extracted whole wheat 

flour 8-80 c 

Mean 44-32 

LSD(1%) 22-56 

(5%) 16-54 

*Same notations as in table 3. 

Low productivity by females compelled to feed on flour extracted in water, 
chloroform or ether, despite these diets supplemented with yeast, might possibly be 
due to this cereal getting depleted, consequent to such treatments, of a variety of 
useful substances like carbohydrates, proteins, lipids and vitamins, many of which 
are likely to be of great nutritional value for these beetles in regulating their total egg 
output as in several insects (Wigglesworth 1960; Johansson 1964; Engelmann 1970; 
Adiyodi and Adiyodi 1974; Nayar 1977). It was, therefore, considered proper to 
enquire into this issue also wherein the egg laying capabilities of these tenebrionids 
fed during their adult lives on one of certain specific organic nutrients enriched with 
yeast were compared with those of counterpart individuals ingesting yeast- 
containing normal whole wheat flour and the data presented in table 10. 

Although normal whole wheat flour proved to be the best amongst the various 
foods tested enabling the females to exhibit maximum oviposition (P<0-01), egg 
output by individuals fed on starch was significantly higher (P<0-01) than that 
recorded from females in pairs that ate sucrose, fructose, cholesterol or a mixture of 
certain vitamins of B complex. A similar state of affairs existed when these beetles 
ingested vitamin-free casein instead of cholesterol or B complex vitamin mixture, the 
protein nutrient also helping, though to a relatively less extent, the mateds to release 
significantly more eggs (P<0-05) than the disaccharide or the ketohexose mono- 
saccharide compounds. 

The lowest egg yield value obtained from mated females of T. castaneum where the 
food of both sexes consisted of yeast supplemented vitamin B complex mixture (table 
10) was suspected to be the outcome of one or the other essential vitamin components 
occurring in alarmingly low proportions in the mixture. Preliminary study involving 
casual observations indicated an increased egg laying by the copulated females if the ' 
beetles ingested vitamin B complex mixture containing higher amount of folic acid. 
This led to an examination of this phenomenon in greater detail when the folic acid 
content in the vitamin food was augmented by 100 times (63 /xg/g of yeast diet) and 
the number of eggs laid by females in couples fed on yeast-mixed B complex mixture 
having low or high folic acid was compared with that deposited by mateds 



Earias and Tribolium reproduction 163 

Table 10. Number of eggs laid during a 20-day period, 
commencing from the day following emergence, in mated 
females of T. castaneum in pairs fed on whole wheat flour or on 
different organic nutrients during their adult lives (data pooled 
from 5 females per test)*. 



Diet (all excepting vitamin B 
complex mixture**) enriched 
with yeast 


Mean number of total 
eggs laid 


Whole wheat flour 


57-20 a 


Starch 


19-60 b 


Vitamin-free casein 


19-OOb 


Glucose 


940 be 


Sucrose 


4-80 c 


Fructose 


4-40 c 


Cholesterol 


2-40 c 


Vitamin B complex mixture** 


l-80c 


Mean 


14-82 


LSD(1%) 


14-62 


(5%) 


10-87 



*Same notations as in table 3. 

**For details concerning preparation and composition of this 

diet, see 2. 



Table 11. Number of eggs laid during a 20-day period, commencing 
from the day following emergence, by mated females of T, castaneum in 
pairs fed during their adult lives on whole wheat flour or on vitamin B 
complex mixture** having low or high folic acid content (data pooled 
from 5 females per test)*. 

Mean number of 
Diet total eggs laid 

Whole wheat flour + 5% yeast 57-20 a 
Vitamin B complex mixture** with high folic 

acid content (63 jug/g of total diet) 43-40 a 
Vitamin B complex mixture** with low folic 

acid content (0-63 jug/g of tojtal diet) 1-80 b 

Mean 34-13 

LSD(1%) 21-53 

(5%) 15-36 

*Same notations as in table 3. 
**Same notations as in table 10. 



naintained on whole wheat flour plus yeast. A significantly greater egg laying was 
loticed in females in tests where the beetles fed on yeast vitamin B complex 
nixture with increased folic acid content (P<001) (table 11). Such a shoot-up in the 
jgg number of these females brought them, statistically, at par with individuals 
vhose adult food comprised normal whole wheat flour mixed with yeast (P > 0-05). 
All egg yield data from mated females of T. castaneum put to variable nutritional 
experiences, as outlined above, were obtained when yeast was never omitted in the 
maginal food of these beetles. Whether such inclusion of yeast in a diet fed by these 
nsects is mandatory to improve the egg output of females and whether individuals 



164 S S Krishna 

that ingest only yeast can exhibit reproductive efficiency similar to those eating food 
with or without this ingredient are questions demanding clearer answers though 
Sokoloff and Ho (1962) were the first to throw some light on this matter. A probe 
into these issues made in the present investigation indicated a definite rise in the egg 
output by females if they belong to pairs whose whole wheat flour diet, instead of 
being offered as such, was supplemented with yeast (P<005) (table 12). However, 
yeast alone as an adult food was miserably incompetent to induce a greater 
oviposition in these mateds in comparison to even those that were maintained on 
plain whole wheat flour (P<001). 

Results concerning the effect of different oilseeds or whole wheat flour 
supplemented with yeast fed by adults of this pest on the insect's oviposition are 
displayed in table 13. A significantly higher number of eggs were deposited by 
females associated with pairs that ingested groundnut or cotton seed supplemented 
with yeast instead of other oilseeds. No statistical difference was observed in the 
oviposition between beetles maintained on groundnut and whole wheat flour both 
fortified with yeast, the latter diet, nonetheless, stimulating a markedly greater egg 

Table 12. Number of eggs laid during a 20-day period, commencing 
from the day following emergence, by mated females of T. castaneum in 
pairs fed during their adult lives on whole wheat flour with or without 
yeast or on yeast alone (data pooled from 5 females per test)*. 



Diet 


Mean number of 
total eggs laid 


Whole wheat flour + 




5% yeast 


57-20 a 


Whole wheat flour 


36-80 b 


Yeast 


1-60 c 


Mean 


31-86 


LSD(1%) 


20-47 


(5%) 


14-67 



*Same notations as in table 3. 

Table 13. Number of eggs laid during a 20-day experi- 
mental period, commencing from the day following 
emergence, in mated females of T. castaneum in pairs fed 
on different oilseeds or whole ^wheat flour during their 
adult lives (data pooled from 5 females per test)*. 



Diet (enriched 

with 5% yeast) 


Mean number of 
total eggs laid 


Whole wheat flour 


57-20 a 


Ground nut 


42-20 ab 


Cotton seed 


35-00 b 


Sesamum 


17-40c 


Linseed 


15-80c 


Mustard 


4-20 c 


Toria 


4-20 c 


Mean 


25-14 


LSD(1%) 


29-84 


(5%) 


22-12 


*Same notations as in tahle 1 



Earias and Tribolium reproduction 165 

laying in comparison to cotton seed (P<005) or remaining oilseeds (P<O01). But 
sesamum, in which postembryonic development of these beetles was unsuccessful 
(Pajni and Virk 1978), when eaten by these insects during their adult lives, facilitated 
the mated females to lay eggs which, interestingly, was about 50% of that recorded 
from females in pairs whose food was cotton seed plus yeast. 

Amongst the various yeast-supplemented spices tested, only coriander enabled 
these beetles to be somewhat productive the mean number of eggs laid per female 
being 10-8. All the remaining diets in this category proved incompetent even to 
sustain longevity of these tenebrionids till the close of the 20-day experimental 
period. 

Egg viability in all the trials arranged here was 100%. 

4. Discussion 



4.1 E. vittella 

A conspicuous contrast in the reproductive efficiency of this pest was noticed 
between females associated with pairs reared on tender okra seeds (0-8 days old) and 
those in couples grown on epicarp of this fruit or on ovary of shoeflower. A profound 
influence of larval nutrition on egg production in the adult (Wiggles worth 1960) is 
implicit in these findings. The considerably high fecundity exhibited by the moths 
developed on seeds is evidently due to the effect of a superior nutritional quality of 
the diet, at least in regard .to free amino acids and water-soluble proteins (Mani et al 
1986) which, in addition to accelerating the overall development (Mehta and Saxena 
1973; Vishwapremi and Krishna 1974a), boosts the reproductive potential as well. 
These nutrient reserves are plausibly accumulated more readily in relatively larger 
quantities during larval life by the adult females reared solely on seeds and 
subsequently utilized by them resulting in their greatly enhanced egg output. The fact 
that the number of eggs laid by females developed on whole fruit, in which such seeds 
were also present, was still significantly less than that deposited by individuals reared 
only on seeds separated from the fruit lends additional support to this postulation. 
That such nutrition becomes an obligatory requirement for the caterpillars, at least 
for the first 3 days of their lives, to enable the resultant females to become maximally 
productive can be inferred by comparison of egg yield and egg hatchability values 
obtained from these individuals, whose subsequent larval and pupal phases were 
allowed to continue on ripened okra seeds (9-11 days old), with those recorded from 
females associated with pairs held on other single or mixed dietary regimens for 
different prescribed periods of their postembryonic development. Further, the 
mandatory need for both sexes of a copulating pair to avail nutrients from okra 
seeds during their larval development to facilitate this noctuid species to achieve 
highest reproductive potential (as manifested through significant increments in 
mean egg output and mean egg viability in the female belonging to this test couple) 
clearly indicates that not only in females but also in males is there an apparent impact 
of nutrition on the reproductive activity, once considered to be of negligible 
magnitude in insects (Wiggles worth 1960). 

So far as the role of adult nutrition in the regulation of reproduction in E. vittella 
was concerned, the results showed a marked elevation in egg deposition when the 



166 S S Krishna 

adult moths were fed on a sugar solution instead of plain water, although they were 
all reared under identical conditions and did not have any pronounced difference m 
longevity or in length of oviposition period. This unquestionably suggests that 
carbohydrate nutrient is very essential during imaginal period in promoting the egg 
laying capacity of mated females as in the butterfly; Colias philodice eury theme (Stern 
and Smith 1960) plausibly through better utilization of dietary protein reserves in 
yolk formation during egg production (Engelmann 1970). The greatest contrast in 
the aggregate number of eggs deposited between raffinose- and glucose-fed moths 
further implies that, like in the syrphid Sphaerophoria scutellaris (Lai and Haque 
1955), different sugars variably regulate the reproductive potential of the spotted 
bollworm. The higher egg output by females ingesting fructose or galactose instead 
of glucose, notwithstanding that all the 3 sugars are monosaccharides, corroborates 
this interpretation. The maximum egg output by moths fed on raffinose can then 
reasonably be assumed to be due to the combined effect of fructose, galactose and 
glucose (all of which will be obtained from the ingested raffinose subsequent to its 
complete hydrolysis) on the reproduction of this pest. The fact that eggs deposited by 
moths that imbibed raffinose in adult stage but reared on epicarp diet was 
significantly greater than those laid by seed-reared and glucose-ingested individuals 
is a pointer to this possibility and highlights the importance of this trisaccharide as 
an efficiently compensating nutritional factor in the diet of this insect. 

Although the need for a carbohydrate nutrient during adult life to realize full 
fecundity in females has now been experimentally established in E. vittella, it is still 
not known whether variations in the effect of different sugars ingested by the adults 
'on the female's oviposition are due to superior nutritional value of some of these 
saccharides or on account of disparities in food quantities consumed. Also the 
question whether the moths in nature obtain through feeding all the necessary 
sugars, quality- and/or quantitywise, from the foliage or flowers of one or more of the 
plants naturally infested by them or they are impelled by instinct to visit certain 
other plants falling outside their normal host range to fulfil their carbohydrate 
requirements for reproduction is still open. 



4.2 T. castaneum 

The possession of more heavily weighing ovaries and occurrence of higher egg 
output by mated females, held right from their birth, on whole wheat flour 
supplemented with yeast instead of on similarly enriched semolina clearly indicate 
that the former cereal foodstuff is the most favourable diet for T. castaneum so far as 
its reproductive potential is concerned an observation which supports the earlier 
findings of Good (1933) and Hamalainen and Loschiavo (1977) who, in their 
publications have, however, compared the fecundity of this insect on natural and 
enriched whole wheat flour with cereal products other than semolina. 

The observation that whole wheat flour reinforced with yeast, as a food is 
relatively nutritionally superior to semolina to effectuate the red flour beetle mated 
females to lay more eggs evidently led to an examination of the ability of these 
females to oviposit if this flour, after being extracted in one or the other of different 
solvents, was provided as adult food to reproductive pairs with addition of yeast. It 
was found that females in pairs fed on 100% ethanol-extracted whole wheat flour 



Earias and Tribolium reproduction 167 

were capable of depositing the highest number of eggs significantly surpassing the 
egg output figures obtained not only from counterpart mateds ingesting this cereal 
food subsequent to extraction in water, ether or in chloroform but also, interestingly, 
even from those eating yeast-added normal whole wheat flour. While no forthright 
statement can be made presently to explain this unexpected shootup in the number 
of eggs laid by mateds fed during their adult lives on ethanol-extracted whole wheat 
flour, it is, however, presumed that such a chemical treatment of this cereal aided in 
the removal of certain not yet characterized ingredients that possibly function as 
natural checks for the females in their reproduction, specifically in relation to 
oviposition. This postulation may also serve as a basis for explaining, in part, the 
occurrence of poor egg laying in adult females associated with couples ingesting 
water-, ether- or chloroform-extracted whole wheat flour wherein such detrimental 
ethanol-soluble constituents were fully present with the simultaneous depletion of 
several nutritionally important chemical compounds, specially starch and casein, 
both of which were found to be very important in the reproduction of this insect 
.(table 10). If this assumption is correct, it would not be out of place here to suggest 
that future investigations into the reproductive biology of T. castaneum may be 
focussed towards chemical characterization and identification of these ethanol- 
soluble constituents and their judicious application in pest management programmes 
aimed at limiting the population build-up of this beetle on stored commodities. The 
poor fecundity (in terms of egg deposition scores) exhibited by the mated females of 
this species when both sexes, as adults, were fed on water-, chloroform- or ether- 
extracted whole wheat flour enriched with yeast, notwithstanding ingestion of yeast- 
added whole wheat flour during their larval life, indicates absence of any benefit 
provided to the imagines in their reproduction via larval nutrition and thus supports 
the inference drawn along similar lines by Reynolds (1944) in his studies on 
T. destructor. 

Oviposition was significantly curtailed if the adult food of the mated females 
consisting only of yeast-supplemented B complex vitamins had folic acid content as 
low as 0-63 ug instead of 63 ug/g of total diet. It appears that folic acid in appreciable 
concentration is also a necessary constituent in the adult diet of T. castaneum for 
aiding the beetle's reproduction like other components of B vitamins such as 
riboflavin, thiamin and niacin, all 3 of which have been reported by Hamalainen and 
Loschiavo (1977) to increase the fecundity of this species. 

Addition of yeast to whole wheat flour given as adult diet caused a marked 
improvement in the egg output by the mated females. From this it is inferred that 
yeast, in which existence of some growth factors necessary for normal development 
of T. confusum has already been discovered (Charbonneau and Lemonde 1960a, b), 
presumably contains also certain as yet unidentified components responsible, 
additively, for the promotion of oviposition in T. castaneum. 

The reproductive potential of these beetles, subsequent to feeding them on 
different selected oilseeds or spices in their adult lives, did not always coincide with 
the comparative dietary efficiency of these products for the larval growth of this 
insect (Pajni and Virk 1978). This implies the occurrence of variability in the 
nutritional competency of these materials insofar as larval development and adult 
reproduction in this pest are concerned. 

A 100% viability of eggs in all test conditions clearly indicates absence of any effect 
of nutritional difference on egg hatchability in this tenebrionid species. 



168 S S Krishna 

Acknowledgement 

This work was supported by funds from the University Grants Commission and 
Indian National Science Academy, New Delhi. 

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Printed in India. 



Relation between feeding and egg production in some insects 

J MUTHUKRISHNAN and T J PANDIAN* 

Department of Zoology, APA College, Palni 624 602, India 

*School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India 

Abstract. Food consumption and fecundity of insects vary with life style and feeding 
pattern. In general, species which feed during the larval and adult stages and maintain a 
smaller biomass, allocate a higher percentage of the ingested energy to egg production (e.g. 
Oryzaephilus surinamensis; 34-4%). Species which grow larger but feed at lower rates and 
pass through extended adult life span display very low egg production efficiency (e.g. 
Poecilocerus pictus; 0-7%). A few others feed at faster rates during the larval period, shorten 
the adult phase and allocate fairly a high percentage of the ingested energy to egg 
production (e.g. Bombyx won'; 5-8%). Food quality regulates food consumption and thereby 
significantly influences egg production in several polyphagous insects. Prey density 
influences food consumption and fecundity of predators. Regression of fecundity on blood 
meal ingested for two hemipterans and two dipterans revealed that the dipterans are 
autogenous and are relatively independent on adult blood meal for oviposition in 
comparison with the hemipterans. 

Keywords. Feeding pattern; fecundity; egg production efficiency. 

1. Introduction 

Insects follow a variety of life styles and feeding patterns, which play a major role in 
determining the strategy of growth and reproduction (Enders 1976; Lawton and 
McNeill 1979). South wood et al (1974) considered that the generation time of an 
individual determines its reproductive strategy. Life span of insects varies from a few 
days in several lepidopterans and parasitic hymenopterans to over 17 years in cicada 
(Richards and Davies 1977). Some are short lived and are smaller in size (e.g. 
parasites, aphids, scale insects etc), while a few others are long lived and large sized 
(e.g. scarabaeid beetles, grasshoppers and locusts etc). A majority of the insects feed 
throughout life (e.g. most of the ametabolous and hemimetabolous insects and a few 
holometabolous insects), while several lepidopterans, ephemeropterans and 
chironomids feed only during the larval period. While a few sanguivores are 
unautogenous and require an adult blood meal to commence oviposition (e.g. Aedes 
aeyypti), several others are autogenous and are independent of adult blood meal for 
oviposition. Egg production is ultimately an aspect of conversion of ingested food. 
Therefore, biotic and abiotic factors, which are known to alter the rate and efficiency 
of food utilization in females also influences her fecundity (Muthukrishnan and 
Pandian 1987). Although a good number of publications report fecundity of insects, 
very few alone relate to feeding with energy allocation to egg production. This paper 
aims to critically evaluate the impact of feeding on egg production in some insects. 

2. Materials and methods 

Following the gravimetric procedure described by Waldbauer (1968), food 
consumption, growth and egg production in Mantis religiosa, Coccinella 



172 



J Muthukrishnan and T J Pandian 









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Relation between feeding and egg production in some insects 



173 



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174 J Muthukrishnan and T J Pandian 

transversalis, Monochilus sexmaculatus and Cicindela cautena were estimated in 
terms of energy. However, rest of the information presented in this paper were 
collected from pertinent publications. From a survey of over 100 publications, about 
30 were selected; the others which do not provide quantitative (mass or energy) data 
for both food consumption and fecundity were not considered. Information 
presented here pertain to a wide range of insects following different life styles and 
feeding patterns. The data collected have been discussed in the light of theoretical 
information provided by Calow (1977). 



3. Effect of life style and feeding pattern 

Table 1 presents data on food consumption and energy allocation to egg production 
in a few insects, which follow different life styles and feeding patterns. Egg production 
efficiency is expressed as percentage of ingested energy allocated to egg production; 
the efficiency ranges from 0-7% in the grasshopper Poecilocerus pictus to 34 and 42% 
in the granivorous Oryzaephilus surinamensis and the juice feeding Aphis fabae, 
respectively. The wide range in the efficiency may partly be attributed to the life style 
(living for longer or shorter durations, and growing smaller or larger) and feeding 
pattern (feeding throughout or part of the life span). The primitive collembolans 
Orchesella cincata and Tomocerus minor moult throughout life comprising 
intermoult periods of active feeding and reproduction, and short non-feeding moult 
periods. About 5 and 7% of the ingested energy (ca 38 J) is allocated by them 
respectively, to egg production. T. minor is metabolically less active than O. cincata 
and hence affords to allocate a higher percentage of ingested energy to egg 
production (Testerink 1982). Of the two species of grasshoppers considered, Oxya 
velox displays egg production efficiency of 4-1% as against 0-7% by P. pictus. Al- 
though their life span is around 250 days, energy costs of growing larger (6-9 g in 
P. pictus vs 0-3 g in O. velox) and maintenance of a larger adult biomass for a longer 
duration (> 105 days in P. pictus vs <75 days in 0. velox) have restricted the egg 
production efficiency of P. pictus to the lowest level (0-7%). Metabolic cost of 
maintenance of larger biomass of adult for a longer duration depletes the energy 
available for egg production (Calow 1977). Although the scarabaeid beetle Rhopaea 
verreauxi lives longer (924 days) than P. pictus and attains a biomass of 3-3 g, it 
allocates 4-8% of the ingested energy to egg production. It does not feed as adult but 
converts most part of the energy accumulated during the larval period into egg. On 
the other hand, P. pictus allocates a part of the ingested energy during nymphal 
period to somatic growth and depends mostly on the energy ingested during the 
adult period for maintenance and egg production. The strategy of the moth 
Cyclophragma leucosticta is similar to that of R. verreauxi. Despite growing as large 
as 5-1 g and passing through a larval period of 107 days, C. leucosticta allocates 3-8% 
of the ingested energy to egg production. Bombyx mori spins an expensive pupal case 
and yet manages to spare 5-8% of the ingested energy for egg production. 
Granivorous beetles like Sitophilus granarius, O. surinamensis and Cryptolestes 
ferrugineus adopt a different strategy. They resort to shorten the larval period 
susceptible to parasitic infection and prolong the adult period to acquire sufficient 
energy and nutrients and maximise egg production. Shortening the larval period to 4 
days and decreasing the adult biomass to 13-9 mg have helped the aphidophagous 



Relation between feeding and egg production in some insects 175 

Jadybird beetles C. transversalis and M. sexmaculatus to allocate as much as 29-0 and 
22-7% of the ingested energy to egg production. Ingestion of amino acid-rich phloem 
sap enables A,fabae to enhance its egg production efficiency to 41-9%. 

4. Effect of quantity of food 

In most hemimetabolous insects, quantity of food ingested during the adult period 
plays a major role in reproduction and hence restriction of adult feeding is likely to 
affect egg production efficiency. Table 2 provides data on food consumption and egg 
production by the milkweed bug Oncopeltus fasciatus receiving 100, 50 and 25 mg 
dry seeds/week/pair (Slansky 1980). A female in 100 mg ration group passed through 
interoviposition periods of 2-3 days and oviposited 1217 eggs weighing 119mg in 
total. Owing to the extension of the interoviposition period to 4-7 and 10-0 days and 
the consequent increase in the metabolic cost of maintenance, egg production 
efficiency of females in the 50 and 25 mg ration groups decreased to 21-1% and 
7-7%, respectively. Corpora allata of females receiving restricted rations are also 
likely to have not been sufficiently activated resulting in decreased fecundity (Ralph 
1976). Restriction of ration may also directly interfere with egg production and 
decrease fecundity (Walker 1976). Mathavan (personal communication) has obtained 
a highly significant correlation between feeding rate and fecundity on the one hand 
and faeces egested and fecundity on the other for the silkworm B. mori. The simple 
linear regression equations especially the one between faeces egested and fecundity 
developed may be useful to predict fecundity of B. mori (figure 1). 

Discussing stability and resilience in predator prey models, Beddington (1976a, 
b) showed that prior to commencement of oviposition, a predator should consume 
sufficient energy to meet its metabolic demand; once this demand is met, fecundity of 
the predator holds a definite relation to prey ingested by it. For instance, Coccinella 
undecempunctata aegyptiaca consumes about 25 aphids/day before commencing 
oviposition; thereafter, with increasing prey consumption to 40 and 75 aphids/day, 
fecundity increases to 60 and 160 eggs/female (table 3). Dixon (1959) has also 
reported similar relation for the beetle Adalia decempunctata (table 3). 

5. Effect of food quality 

In addition to differences in chemical composition, natural food materials differ in 
their capacity as phagostimulants. Consequently, quantities of food consumed, 

Table 2. Effect of ration levels on food consumption and egg production in O. fasciatus 
fed on air-dried seeds of Asclepia syriaca for a period of 13 weeks after eclosion (from 
Slansky 1980; modified). 

Ration (mg/seed/week/$ $) 



Parameters 


100 


50 


25 


Consumption (mg dry wt/$ $) 
Inter-oviposition period (day) 
Fecundity (egg/female) 
Biomass of eggs produced (mg dry wt/$) 
Egg production efficiency (%) 


422-4 
2-3 
1217 
119-0 
27-9 


268-2 
4-7 
576 
59-4 
2M 


189-8 
10-0 
131 

14-6 

7.7 



176 



J Muthukrishnan and T J Pandian 



( ) Feeding rate(mg/g live larva/day) 
140 220 300 



600 - 



500 



o 

400 



30O 



o 
z 



200 



100 



Y= -230-4-i-2-609X 

r = 0-997 / 





/ YO8-734+0.205 
/ r = 0-943 



400 1200 2000 
(-_..) Faeces (mg dry) 



Figure 1. Fecundity of B. morl as functions of (A) feeding rate and (B) faeces egested 
(Mathavan, personal communication). 



Table 3. Fecundity in relation to prey density in 
C. u. aegyptiaca and A. decempunctata. 



C. u. aegyptiaca" 


A. decempunctata b 


Prey density 

(No./?) 


Fecundity 

(egg/?) 


Prey density 

(No./?) 


Fecundity 

(egg/?) 


30 

35 
40 
55 
60 

75 


10 
30 
60 
120 
130 
160 


3 
7 
15 
23 


3 
13 
18 
15 



"Data from Beddington et al (1976a). b Data from Dixon (1959). 

assimilated and converted by polyphagous insects vary with quality of food (Soo 
Hoo and Fraenkel 1966; Muthukrishnan and Rajeeya 1979). Such differences in food 
utilization account for the variations in the fecundity of a number of polyphagous 
insects. For instance, fed ad libitum on Solarium tuberosum and Mamestra configurata 
consumed 388 mg (dry) of food and produced 279 eggs after emergence (Bailey 1976). 
Feeding on Brassica campestris enhanced its consumption to 498 mg and fecundity 
to 756 eggs (table 4). Young et al (1950), East (1977) and Ottens and Tood (1979) 
have reported wide variations in the fecundity of whitefringed beetles Graphognathus 
spp. fed on leguminous and graminaceous plants. Fecundity of the beetle varied from 
4-26 eggs/female while feeding one or the other of 12 different species of grass to 
153-631 or 1450 eggs/female for those feeding leguminous or malvaceae plants 



Relation between feeding and egg production in some insects 



111 



Table 4. Effect of food quality on food consumption and 
fecundity of M. configurata (from Bailey 1976; recalculated). 



Host plant 


Food consumption 
(mg dry wt/larva) 


Fecundity 

(egg/?) 


S. tuberosum 


388 


279 


Cheuopidium album 


448 


411 


B. campestris 


498 


756 


Brassica napus 


494 


838 , 


Artificial diet 





1367 



80 



70 



40 



20 



1 



30 



I 

D 

z 



20 



10 




Y= 6-8 +23 -885 
r=0.998 



R. prolixus 



* -4-48 +0-12 X 
r= 0-69 



T- niqrovittotus _ 



Y = 46-2+3-972 X 
r=0-85 



20 



40 



6O 



Tr. infestans 




' Y=~33-35+0-0886X 
r= 0-938 



J 1 I I I 



200 400 900 1300 
Blood meal { mg /female) 



275 
225 

175 

125 
1OO 

SO 
60 



- 4O 



I700 



Figure 2. Fecundity of some sanguivorous insects as a function of blood meal consumed. 
(A) A. aegypti (Roy 1936); (B) T. nigrovittatus (Magnarelli and Stoffolano 1980); (C) 
R. prolixius (Patterson 1979); (D) T. infestans (Regis 1979). 



(Ottens and Tood 1979). However, food consumption in the beetles feeding. -on- 
different host plants was not estimated by the authors and hence fecundity could not 
be related with quantity of food consumed. 



6. Autogeny 

By feeding the stable fly Stomaxys calcitrans on a blood meal mixed with soybean 
trypsin inhibitor, Spates (1979) demonstrated the importance of protein for egg 
production in sanguivores. However, arctic mosquitoes such as Aedes impiger and 
A. nigriceps as well as A. detritus inhabiting the deserts of Sahara and Tunisia, where 



178 J Muthukrishnan and T J Pandian 

chances of finding mammalian host are remote are autogenous and produce eggs 
without having to consume a blood meal after emergence (Corbet 1964). Larval 
nutrition is one of the major factors controlling autogeny in insects (Friend et al 
1965). For instance, the bug Rhodnius prolixus fed on full blood meal at each 
nymphal instar oviposits 51 eggs after ingesting first adult blood meal compared 
with 18 eggs by that fed on partial meals during nymphal period (Patterson 1979). 
Fecundity of the bug Triatoma infestans ingesting 930-1510 mg of blood meal after 
eclosion varies from 50-95. On an average about 16-6 mg of blood is required for the 
production of an egg (Regis 1979). Magnarelli and Stoffolano (1980) fed Tabanus 
nigrovittatus on different quantities of blood (0-60 mg) and observed a linear relation 
between quantity of meal and fecundity. They have demonstrated that the 
autogenous and anautogenous fecundity of the fly do not differ significantly. Figure 2 
shows the relation between blood meal ingested and fecundity for two dipterous and 
two hemipterous sanguivores. The negative intercept on y of the regression lines for 
the hemipterous R. prolixus and T. infestans clearly indicates that blood 
consumption after eclosion is obligatory for the initiation of oviposition; on the other 
hand, the positive intercept for the dipterous A. aegypti and T. nigrovittatus indicates 
their relative independence on adult blood meal for oviposition of at least a few eggs. 

Acknowledgement 

Financial assistance to one of us (JM) by the University Grants Commission (F.23- 
62/83 SR II), New Delhi is gratefully acknowledged. 

References 

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Beddington J R, Free C A and Lawton J H 1976a Concepts of stability and resilence in predator-prey 

models; J. Anim. Ecol 45 791-816 
Beddington J R, Hasseil M P and Lawton J H' 1976b The components of arthropods predation. II 

Predator pate of increase; J. Anim. Ecol. 45 165-185 
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(Berlin) 55 62-68 

Calow P 1977 Ecology, evolution and energetics; a study in metabolic adaptations; Adv. Ecol. Res. 10 1-60 
Campbell A and Sinha R N 1978 Bioenergetics of granivorous beetles, Cryptolestes ferrugineus and 

Rhyzopertha dominica (Coleoptera: Cucujidae and Bostrichidae); Can. J. Zoo/. 56 624-633 
Campbell A, Singh N B and Sinha R N 1976 Bioenergetics of the granery weevil, Stiphilus granarius (L.) 

(Coleoptera: Curculionidae); Can. J. Zoo/. 54 786-798 
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maculatus Fab.\ Ph.D. thesis, Madurai Kamaraj University, Madurai 

Corbet P S 1964 Autogeny and oviposition in Arctic mosquitoes; Nature (London) 203 669-670 
Delvi M R 1972 Ecophysiological studies on chosen arthropods, Ph.D. thesis, Bangalore University, 

Bangalore 
Delvi M R and Pandian T J 1971 Ecophysiological studies on the utilization of food in the paddy field 

grasshopper Oxya velox; Oecologia (Berlin) 8 267-275 
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Adalia decempunctata (L.); J. Anim. Ecol. 28 259-281 
East R 1977 Effects of pasture and forage crop species on longevity, fecundity and oviposition rate of adult 

white fringed weevil Graphognathus leucoloma (Boheman); N.Z. J. Exp. Agric. 5 177-181 
Enders F 1976 Size, food finding and Dyar's constant; Environ. Entomol. 5 1-10 



Relation between feeding and egg production in some insects 179 

Friend W G, Choy C T H and Cartwright E 1965 The effect of nutrient intake on the development and the 

egg production of Rhodnius prolixus Stahl (Hemiptera: Reduvidae); Can. J. Zooi 43 891-904 
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Lawton J H and McNeill S 1979 Between the devil and the deep blue sea: on the problem of being a 

herbivore; in Population dynamics (eds) R M Anderson, B D Turner and L R Taylor (Oxford: 

Blackwell) pp 223-244 
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(Berlin) 32 367-376 
Magnarelli L A and Stoffolano J G 1980 Blood feeding, oogenesis and oviposition by Tabanus 

nigrovittatus in the laboratory; Ann. Entomol. Soc. Am. 73 14-17 
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University, Madurai 
Muthukrishnan J and Rajeeya A M 1979 Effect of food quality on food utilization in Periplaneta 

amcricana\ Indian J. Anim. Res. 13 98-102 
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whitefringed beetle; Ann. Entomol. Soc. Am. 72 837-839 
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25311-314 
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175 
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Kamaraj University, Madurai 
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parameters; Am. Nat. 108 791-804 
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341-351 

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Entomol. 5 599-603 
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 181-184. 
Printed in India. 



Nutrient modulation of hormone production: Dietary essential fatty 
acids, tissue prostaglandins and their probable role in insect reproduction 

V K K PRABHU and MARIAMMA JACOB 

Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695 581, India 

Abstract. The essential polyunsaturated fatty acids required by insects in their food appear 
to be needed for prostaglandinogenesis. Prostaglandins themselves are likely to be widely 
distributed in insects playing perhaps an hitherto unsuspected important role as in 
reproduction. 

Keywords. Polyunsaturated fatty acids; arachidonic acid; prostagladins; reproduction; 
insects. 



Hobson (1935) found that cholesterol was an essential nutrient for blowfly 
larvae and Fraenkel and Blewett found that the unsaturated fatty acid linoleic acid 
was required for proper development and reproduction in many insects (Fraenkel 
and Blewett 1945, 1946). They also found that arachidonic acid played an important 
role in the metabolism of insects (Fraenkel and Blewett 1947). Linolenic acid was 
found to be as effective as linoleic acid, but oleate was ineffective in many insects as 
in Ephestia kuhniella, but in others as in Tenebrio, linoleate was not required. 
Apparently, linoleate or linolenate was synthesized by the animal or the symbionts 
harboured by the insect. This work was followed by a spate of information stressing 
the requirement of various long chain poly unsaturated fatty acids in the diet of 
various insects for their normal physiological maintenance. From the vast literature 
which has accumulated, it was clear that insects, as mammals, cannot synthesize 
linoleate or linolenate from simpler precursors. However, their functional role in 
normal growth and reproduction remained largely unknown. Chippendale's work on 
Trichoplusia ni (Chippendale et al 1964) showed the necessity of using pure samples 
for nutritional studies as contaminants had nutritional value. They found that 
linolenate was an essential nutrient, which could not be replaced by linoleate. It is 
now well-established that sterols are indispensible nutrients for all insects, and also 
require dietary polyunsaturated fatty acids wherever they have been studied critically 
(Chippendale 1972; Dadd 1973; Downer 1978). 

Sterols are known to function as structural components of insect membranes, but 
perhaps a more important role they play is as precursors of the moulting hormone 
ecdysone (Clayton 1964) and this function of dietary sterol requirement in insects is 
now well established. However, functional significance of the requirement of 
very small amount of essential poly unsaturated fatty acids is not yet well 
understood. As the quantity of these substances required by insects is extremely 
small they do not appear to be linked with normal energy metabolism. 

In the cabbage looper T. ni, nutritional studies have shown that either linoleate or 
linolenate promoted larval growth, linolenate was essential for normal wing 
development (Chippendale et al 1964). Since linoleate and linolenate were 
preferentially incorporated into phospholipids, a structural role for them in 
membrane phospholipids was postulated (Grau and Terriere 1971). Dietary 



182 V K K Prabhu and Mariamma Jacob 

requirement of poly unsaturated fatty acids for successful pupal eclosion, wing 
expansion, larval growth and reproductive capacity has been detected in many 
groups of insects (Dadd 1973, 1985; House 1974; Downer 1978). It is difficult to 
completely eliminate trace amounts of these substances from prepared diets and 
hence some of the earlier claims of nonessential nature of poly unsaturated fatty 
acids would require confirmation. These substances may also pass on from mother to 
offspring; the micro-organisms in the insects may also be involved in the synthesis of 
these compounds. It is increasingly becoming clear that arachidonic acid, the poly 
unsaturated fatty acid precursor of prostaglandins in vertebrates, is required for 
normal growth and development and accelerated larval growth in Trogoderma 
granarium (Pant and Pant 1961) and is essential for emergence of viable mosquito 
adults (Dadd and Kleinjan 1978) and for eclosion and flight activity of many species 
of mosquitoes (Dadd and Kleinjan 1979; Dadd 1985). 

In vertebrates arachidonic acid and the related C20 polyunsaturates, homo-y- 
linolenic and eicosapentanoic acids are precursors of prostaglandins and local hormone- 
like substances. Several long chain poly unsaturates including arachidonic acid, have 
also been detected in many species of insects recently (Dadd 1981). Prostaglandins 
are hormone-like ubiquitous substances whose synthesis and functions are well 
established in vertebrates (von Euler and Eliasson 1967). Recent detection of 
prostaglandins and prostagladin synthetase in insects (Destephano and Brady 1977) 
in the cricket Acheta domesticus and further work on this insect points to the 
likelihood of the important role these compounds might be playing in insect 
reproduction. Prostaglandins have also been detected in the cricket Teleogryllus 
commodus (Loher et al 1981), Bombyx mori (Setty and Ramaiah 1979) and in Musca 
domestica (Wakayama et al 1980). In all these species, the compound has been 
found to occur primarily in reproductive tissues though it is also found in other 
tissues (Murtaugh and Denlinger 1982). 

Prostaglandin synthetase was found for the first time in insects in the 12100 g 
pellet of reproductive tract homogenate of the male cricket A. domesticus 
(Destephano et al 1976). The supernatant and microsomal fractions were found to be 
inactive. Subsequent studies on this animal showed that this enzyme was localised in 
the testes, seminal vesicles and spermatophores (Destephano and Brady 1977). Non- 
steroidal anti-inflammatory drugs which are known to inhibit enzymes of the 
prostaglandin synthetase complex (indomethacin, phenyl butazone and 
acetaminophen), appeared to inhibit prostaglandinogenesis from arachidonic acid in 
Culex pipiens when the larvae were reared in media containing the inhibitors and 
arachidonic acid (Dadd and Kleinjan 1984). In Acheta domesticus the enzyme was 
absent in bursa copulatrix, spermatheca, spermathecal canal and oviducts from 
virgin females, but in mated females substantial enzyme activity was detected in these 
organs. Destephano and Brady (1977) concluded that the female cricket received the 
enzyme at the time of mating through spermatophore. PGE 2 -like material was found 
in this animal in the testis and to a lesser extent in the remainder of the male 
reproductive tract, but was not present in the virgin female reproductive tissues. 
However, on mating, the reproductive tissues acquired PGE 2 -like material. Injection 
of PGE 1? PGE 2 and PGF 2a stimulated oviposition by virgin females. They also 
observed that the PG synthetase inhibitor N-acetyl-p-aminophenol suppressed 
oviposition in mated females. The authors concluded that post-copulatory PG 
biosynthesis in the female reproductive tract might be partially responsible for 



Nutrient modulation of hormone production 183 

triggering oviposition in this animal. These observations of Destephano and Brady 
(1977) have been confirmed essentially by Loher et al (1981) in Teleogryllus 
commodus. In addition they found 7 polyunsaturated fatty acids in the spermatheca 
of virgin female which included 2-2% (of the total fatty acids) of arachidonic acid, the 
precursor of PGE 2 , and to a lesser amount precursors of PGEi and PGE 2 and very 
high concentration of linolenic acid (36-9%). In vitro incubation studies showed that 
the spermatophore and the female's spermatheca contents incubated with labelled 
arachidonic acid as substrate, produced PGE 2 and PGF 2a , the former about twice 
the rate of the latter. They also found that nanogram quantities of PGE 2 injected 
into the oviduct stimulated mating-induced egg release. Setty and Ramaiah (1979) 
isolated PGE l5 PGE 2 and PGF la and PGF 2a from reproductive tissues including 
testes, vasa deferentia, seminal vesicles and ejaculatory ducts of the silk moth B. mori. 
Both crude and purified PGE and PGF a fractions were found to have biological 
activity when tested using guinea pig ileum preparation in vitro. As opposed to the 
above findings especially of Destephano and Brady (1977) and of Loher et al (1981) 
in crickets, though low levels of prostaglandin-like compounds were detected in 
reproductive tissues of the cabbage looper T. ni adult males and females which 
increased 3 folds (PGE 2 ) and 2 folds (PGF 2a ) in the female reproductive tissues after 
mating, injected PGE 1? PGE 2 and PGF 2a had no effect on either pheromone 
production, calling behaviour or oviposition in this animal. The cricket, 
prostaglandin synthetase inhibitor N-acetyl-p-aminophenol in the diet of adult 
females did not alter calling behaviour of virgin or mated female of this species, 
though in the larval diet the treatment lengthened larval stadia but ha4 little effect 
on reproductive activities (Hagan and Brady 1982). This could be bacause a 
prostaglandin different from that injected might be the active one in this species. It 
has to be borne in mind that prostaglandins are a large family of compounds 
differing in their action from species to species and from one tissue to another, 
sometimes with diametrically opposite effects; their action also depends often upon 
the physiological state of the organ and tissues. Indeed more extensive and detailed 
work is needed on insects to clarify the significance of prostaglandins in reproduction 
and perhaps on other activities of insects. 

References 

Chippendale G M 1972 Insect metabolism of dietary sterols and essential fatty acids; in Insect and mite 

nutrition (ed) J G Rodriguez (Amsterdam, London: North Holland Publishing Company) pp 423-435 
Chippendale G M, Beck S D and Strong F M 1964 Methyl linolenate as an essential nutrient for the 

cabbage looper, Trichoplusia ni (Hubner); Nature (London) 204 710-711 
Clayton R B 1964 The utilization of sterols by insects; J. Lipid Res. 5 3-19 
Dadd R H 1973 Insect nutrition: Current developments and metabolic implications; Annu. Rev. Entomol 

18381^20 
Dadd R H 1981 Essential fatty acids for mosquitoes, other insects and vertebrates; in Current topics in 

insect endocrinology and nutrition (eds) G Bhaskaran, S Friedman and J G Rodriguez (New York; 

Plenum Press) pp 189-214 
Dadd R H 1985 Nutrition: Organisms; in Comprehensive Insect Physiology, Biochemistry and 

Pharmacology: Regulation: Digestion, Nutrition, Excretion (eds) G A Kerkut and L I Gilbert (Oxford, 

New York, Toronto, Sydney, Paris, Frankfurt: Academic Press) Vol. 4 pp 313-390 
Dadd R H and Kleinjan J E 1978 An essential nutrient for the mosquito Culex pipiens associated with 

certain animal-derived phospholipids; Ann. Entomol. Soc. Am. 71 794-800 
Dadd R H and Kleinjan J E 1979 Essential fatty acid for the mosquito Culex pipiens: arachidonic acid; J. 

Insect Physiol. 25 495-502 



184 V K K Prabhu and Mariamma Jacob 

Dadd R H and Kleinjan J E 1984 Prostaglandin synthetase inhibitors modulate the effect of essential 

dietary arachidonic acid in the mosquito Culex pipiens; J. Insect PhysioL 30 721-728 
Destephano D B and Brady U E 1977 Prostaglandin and prostaglandin synthetase in the cricket Acheta 

domesticus; J. Insect PhysioL 23 905-911 
Destephano D B, Brady U E and Woodall L B 1976 Partial characterization of prostaglandin synthetase 

in the reproductive tract of the male house cricket Acheta domesticus; Prostaglandins 11 261-273 
Downer R G H 1978 Functional role of lipids in insects; in Biochemistry of insects (ed) M Rockstein 

(New York, San Francisco, London: Academic Press) pp 58-92 
Fraenkel G and Blewett M 1945 Linoleic acid, a-tocopherol and other fat soluble substances as 

nutritional factors for insects; Nature (London) 155 392 
Fraenkel G and Blewett M 1946 Linoleic acid, vitamin E and other fat soluble substances in the nutrition 

of certain insects, Ephestia kuhniella, E. elutella, E. cautella and Plodia interpunctella; J. Exp. Biol 22 

172-190 
Fraenkel G and Blewett M 1947 Linoleic acid and arachidonic acid in the metabolism of the insects, 

Ephestia kuhniella and Tenebrio molitor; Biochem. J. 41 475-478 
Grau P A and Terriere L C 1971 Fatty acid profile of the cabbage looper, Trichoplusia ni, and the effect of 

diet and rearing conditions; J. Insect PhysioL 17 1637-1649 
Hagan D V and Brady U E 1982 Prostaglandins in the Cabbage looper, Trichoplusia ni', J. Insect PhysioL 

28 761-765 

Hobson R P 1935 On a fat soluble growth factor with cholesterol; Biochem. J. 29 2023-2026 
House H L 1974 Nutrition; in The Physiology oflnsecta (ed) M Rockstein (New York, London: Academic 

Press) Vol. 5 pp 1-62 
Loher W, Ganjian I, Kubo I, Stanley Samuelson D and Tobe S S 1981 Prostaglandins: their role in egg 

laying of the cricket Teleogryllus commodus; Proc. Watl. Acad. Sci. 78 7835-7838 
Murtaugh M P and Denlinger D L 1982 Prostaglandins E and F 2a in the house cricket and other species; 

Insect Biochem. 12 599-603 
Pant N C and Pant J C 1961 Nutritional studies of Trogoderma granarium. V. Studies on lipid 

requirements; Indian J. Entomol. 14 71 
Setty B N Y and Ramaiah T 1979 Isolation and identification of prostaglandins from the reproductive 

organs of male silk moth Bombyx won; Insect Biochem. 9 613-617 

von Euler U S and Eliasson R 1967 Prostaglandins (New York, London: Academic Press) p 164 
Wakayama E J, Dillwith J W and Blomquist G J 1980 In vitro biosynthesis of prostaglandins in the 

reproductive tissues of the male house fly Musca domestica; Am. Zool. 29 904 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 185-193. 
Printed in India. 



Assessment of neuroendocrine mechanisms of feeding and reproduction 
in phytophagous insects 

D MURALEEDHARAN 

Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695 581, India 

Abstract. In acridids during feeding, the central nervous system receives information 
through sensilla which in turn integrated incoming information giving the appropriate 
motor output. Hormones and haemolyph factors like osmotic pressure also limit meal size. 
In the red cotton bug, Dysdercus cingulatus hormones from the nqurosecretory cells of the 
brain induced feeding and the protein food ingested in turn stimulated midgut enzyme 
production while juvenile hormone has little influence either on feeding or enzyme 
production. In lepidopterans both brain hormone and juvenile hormone seemed to 
stimulate feeding and the amount of food ingested induced enzyme production. Midgut 
endocrine cells have also been demonstrated in several lepidopteran species. 

Reproduction involving vitellogenesis, spermatogenesis, oviposition, accessory glands 
activity and mating behaviour is also under the regulatory control of several hormonal 
principles such as juvenile hormone, ecdysone, oostatic hormone etc. However, a detailed 
.analysis of the literature available on the topic reveals that these regulatory mechanisms are 
tissue and species dependent. 

Keywords. Feeding; reproduction; neuroendocrines; vitellogenesis; digestion. 

1. Introduction 

The fundamental nutritional endeavour of any organism is to enumerate their food 
substances to the simplest utilisable chemical form, to distinguish those which are 
essential rather than merely usable and to determine the relative amount of each 
substance that are optimally needed. In that perspective, different physiological 
processes like feeding, digestion and absorption constitute their own specific roles in 
preparing the insect finally for meeting their needs for energy and progeny production. 
Various aspects of insect feeding have been discussed in detail in some of the recent 
reviews (Gelperin 1971; Truman and Riddiford 1974; Barton Browne 1975; Bernays 
1985). The regulation of feeding is part of a larger mechanism for metabolic 
homeostasis. In addition to classical hormones as brain hormone (BH), juvenile 
hormone (JH) and ecdysone, several of the other endocrine principles are also found 
to influence many of the physiological processes related to feeding, nutrition and 
reproduction in insects. Most of the insects continue to feed throughout their life 
thus supplying the essential materials for body maintenance, growth and repro- 
duction. Reproduction is the culmination of so many of the sequential processes 
such as sexual differentiation, gametogenesis, mating, oviposition etc which are again 
under the influence of different hormones. Many of the recent reviews have high- 
lighted these aspects in sufficient detail (Engelmann 1970; de Wilde and de Loof 
1973; Raabe 1982; Davey 1984; Koeppe et al 1985; Hagedorn 1985). 

2. Feeding and digestion 

2.1 Acridids 

Sectioning of the posterior pharyngeal nerves in Locusta migratoria in front 



186 D Muraleedharan 

of the foregut region resulted in hyperphagia while sectioning other nerves to 
the foregut did not show this effect (Bernays and Chapman 1973). During feeding, the 
central nervous system (CNS) of these insects receives information from a large 
number of sensilla each of which contain numerous physiologically different receptor 
cells. CNS integrates the incoming information with input from other sensory system 
to give the appropriate motor output. The pharyngeal nerves supply, the anterior 
part of the foregut, the last part to be filled during a meal. Here there is a network of 
nerves containing about 10 large cells probably being the perikarya of stretch 
receptors and the input from these cells indicating complete fullness of the foregut 
which is the signal for cessation of feeding (Bernays 1985). Haemolymph factors may 
also limit meal size. If the osmotic pressure in the haemolymph was elevated without 
volume change about 20 min before feeding, meal size in Locusta larvae reduced 
substantially suggesting a control by osmotic pressure on feeding (Bernays and 
Chapman 1974). However, an alteration of nutrient concentration and osmotic 
pressure during the course of a meal through an implanted cannula had no effect on 
the size of that meal. An extract of corpora cardiaca (CC) also did not have any effect 
on the meal size. Further, crop stretching switches off feeding and results in the release 
of one or more hormones from the storage lobes of CC. One such hormone is the 
diuretic hormone influencing the absorption of water from the food thus increasing 
the haemolymph volume (Mordue 1969). A hormonal principle also causes the pores 
on the terminal sensilla of the palps to close so that the sensilla are no longer 
functional. This effect as measured by the changes in electrical resistance across the 
tips of the palps persists for at least an hour in locusts which have had a large meal 
following a period of deprivation. Two hours later, the sensilla are again fully 
functional and injections of CC storage lobe extract showed that the change is 
concentration dependent (Bernays and Mordue 1973). The hormonal principle from 
the CC storage lobes enhanced the movements of the gut as well as in locusts (Cazal 
1969). 

In Melanoplus sanguinipes, a massive release of neurosecretion from brain median 
neurosecretory cells (MNC) and CC occurs within 20-30 min of feeding (Dogra and 
Gillott 1971). Release of neurosecretion is initiated by stretch receptors on the fore- 
gut so that frontal ganglionectomy or certain other operations on the stomodaeal 
nervous system prevents it since the operation results in an interference with a large 
number of sensory, motor and relay pathways. The movement of food back from the 
buccal cavity is reduced and it also prevents the forward flow of enzymes from the 
midgut (Anstee and Charnley 1977). Estimations of dry food eaten by Locusta show 
that the amount consumed by allatectomised males does not differ significantly from 
that consumed by their controls (Strong 1975). Also implantation of additional 
corpora allata (CA) in the larvae of L. ?nigratoria did not alter food consumption 
(Beenakkers and Van Den Broek 1974). Hence JH seems to have very little 
regulatory influence on feeding activity in the Orthopterans. 

But midgut enzyme activity is clearly related to food intake in almost all the 
orthopterans studied and Dogra (1974) in the light of available literature concludes 
that in this group of insects enzyme production is regulated by a neurosecretion 
released from brain as a result of crop filling or foregut activity. The timing of release 
of neurosecretion in Melanoplus is such that it could regulate protease activity 
increase. According to Anstee and Charnley (1977) the enzyme production in locusts 
is regulated by the total volume of food ingested acting via the neurosecretory 
system. Cauterisation of the MNC of brain in the adult Gomphocerus rufus brought 



Assessment of neuroendocrine mechanisms in phytophagous insects 187 

down the protease level significantly (Loher 1965). Release of neurosecretion is 
initiated by stretch receptors on the foregut so that frontal ganglionectomy or certain 
other operations on the stomodaeal nervous system prevents it (Clark and Langley 
1963) and the gut carbohydrase activity is also reduced (Bernays and Chapman 
1973). Neither ovariectomy nor allatectomy affected protease activity in 
grasshoppers (Dogra 1974) and even in the starved insects a residual level of enzyme 
activity was detected. Khan (1964) observed an increase in enzyme activity in locusts 
after moulting and hence some enzyme activity is assumed even in the absence of 
feeding. Neck ligature performed within 24 h of moulting also reduced invertase 
activity in the insects even though injection of blood from fed donors did not elevate 
the enzyme activity in starved recipients. 



2.2 Red cotton bug 

We in our laboratory have tried to delineate the endocrine mechanisms on feeding 
and digestion in the female red cotton bug, Dysdercus cingulatus. Extirpation of 
MNC soon after their emergence retarded feeding activity while implantation of 
active MNC from other donors restored this effect to a substantial level 
(Muraleedharan and Prabhu 1979a). Hence the neurosecretory principles from the 
brain seem to stimulate food consumption in this bug. But JH was found to have 
no significant influence on food consumption in this insect (Muraleedharan and 
Prabhu 1981). Also ovariectomy in these insects resulted in hyperphagia and 
hyperactivity of median neurosecretory cells in the brain. Also injection of methanol 
extract of active ovaries retarded rate of feeding in ovariectomised Dysdercus which 
tempted us to conclude that in this insect some ovarian factor (probably ecdysone) 
inhibited the activity of MNC resulting in the reduced feeding rate (Muraleedharan 
1984). Midgut protease and invertase activities were also very low in MNC-ablated 
cotton bugs while implantation of MNC induced enzyme production. Since casein 
was found to stimulate protease activity (Muraleedharan and Prabhu 1978) and 
sucrose fed animals showed only residual levels of protease and invertase activities 
(Muraleedharan and Prabhu 1979b) it became also apparent that in D. cingulatus 
ingested food especially proteins stimulated midgut enzyme production through a 
secretogogue mechanism (figure 1). 




FOOD CONSUMPTION 
(Protein?) 

Enzyme production 
Figure 1. Probable regulatory mechanism of feeding and digestion in D. cingulatus. 



188 D Muraleedharan 

2.3 Lepidopterans 

Our own studies in the late instar caterpillars of the teak defoliator, Hyblaea puera 
demonstrated that implantation of fresh and active brain to raise the insect's own 
neurosecretion stimulated feeding. Additional supply of JH (ZR515-JHa) through 
food. also induced feeding (Muraleedharan and Prabhu 1981). Higher levels of 
midgut amylase activity also were noticed in both the above experimental groups of 
caterpillars. Hence it seems that in this insect both MNC-hormone and JH induced 
feeding and the food ingested in turn must have stimulated amylase production via a 
secretogogue mechanism. However, these findings do not confirm whether the 
MNC- hormone stimulated feeding directly or through its trophic action on corpora 
allata since MNC is known to secrete an allatotropic hormone. In the larvae of the 
common cutworm Spodoptera litura also JH seem to stimulate feeding since Tojo et 
al (1985) demonstrated that application of both JHI and JHII increased feeding 
activity while allatectomy decreased the duration of final feeding period in final 
instar larvae. In Bombyx mori also Calvez (1981) demonstrated the involvement of 
JH in both obligatory and facultative feeding period. Caterpillars of Achoea Janata in 
which allatectomy has been performed, consumed very small amount of food 
compared to that consumed by their sham-operated controls and the operation 
disrupted the normal circadian feeding rhythm as well (George Abraham and 
Muraleedharan 1987). However, application of an anti-allatin, Precocene-II on the 
above larvae brought down food consumption even though with little effect on the 
feeding rhythm. Hence it is apparent that in A. Janata also JH stimulated feeding. In 
the same larvae treated with Precocene-II, midgut protease activity (Reema Mathews 
and Muraleedharan 1987) and midgut carbohydrases (Raman and Muraleedharan 
1987) were found to be reduced to a significant level while additional supply of JHa 
(ZR 515) by topical application elevated the enzyme levels. However, treatment of 
JHa and Precocene-II had no significant influence on the enzyme activity rhythms. 
In the tobacco horn worm, Manduca sexta larvae, starvation stimulated CA activity 
with the induction of supernumerary moult (Jones et al 1980) and according to 
Bhaskaran (1981) both CA and MNC are necessary for the induction of a 
supernumerary moult in this larva and he suggests that starvation activates brain to 
synthesise/or release more allatotropic hormone. 

Electron microscopic studies of the midgut region in 5 different species of 
lepidopterans, B. mori, Pieris rapae, Papilio xultus, Samia cynthia ricini, Lymantria 
dispar and Galleria mellonella have demonstrated the presence of gut endocrine cells 
with an abundance of neuroendocrine granules which are dispersed through out the 
epithelia of the larval, pupal and adult midgut (Endo and Nishiitsutsuji-Uwo 1981). 
The hormonal principles from these cells may have a stimulatory effect on enzyme 
secretion as is seen in vertebrates. 



3. Reproduction 

Raabe (1982) opined that JH and ecdysone act during certain phases of reproduction 
in insects, but in these cases as in all functions neurohormones play a pivotal role 
and are involved in all mechanisms related to reproduction. JH is found to be both 



Assessment of neuroendocrine mechanisms in phytophagous insects 189 

tissue and species specific in insects (Koeppe et al 1985) and in the majority of insects 
JH is the gonadotropin regulating vitellogenesis. In addition it has been found to be 
involved in other reproductive phenomena like early events in gonads, control of 
accessory glands, reproductive behaviour etc. 



3.1 Vitellogenesis 

There remains a group of observations indicating the involvement of neuroendocrine 
factors in the process of vitellogenesis in some of the phytophagous insects (Girardie 
1966; Minks 1967). McCafferey (1976) demonstrated that electrocoagulation of 
MNC from brain prevents vitellogenesis and implantation of CA into these operated 
insects did not result in the restoration of egg production completely even though 
a few follicle cells deposited yolk in locusts. Similarly in N. cinerea decapitation in 
females did not prevent egg production in the normal fashion if CA has been 
implanted into these (Luscher 1968). In D. cingulatus extirpation of MNC and their 
reimplantation showed that these cells stimulated vitellogenesis. But subsequent 
studies in allatectomised females appeared to demonstrate that CA also stimulated 
yolk deposition. Jalaja and Prabhu (1977) in the light of the above findings 
concluded that in this plant bug, CA-hormone (JH) stimulated vitellogenesis and the 
MNC had a trophic effect on CA activity thus indirectly stimulating vitellogenesis. 
CA have been found to be indispensable for yolk deposition in many other 
phytophagous insect species as well (Highnam et al 1963; Pener 1965; Strong 1965; 
Joly 1968). The most conclusive evidence that JH regulates vitellogenin synthesis 
came from the elegant in vitro demonstration in locusts by Wyatt et al (1976). They 
cultured fatbodies from allatectomised females in the medium containing hormonal 
analogues. Both CA and MNC from brain stimulated protein synthesis in the 
fatbodies of adult females of D. cingulatus (Raji Raghavan and Muraleedharan 1985). 
Extirpation of either the brain or CA from the female pupae of M. sexta hampered 
post emergence egg maturation in the emerging adults (Siew and Gilbert 1971). In 
fact vitellogenesis starts even at the pupal stages in most of the lepidopterans and 
allatectomy performed in pupae or adult stage does not affect the process in 
Hyalophora (Williams 1952) and in Philosamia (Ichikawa and Nishiitsutsuji-Uwo 
1959). The exact mechanism of action of brain hormone on vitellogenic process is 
still unknown. Some suggest that their action on yolk deposition is through protein 
metabolism. But in locusts the passage of certain haemolymph proteins into the 
oocytes is favoured by MNC while the passage of other proteins is favoured by CA. 
The ovaries and haemolymph of different kinds of reproductively competent adult 
females contain ecdysteroids. In Locusta ecdysone is being synthesised from 
cholesterol in the follicle cell sheath surrounding the terminal oocyte which is being 
controlled by a neurohormonal factor (Hoffmann et al 1980). In these insects 
ecdysone has been detected towards the end of egg maturation and just before 
oviposition and according to Hagedorn (1985) ovarian ecdyspne can play an 
important role in the control of insect vitellogenesis. In many of the haematophagous 
insects especially in mosquitoes ovary has been found to produce a releasing factor 
that permits egg development neurosecretory hormone (EDNH) to be released after 
a blood meal (Borovsky 1982; Lea and Van Handel 1982). 



190 D Muraleedharan 

3.2 Oostatic hormone 

In many of the insect species, ovary has been found to exert a feedback control th; 
results in an inhibition of vitellogenesis under certain conditions when ripe oocyt 
cannot be laid (Engelmann 1957; Nayar 1958; Adams et al 1968; Meola and Lea 197 
Huebner and Dayey 1973). Vitellogenesis inhibition is due to a substance comir 
from the ovary an anti-gonadotropin to act through pars intercerebral 
neurosecretory cells. 

3.3 Spermatogenesis 

Control mechanisms of Spermatogenesis in insects have not been well understoo 
However, it has been found that in silkworms especially in H. cecropia and in Sam 
cynthia Spermatogenesis is under the influence of ecdysone (Schmidt and Willian 
1953). The regulation of spermatogonial mitosis in Samia cynthia (Kambysellis ar 
Williams 1971) and in Locusta (Dumser 1980) is found to be under a stimulato: 
control of ecdysone. Also Ambika and Prabhu (1978) have demonstrated that J 
directly stimulated transformation of spermatocytes into spermatids and sperms. 

3.4 Oviposition 

In most cases oviposition in insects takes place after mating and the brain plays 
important co-ordinating role in this process. It has been found that the mat< 
females of B. mori contains a substance that induces oviposition in an unmat< 
female (Moika 1941). Also in the bug Iphita limbata (Nayar/1958) and in Schistocen 
gregaria (Okelo 1971) premature egg laying occurred when the haemolymph 
ovipositing females was injected into the females with nearly mature eg 
According to Raabe (1982) the oviposition factor has been present throughout tl 
CNS whose production and release are controlled by the cephalic nerve centres. Tl 
specific action of this factor has also been found to be on the genital valves. In tl 
grasshopper Melanoplus brain extracts stimulated egg laying (Friedal and Gillc 
1976). 

3.5 Accessory glands 

In most of the female orthopterans, a number of accessory sex glands and organs a 
associated with oocyte maturation, fertilization and encapsulation. These accesso 
glands include the left and right colletrial glands, the spermatheca, the vestibul 
organ, the dermal glands, the ovipositor, the valvula and the genital atrium. The on 
accessory sex gland known to be regulated by CA is the left colletrial gland and eve 
here the exact mechanism of its control is uncertain. In orthopterans, it has bet 
found that JH regulates either directly or indirectly by inducing protein synthesis 
by stimulating protocatechuic acid accumulation and calcium oxalate cryst 
formation (Raabe 1982). In acridids like Gomphocerus JH deprivation by allatecton 
prevented normal fluid accumulation in accessory glands (Loher 1965). Howevc 
very little information is available concerning the role of JH in the development 
male accessory glands. 



Assessment of neuroendocrine mechanisms in phytophagous insects 191 
3.6 Mating behaviour 

Several of the morphological and behavioural changes associated with the onset of 
mating have been found to be under the influence of JH. The experiments performed 
by Riddiford and Williams (1971) on the great saturniid moth, Anther ea poly f emus 
and on H . cecropia revealed the action of corpora cardiaca and corpora allata in the 
production of female pheromones and in calling behaviour. 

Even though a lot of information is available on the endocrine mechanisms 
operative in different species of insects on feeding and reproduction, a lot of gaps in 
respect of the exact mechanisms of action of various hormonal factors in the context 
of both the above processes need further ingenius and accurately designed 
experiments utilising the modern technological developments. Another important 
factor to be considered is that these regulatory mechanisms in different groups of 
insects seem to be different as evidenced by the available literature on the topic. 

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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 195-206. 
Printed in India. 



Nutritional value of malvaceoos seeds and related life-table analysis in 
terms of feeding and reproductive indices in the dusky cotton bug, 
Oxycarenus laetus Kirby (Hemiptera: Lygaeidae) 

K RAMAN 

Entomology Research Institute, Loyola College, Madras 600 034, India 

Abstract. Seed host diversity and subsequent impact of the nutritive value of different 
malvaceous host plant seeds determined the life-history consequences and the seed-use 
pattern of the dusky cotton bug, Oxycarenus laetus Kirby. Significant differences were 
observed in the feeding, food utilization and reproductive success of the bugs depending 
upon the type of seed food utilized as well as its biochemical profile particularly proteins, 
nitrogen, carbohydrates, phenols and lipids. Analysis of the different life-table statistics such 
as developmental time, maturation time, net reproductive rate (R ), and the intrinsic rate of 
increase (r m ) revealed seeds of crop plants to be better utilized when compared to 
malvaceous weed hosts. 

Keywords. Nutritional value of seeds; life-table analysis; feeding and reproductive indices; 
dusky cotton bug; Oxycarenus laetus. 



1. Introduction 

Feeding and reproduction in insects are very closely related to nutritional factors, the 
qualitative and quantitative aspects of which have an impact not only on fecundity, 
but also on the rates of growth and development. Johansson (1958) observed that the 
quality and quantity of food influence the pre-oviposition period and fecundity in 
Oncopeltus fasciatus (Dallas). Chaplin and Chaplin (1981) studied the growth of 
O. fasciatus on the seeds of Asclepias spp. and attributed that the seed nitrogen 
content and seed cardenolide content were the important parameters accounting for 
87-96% of the variability in growth dynamics of the bugs. The dusky cotton bug, 
Oxycarenus laetus Kirby is particularly associated with several malvaceous crop and 
weed seeds for its growth, development and reproduction (Thangavelu 1978). 
Observations by Ananthakrishnan et al (1982) showed that the diversity in different 
malvaceous seed hosts induced differential preference in 0. laetus with respect to 
orientation and seed use pattern. A correlation of the nutritional values of different 
malvaceous crop and weed hosts with the life-history traits of O. laetus involving 
feeding and reproductive indices, appear essential for a better understanding of the 
reproductive biology of these insects. 



2. Materials and methods 

Adults and nymphs of 0. laetus were maintained in the laboratory on various 
malvaceous host plant seeds. Fresh seeds were provided on every alternate day and 
moisture requirements were met by placing wads soaked in water inside the 
container. 



196 K Raman 

2.1 Growth, fecundity and food utilization studies 

The comparative rate of development, growth rate and fecundity rates of O. laetus on 
different malvaceous crop and weed hosts were computed as described by 
Ananthakrishnan et al (1982). To assess the impact of food on the development of 
ovaries, paired ovaries of females (3 days after eclosion) were dissected in insect 
ringer and the number of mature oocytes/ovary as well as the percentage ovarian 
indices (calculated as the ratio of the total ovariole length and length of the basal 
oocyte) were calculated. In addition, the quantity of different seed food utilized was 
also assessed based on the methods of Saxena (1963). 

2.2 Life-table analysis of O. laetus 

Laboratory cultures of O. laetus continuously reared on different malvaceous host 
plant seeds for atleast two generations were used for life-table parameter studies. All 
the experiments were carried out at 32-5C05, 85% RH and 13L:11D 
photoperiod. When nymphs reached the 5th instar stage, the cultures were 
monitored daily and newly eclosed adults were removed from the culture, paired 
with a member of the opposite sex and 25 such pairs were so monitored. Date of 
laying of each batch of eggs (clutch) and the number of eggs per clutch were recorded 
for each female. To determine the time of development from egg to mature adult, a 
sample of 50 eggs from each test population was monitored separately. Time of 
hatching and duration from eclosion to adult emergence were recorded which when 
summed up yielded the average interval from birth to eclosion. This value was 
added to the maturation time (eclosion to first oviposition interval) for each female 
to give the value of absolute age. The various population statistics followed in the 
present study was then computed as per the methods of Dingle and Baldwin (1983). 

2.3 Biochemical analysis 

The different malvaceous host plant seeds were analysed for various biochemical 
components. The total carbohydrates were estimated by the methods of Dubois et al 
(1956), proteins by Lowry et al (1951) and phenols by Bray and Thorpe (1954). The 
nitrogen was analysed by using Kjeldahl's apparatus following the procedure of 
Vogel (1963). For the estimation of total lipid content of seeds, 5 g of the material 
was ground and extracted in petroleum ether for 24 h using a Soxhlet apparatus. The 
total quantity of lipids was then estimated by gravimetric method. To the total lipids 
extracted from different seeds, 10 ml of ethanol, 3 ml of 28% NH 4 OH, 25 ml of 
petroleum ether and 25 ml of diethyl ether were added to a separating funnel, shaken 
for 5 min and allowed to stand for 20 min. After draining the bottom phase, the ether 
phase was dried to which 3 ml of 0-5 N NaOH in methanol was added and heated in 
a steam bath for 15 min. To this 5 ml of water was added followed by 2 N HC1. After 
dissolving the material, the pH was adjusted to 2. The fatty acid methyl esters were 
then extracted into 5 ml of petroleum ether and 5 ml of diethyl ether from the 
acidified methylated lipid extract. Fatty acid profiles were then analysed using a 
Hewlett Packard HPLC system fitted with a Hypersil ODS 5 um column with water 
and acetonitrile as mobile phase at a flow rate of 045 um as per the gradient 



Feeding and reproductive indices in O. laetus 



197 



programme of Schuster (1985). The different fatty acids were identified according to 
their retention time which was detected at two wavelengths (210 and 230 nm). 

3. Results 

3.1 Comparative growth rate, development and longevity of O. laetus 

Observations on the comparative growth rate, duration of post-embryonic 
development and longevity of O. laetus showed significant variations when fed with 
the seeds of different malvaceous host plants (table 1). The growth rate (GR) was 
maximum (29-75) on Gossypium hirsutum and was also significantly different from 
other seed diets. The GR for Hibiscus sabdariffa var albus (26-32) was on par with H. 
s. var rubber (25-38). Similarly, there were no significant differences in the GR among 
Abelmoschus esculentus, Abutilon crispum and A. indicum. Data on the time taken for 
post-embryonic development indicated that on G. hirsutum, O. laetus took only 21 
days to complete the life-cycle as against other hosts, wherein a longer time was 
required. The longevity of adults also varied significantly when fed with the seeds of 
various malvaceous host plants, seeds of crop plants enabled the bugs to live longer 
compared to those of weed hosts. Females and males maintained on G. hirsutum 
lived for 48-50 and 26-45 days respectively. These observations revealed that seeds of 
malvaceous crop plants were superior compared to those of weed hosts in supporting 
better growth and development of O. laetus. 

3.2 Food utilization pattern of 0. laetus 

The food utilization patterns of O. laetus on different malvaceous seed hosts are 
furnished in table 2. Computation of dry weight of food ingested and utilized by the 



Table 1. Comparative growth index, duration of post-embryonic development and 
longevity of O. laetus on different malvaceous host plants. 



Host plants 


Growth rate 


Duration of 
post-embryonic 
development (days) 


Mean longevity (days) 


Males 


Females 


Crops 










G. hirsutum 


29-75 a 


21-00 a 


26-45 a 


48-50 a 


H. s. var albus 


26-32 b 


21-47 a 


23-15 b 


42-40 b 


H. 5. var rubber 


25-38 b 


22-00 b 


23-00 b 


42-10 b 


A. esculentus 


19-75c 


22-12 b 


23-00 b 


40-10 b 


Weeds 










A. crispum 


19-23 c 


22-40 b 


22-75 b 


38-00 c 


A. indicum 


18-20c 


23-60 c 


22-75 c 


37-75 c 


H. vitifolius 


16-72 d 


23-60 c 


21-00 c 


32-50 d 


S. rhombifolia 


16-30d 


23-60 c 


20-00 c 


32-00 d 


S. rhomboidea 


14-85 d 


24-20 d 


19-OOc 


32-00 d 


H. panduraeformis 


14-25 d 


24-54 d 


19-OOc 


31-50 d 



Mature dry seeds were provided as diets. Values represent mean of 5 replicates. 
GR: Number of 5th instars moulting into adults and duration for moulting. 
Means followed by a common letter are not significantly different at 1% (DMRT). 



198 K Raman 



Table 2. Food utilization of O. laetus on certain malvaceous host plants. 



Host plants 


Dry wt. of food 
ingested (mg/ 
insect/day) 


Dry wt. of 

excreta (mg/ 
insect/day) 


Dry wt. of 
food utilized 
(mg/insect/day) 


Proportion 
of food 
utilized 


G. hirsutwn 


*2-0530-ll 


0-274 0-041 


1-7790-391 


0-863 


H. s. var dibits 


1-832 0-041 


0-263 0-001 


1-5690-012 


0-856 


H. s. var rubber 


1-8300-140 


0-262 0-012 


1-568 0-001 


0-857 


A. esculentus 


1-406 0-032 


0-244 0-0 11 


1-161 0-011 


0-826 


A. crispum 
A. indicum 


1-403 0-0 11 
1-404 0-050 


0-242 0-101 
0-242 0-021 


1-161 0-101 
1-1620-011 


0-827 
0-828 


H. vitifolius 
S. rhombifolia 
S. rhomboidea 


1-2130-132 
0-932 0-020 
0-849 0-020 


0-241 0-0 11 
0-241 0-0 12 
0-21 9 0-021 


0-972 0-014 
0-691 0-01 3 
0-630 0-011 


0-801 
0-741 
0-742 


H. panduraeformis 


0-8370-110 


0-216 0-013 


0-621 0-112 


0-742 



*Represent MeanS.E. Dry mature seeds were provided as diets. 

5th instar of O. laetus revealed that it was highest for G. hirsutum (2-053 mg/day and 
1-779 mg/day respectively) as against other seed diets. Also, seeds of crop plants were 
better ingested and utilized by 0. laetus when compared to those of malvaceous weed 
hosts. The proportion of food utilized also exhibited a similar trend wherein crop 
plant seeds were much more preferred compared to weed hosts. These results 
confirm that seeds of crop plants were of better quality than those of weed hosts. 

3.3 Impact of seed diet on the fecundity of O. laetus 

The mean number of eggs laid by O. laetus when fed with the seeds of different host 
plants are indicated in figure 1. The fecundity was highest (3 1-50 eggs/female) when 
fed with G. hirsutum followed by 27-50, 26-35 and 25-00 eggs/female on H. s. var albus, 
H. s. var rubber and A. esculentus. Similarly, when G. hirsutum was the seed diet, the 
fecundity index was significant and maximum (10-45) compared to other seed diets. 
Computation of percentage ovariole index as well as the mean number of 
oocyte/ovary revealed that seeds of G. hirsutum to be the most preferred host. 

3.4 Life-table parameters of O. laetus 

The life-table and population consequences of 0. laetus on 3 seed hosts viz 
G. hirsutum, A. esculentus and A. crispum (weed) were studied. The total develop- 
mental time of O. laetus on these hosts was found to be 21, 22 and 23 days respectively. 



3.4a Survivorship: Figure 2 depicts survivorship of l x which is the proportion of indi- 
viduals alive at birth (age 0) and still alive at age x. When reared on G. hirsutum the 
mortality of females was least upto the 18th day of their eclosion beyond which the 
proportion of females surviving started decreasing gradually upto 38th day. Beyond 
the 40th day the proportion of females surviving declined rapidly and almost all the 
females died by the 50th day of their eclosion. On A. esculentus the mortality of 
females was least upto the 12th day of adult eclosion and rapidly increased 
thereafter. On the weed host, A. crispum the mortality started increasing even from 



Feeding and reproductive indices in O. laetus 



199 



80 



-40 




30 



oocyte 
CU Ovarian index 



Q Eggs/female 
Fecundity index 



16 



1-5 



VO. 



0-5 



GH HSA HSR AE AC A! HV SRF SRA HP 
Host plant 

Figure 1. Impact of seed diets on the fecundity and ovarian indices of O. laetus. 
(GH, G. hirsutum; AE, A. esculentus; HSA, H. s. var albus\ HSR, H. s. var rubber; HV, 
H. vitifolius; AC, A. crispum; AI, A. indicum; SRF, S. rhombifo\ia\ SRA, S. rhomboidea; HP, 
H. panduraeformis). 



1-0 - 



Z 0-5 




J_ 



25 
Age (days) 



50 



Figure 2. Survivorship curves of 0. laetus under the influence of different seed diets. 
(GH, G. hirsutum', AE, A. esculentus; AC, A. crispum). 



200 



K Raman 



the 10th day. However, on A. esculentus and A. crispum all the females died by the 
40th day of their eclosion. 

3.4b Reproduction: The time course of reproduction, as well as its onset, was 
markedly influenced by the type of host plant seeds provided for the female. The 
influence of type and quality of food on the egg pattern are summarized in figure 3 
which gives the egg curves produced with each type of diet. When fed with the seeds 
of G. hirsutum reproduction commenced earlier i.e. on the 8th day after adult 
eclosion, accelerated on the llth and 12th day, decreased on the 13th day and 
reached at its peak on the 16th day with maximum egg production on the 17th day. 
On analysing the egg curve for A. esculentus it was apparent that the onset of egg 
laying commenced only on the 9th day. Beyond the 30th day the number of 
eggs/female decreased considerably upto the 40th day, after which no individuals 
were alive. Rearing on the seeds of the weed host, A. crispum the egg laying started 
only on the 1 1th day of eclosion of female which gradually increased and beyond the 
18th day the number of eggs produced sharply declined. Thus, a comparative 
assessment of the egg curve indicated that the birth rate in female in terms of the 
number of eggs/female to be maximum for G. hirsutum compared to A. esculentus 
and A. crispum. 

3.4c Rate of increase (rj: The influence of different diets on reproduction was 



2 20 
a 



Gh 




^^ x-x-x4zx>o 



25 
Age (days) 



50 



Figure 3. Birth rate of O. laetus under the influence of different seed diets. 
(GH, G. hirsutum; AE, A. esculentus; AC, A. crispum). 



Feeding and reproductive indices in O. laetus 



201 



determined by computation of the population rate of increase. The r m measure 
denote the intrinsic rate of increase of population with a stable age distribution. 
Feeding with cotton seeds gave r m value of 0-1448 followed by 0-1365 and 0-1229 for 
A. esculentus and A. crispum (table 3). Similarly, the net reproductive rate (R ) 
representing the total female births were 640-14, 292-35 and 143-05 respectively on 
these host plants. The different life-table statistics computed for 0. laetus are 
represented in table 3. 

3.5 Nutritive value of malvaceous seeds 

The results of the various biochemical parameters analysed are indicated in table 4. 
Analysis of the total proteins revealed that it was maximum (236-80 mg/g) on 
G. hirsutum as against 1 14 and 107-50 mg/g obtained for H. s. var albus and H. s. var 
rubber. The total protein content of weed hosts were very low when compared to 
crop plants. The estimation of carbohydrates for different seed hosts did not reveal 
marked variations among different host plants. However, it was noted that seeds of 



Table 3. Life-table statistics of 0. laetus on malvaceous host plants. 



Parameters 


G. hirsutum 


A. esculentus 


A. crispum 


Developmental time 


2 1-00 days 


22-00 days 


23-00 days 


Maturation time 


7-00 days 


8-00 days 


10-00 days 


Time to death 


49-00 days 


39-00 days 


37-00 days 


Clutch size 


3-50 


2-70 


1-50 


Interclutch interval 


1-50 days 


2-00 days 


2-50 days 


Number of clutches 


4 1-00 days 


3 1-00 days 


28-00 days 


Age at first reproduction (a) 


28-00 days 


30-00 days 


33-00 days 


Mean number of eggs/female (m v ) 


31-50 


25-00 


21-00 


Net reproductive rate (RJ 


640-14 


292-35 


143-05 


Mean generation time (7) 


44-61 


41-59 


40-36 


Intrinsic rate of increase (/-,) 


0-1448 


0-1365 


0-1229 


Capacity for increase (/;) 


0-1657 


0-1384 


0-1296 


Cohort generation time ( 7',,) 


38-99 days 


41-03 


40-38 



Table 4. Chemical composition of different malvaceous host plant seeds. 



Host plants 


Proteins (mg/g 
dry wl.) 


Carbohy- 
drates (mg/g 
dry wl.) 


Phenols 
(mg/g dry 
wt.) 


Nitrogen 

(o) 


Lipids 
(%) C/N ratio 


G. hirsutiini 


236-80 0-1 3" 


12-00 0-59 


18-75 0-11 


8-73 0-63 


41-200-11 


1-37 


//. s. var alhus 


11 4-00 0-23 


15-00 0-10 


5-25 0-20 


5-140-10 


2 1-08 0-20 


2-92 


H. s. var rubber 


107-50 0-11 


18-00 0-23 


4-750-10 


5-360-15 


24-52 0-23 


3-36 


A. esculentus 


142-5()0-33 


16-00 0-10 


4-25 0-04 


5-62 0-22 


14-05(M1 


2-85 


H. ritifoHus 


6H)0010 


19-00 0-10 


1-75 0-20 


4-64 0-11 


4-50 0-20 


4-09 


A. crispum 


60-00 0-89 


2 1-00 0-1 3 


3-25 0-11 


4-94 0-56 


4-00 0-10 


4-25 


A. indicum 


52-80 0-1 1 


23-50 0-11 


3-05 0-03 


4-73 0-26 


4-1 5 0-03 


6-03 


S. rhombifolia 


49-80 1-41 


2HO0-13 


2-50 0-10 


4-36 0-03 


2-1 5 0-03 


5-16 


S. rhomhoideti 


33-60 0-33 


22-50 0-21 


2-75 0-1 3 


4-lQ0-ll 


2-100-13 


5-49 


H. ptnnlui'cieformis 


23-60 0-23 


20-00 0-1 2 


3-50 0-10 


3-05 0-47 


2-00 0-1 3 


6-58 



"Represent Mean of 3 replications SE. 



202 



K Raman 



G. hirsutum were low in total carbohydrate content as against other host plants. But 
the total nitrogen (8-73%) and phenolic (18-73 mg/g) contents were relatively high. 
Estimation of total lipids also indicated that it was higher for seeds of crop plants 
when compared to weed hosts. The carbohydrate/nitrogen ratio, a measure to 
indicate the degree of susceptibility of host plants to insect attack, were lower for 
crop plants as against weed hosts indicating that seeds of crops were more preferred 
by O. laetus. 

Figure 4 provides the HPLC analysis of the fatty acid methylester profiles of some 
important malvaceous crop and weed seeds utilized by O. laetus. The individual fatty 
acids were identified based on the retention time and the percentage area 
composition of the relative compounds were obtained (table 5). It can be seen that the 
seeds of G. hirsutum contained arachidic, stearic, oleic, palmitic, linoleic and linolenic 
acids. Linolenic acid was not detected in the seeds of A. esculentus and H. s. var albus. 
Similarly, linolenic and arachidic acids were not identified in the seeds of H. s. var 
rubber. Interestingly, seeds of the weed hosts, Sida rhombifolia and S. rhomboidea 
were deficient in most of the important fatty acids. These observations confirm the 
fact that seeds of crop plants are richer in several fatty acids as against those of weed 
plants. 



Fattyacid methyl esters 




_ 
a. o"to o 



\_ 



II i 



"o Si 3* 
a. o In 













Ij 










Jli 


-_J 


^ 


J 


u 



go 

= CLO 









01! 








1 








i 




. 




(/")! 


r 


i . _ 


SJ 


l^ ^ 1 




10 15 20 25 5 10 
Retention time ( min) 



25 



Figure 4. HPLC profile of triglyceride methyl esters in different malvaceous seeds. 



Feeding and reproductive indices in O. laetus 



203 



Table 5. Qualitative profile of triglyceride methyl esters in different malvaceous seeds. 



Retention time 
Host plants (in in) 


Retention time 
Area(%) Host plants (min) 


Area (%) 


G. hirsutum 1-04 


0-038 H. s. var albus 


3-71 


97-623 


1-33 


0-050 


3-82 


1-558 


1-54 


0-020 


12-87 


0-030 


1-77 


0-205 


14-18 


0-154 


7-24 


0-398 


15-16 


0-666 


7-71 


' 033 H. s. var rubber 


2-99 


16-503 


7-83 


0-130 


4-40 


64-581 


10-05 


0-022 


13-07 


7-892 


10-12 


0-014 


15-61 


2-603 


12-08 


0-074 


17-34 


2-645 


15-96- 


3-775 


22-55 


5-775 


17-80 


74-501 






22-47 


20-740 ^* r h m bifoti a 


2-71 


0-722 






2-88 


3-742 


A. esculentus 2-65 


0-300 


3-90 


94-267 


2-96 


1-942 


15-45 


1-269 


3-05 


5-961 






3-54 


3.Q35 S. rhomboid ea 


2-81 


0-675 


3-94 


69-857 


2-97 


5-825 


12-39 


10-164 


4-05 


30-860 


12-64 


2-283 


4-35 


22-196 


12-71 


2-840 


4-51 


.39-907 


15-83 


1-210 


15-35 


0-269 


16-64 


0-933 


15-70 


0-267 


16-97 


0-243 






17-04 


1-208 






19-59 


0-025 







4. Discussion 

Feeding and reproduction in insects are two closely linked physiological processes, the 
magnitude of which is strongly influenced by nutritional factors. Seeds, the prime 
diet of many lygaeid bugs, are also highly nutritious. Species of the genus Oxycarenus 
are primarily restricted to the plant order Malvales which include the families 
Malvaceae, Sterculiaceae and Bombaceae (Adu-Mensah and Kumar 1977). In India, 
the dusky cotton bug, O. laetus utilizes mature and dry seeds of several crop and 
weed hosts (Thangavelu 1978; Ananthakrishnan et al 1982) and in the present study 
it was found that seed host diversity had a profound bearing on the growth, 
development and longevity of the bugs. The type of seed food is known to influence 
the survival, growth and development of several other species of heteropterans also, 
such as the lygaeids, Lygaeus equestris (L) (Kugelberg 1973) and O.fasciatus (Ralph 
1976), pentatomid Piezodorus guildnii (Westwood) (Panizzi and Slansky 1985), and 
mirid Cyrtopeltis tennis Reut. (Raman and Sanjayan 1984). The present investigation 
also showed that seeds of malvaceous crop plants favoured faster development of 
O. laetus thereby attributing the fact that growth rate and growth efficiency increases 
as the suitability of diet increases (Chaplin and Chaplin 1981). On seeds of weed 
hosts the nymphs exhibited a slower growth rate and consequently the moulting 
period was also delayed which indicate that they have to spend additional time in 



204 K Raman 

order to attain the critical body size that would eventually lead to adult ecdysis 
(Blakley and Goodner 1978). 

The utilization of a host plant by an insect is determined not only by its -ability to 
locate and colonize on the prospective host plant, but also on the capacity to ingest, 
assimilate and convert the food into body tissues (Slansky 1982). The food utilization 
pattern of O. laetus indicated that the quantity of food ingested and utilized were 
maximum when maintained on the seeds of G. hirsutum and other crop plants 
compared to weed hosts. The differences in the amount of food ingested and utilized 
from different malvaceous seed hosts also profoundly influenced the reproductive 
performance of adult females. It has also been shown that maximum egg production 
occurred when the bugs were maintained continuously on a diet of cotton seeds only 
and any interruption in its dietary schedule, either by maintaining on water alone or 
by substituting seeds of lower nutritive value, significantly affected the total egg out- 
put (Raman and Sanjayan 1983). The natural food has been found to influence the 
reproduction in seed-feeding heteropterans in several ways. For instance, in 
L. equestris (Kugelberg 1973) and O.fasciatus (Ralph 1976) the length of oviposition 
period, number of eggs laid and longevity of adults were affected by type of seed 
food. Similarly, Ewete and Osisanya (1985) showed that seeds of cotton, okra and 
kenaf profoundly influenced the development, longevity and fecundity of 
Oxycarenus gossypinus Dist. in Nigeria. In addition, inadequate diet or diet of 
inferior quality during 5th stadium irreversibly affected growth, development, 
reproduction and flight capacity in the pentatomid Nezara viridula () (Kester and 
Smith 1984). Computation of age-specific fecundity life-table of O. laetus revealed 
that the type of seed food influenced the time course of reproduction as well as its 
onset. When fed with the seeds of G. hirsutum, egg-laying commenced earlier and 
accelerated rapidly leading to maximum egg out-put. Higher intrinsic rate of increase 
and net reproductive rates were computed for G. hirsuium compared to A. esculentus 
and A. crispum. Similar observations were also reported in the case of the milkweed 
bug O.fasciatus (Klausner et al 1980), pentatomid P. guildnii (Panizzi and Slansky 
1985), and coried Clavigrella tomentosicollis (Stall) (Iheagwam 1982) wherein higher 
reproductive rate, intrinsic rate of increase and net reproductive rates were 
consistently observed when fed with the seeds of preferred host. 

The quality of host plant is known to influence the feeding behaviour of 
phytophagous insects and each species requires a particular quantitative 
composition of nutrients in its diet to complete normal growth and development. 
Interestingly, in the present study a strong relationship was found to exist between 
the food preference of O. laetus and biochemical composition of host plant seeds. 
Even though all the seed hosts belong to the family Malvaceae, it was observed that 
the food utilization varied among different seeds, presumably due to the differences 
in their nutritional composition. Seeds of G. 'hirsutum, the most preferred host, 
contained higher total nitrogen, proteins, lipids and lower carbohydrates and narrow 
C/N ratio. It was stressed that increased nitrogen content increases fecundity, 
longevity and survival rates of phytophagous insects (McNeill and Southwood 1978; 
Chaplin and Chaplin 1981; Ananthakrishnan et al 1982; Raman and Sanjayan 1984) 
while low sugars coupled with high protein content (Beck 1956) favoured greater 
host preference. In the present investigation increased phenolic contents were 
associated with greater preference of 0. laetus probably indicating that their 
concentrations were not substantially high in order to have an inhibitory effect on 



Feeding and reproductive indices in O. laetus 205 

feeding. Seed host preference was also enhanced by increased total lipid content and 
the presence of several fatty acid methyl esters in crop plants. Free fatty acids (non- 
volatile lipids) are also reported to act as biting or feeding stimulants for certain 
insects such as the silkworm Bombyx mori (linolenic acid in mulberry leaves); the fire 
ants Solenopsis sp. (linoleic and linolenic acids) and in the Dermestes beetles (palmitic 
and stearic acids) (Seigler 1983). Also, in O. fasciatus the body lipid requirements 
were found to be met by the unsaturated fatty acids present in the milkweed seeds 
and the fatty acid composition of the bugs was influenced by the type of seed diet 
(Nation and Bowers 1982). In general, it appears that the nutritional quality of the 
diet accounts for the major portion of the variation in rates and efficiency of food 
utilization leading to increased reproductive performance (Scriber and Slansky 1981). 
Hence, the increased food utilization favouring faster developmental rates and higher 
fecundity values of 0. laetus when fed with seeds of crop plants could be attributed to 
the nutritional superiority of the seed food. 



Acknowledgements 

The author is grateful to Prof. T N Ananthakrishnan for encouragement and 
also for critically going through the manuscript. The author also acknowledges the 
help rendered by Dr G Suresh for the biochemical analysis of seed samples using 
HPLC system. Instrumentation facilities were available, thanks to COSIST programme. 

References 

Adu-Mensah K and Kumar R 1977 Ecology of Oxycarenus species (Heteroptera: Lygaeidae) in southern 

Ghana; Biol. J. Linn. Soc, 9 349-377 
Ananthakrishnan T N, Raman K and Sanjayan K P 1982 Comparative growth rate, fecundity and 

behavioural diversity of the dusky cotton bug, Oxycarenus hyalinipennis Costa (Hemiptera: Lygaeidae) 

on certain malvaceous host plants; Proc. Indian Natl Sci. Acad. B48 577-584 
Beck S D 1956 A bimodal response to dietary sugars by an insect; Bull. Entomol. Res. 10 219-228 
Blakley N and Goodner S R 1978 Size-dependent timing of metamorphosis in milkweed bugs (Oncopeltus) 

and its' life history implications; Biol. Bull. 155 499-510 
Bray H C and Thorpe W R 1954 Analysis of phenolic compounds of interest in metabolism; Methods 

biochem. Anal. 1 2752 
Chaplin S B and Chaplin S J 1981 Comparative growth energetics of a migratory and non-migratory 

insect: The milkweed bugs; J. Anim. Ecol 50 407-420 
Dingle H and Baldwin D 1983 Geographic variation in life histories: A comparison of tropical and 

temperate milkweed bugs (Oncopeltufy'm Diapause and life cycle strategies in insects (eds) V K Brown 

and I Hodek (Netherlands: Dr W Junk Publishers) pp 143-165 
Dubois M, Gilles K A, Hamilton J K, Rebers P A and Smith F 1956 Calorimetric determination of sugars 

and related substances; Anal Cham. 28 351-356 
Ewete F K and Osisanya E O 1985 Effects of various diets (seeds) on development, longevity and 

fecundity of the cotton seed bug, Oxycarenus gossypinus Dist. (Heteroptera: Lygaeidae); Insect Sci. 

Appl. 6 543-545 
Iheagwam E U 1982 Effects of host plants (legume) on life and population parameters of Claviyralla 

tomentosicollis (Hemiptera: Coreidae); Rev. Zool Africane 96 912-920 
Johansson A S 1958 Relation of nutrition to endocrine reproductive functions in the milkweed bug, 

Oncopeltus fasciatus D. (Heteroptera: Lygaeidae); Nytt. Mag. Zool. 1 1-132 
Kester K M and Smith C M 1984 Effects of diet on growth, fecundity and duration of tethered flight of 

Nezara viridula; Entomol. Exp. Appl. 35 75-81 
Klausner E, Miller E R and Dingle H 1980 Nerium oleander as an alternate host plant for south Florida 

milkweed bugs, Oncopeltus fasciatus; Ecol. Entomol. 5 137-142 



206 K Raman 

Kugelberg O 1973 Laboratory studies on the effects of different natural foods on the reproductive biology 

of Lygaeus equestris L. (Het: Lygaeidae); Entomoi Scand. 4 181-190 
Lowry O H, Rosebrough N G, Farr A L and Randall R G 1951 Protein measurements with Folin Phenol 

reagent; J. Biol. Chem. 193 265-275 

McNeill S and Southwood T R E 1978 The role of nitrogen in the development of insect/plant 
relationships; in Biochemical aspects of plant and animal coevolution (ed.) J Harborne (London: 

Academic Press) pp 77-98 
Nation J L and Bowers W S 1982 Fatty acid composition of milkweed bugs Oncopeltus fasciatus and the 

influence of diet; Insect Biochem. 12 455-459 
Panizzi A R and Slansky F Jr 1985 Legume host impact on performance of adult Piezodorus guildnii 

(Westwood) (Hemiptera: Pentatomidae); Environ. Entomol 14 237-242 
Ralph C P 1976 Natural food requirements of the large milkweed bug, Oncopeltus fasciatus (Hemiptera: 

Lygaeidae), and their relation to gregariousness and host plant morphology; Oecologia 26 157-175 
Raman K and Sanjayan K P 1983 Quantitative food utilization and reproductive programming in the 

dusky cotton bug, Oxycarenus hyalinipennis (Costa) (Hemiptera: Lygaeidae); Proc. Indian Natl Sci. 

Acad.E49 23 1-236 
Raman K and Sanjayan K P 1984 Host plant relationships and population dynamics of the mirid, 

Cyrtopeltis tennis Reut. (Hemiptera: Miridae); Proc. Indian Natl. Sci. Acad. B50 355-361 
Saxena K N 1963 Mode of ingestion in a heteropteran insect, Dysdercus koenigii (F) (Pyrrhocoridae); J. 

Insect Physiol.941-ll 
Schuster R 1985 Determination of fatty acids in margarine and butter by on-column derivatization; 

HPLC Application (Hewlett Packard) Pub. No. 12-5954-0826 
Scriber J M and Slansky F Jr 1981 The nutritional ecology of immature insects; Annu. Rev. Entomol. 26 

183-211 
Seigler D S 1983 Role of lipids in plant resistance to insects; in Plant Resistance to Insects, (ed) P A Hedin 

(New York: American Chemical Society) pp 303-327 

Slansky F Jr 1982 Insect nutrition: An adaptationist's perspective; Fla. Entomol. 65 45-71 
Thangavelu K 1978 N Some notes on the host specificity of dusky cotton bug, 0. laeius (Heteroptera: 

Lygaeidae); J. Nat. Hist. London 82 481-486 
Vogel A 1963 Determination of nitrogen by Kjeldahl's method; in A Text-book of Quantitative Inorganic 

Analysis including Elementary Instrumental Analysis, (3rd edition) pp 256-257 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 207-215. 
Printed in India. 



Influence of leaf age on feeding and reproduction in Raphidopalpa 
atripennis F. (Coleoptera: Chrysomelidae) 

R S ANNADURAI 

Entomology Research Institute, Loyola College, Madras 600 034, India 

Abstract. The influence of leaf age on the quantitative food intake and the reproductive 
success of the cucurbit specialist Raphidopalpa atripennis F. is provided. The host plant 
influence on the adult longevity and survival percentage were also studied. The differential 
feeding behaviour and the relative preference of this beetle towards mature leaves and better 
reproductive success are attributed to the chemical composition of the host leaf. The indi- 
vidual fatty acid profile of the various hosts as well as the different leaf ages is also 
attempted. 

Keywords. Food utilisation; leaf age; fecundity; longevity; Raphidopalpa atripennis. 



1. Introduction 

^ 

Studies on the impact of age dependent changes in foliage on feeding and 
reproduction in insects have been initiated in such insects as Leptinotar&a decemli- 
neata, Haltica lythri and Plagiodera versicolora on individual host plant (Grison 1957, 
1958; de Wilde et al 1969; Phillips 1976; Raupp and Denno 1980) not to mention of 
isolated instances in several other insects. However, information pertaining to the 
relative preference, comparative food utilization on different host plants and the 
consequent changes in the reproductive ability aspects deserve closer scrutiny. 
Observations on these aspects are discussed here with reference to the cucurbit 
infesting Chrysomelid beetle Raphidopalpa atripennis F. 

2. Material and methods 

Adults of both sexes of R. atripennis collected from such hosts as Luffa acutangula 
Roeb., Luffa cylindrica Roem. and Mukia scabrella Arn. were reared in the labora- 
tory in plastic containers with their respective host leaves. Moisture requirements 
were met by placing a wet cotton wad in the containers. Fresh leaves were provided 
every alternate day. 

2.1 Quantitative food utilisation 

In order to study the quantitative food utilization by R. atripennis leaf discs (7*5 cm 
dia) or entire leaves were placed inside a petri dish over a moist filter paper of the 
same dimension. The test insects were fed with water for 2-3 h by providing a moist 
cotton to clear their guts before the start of the experiment. The initial weight of the 
leaf disc, the filter paper and the test insect were determined. A pair of beetles were 
released into the dish and covered by another petri plate. A control was also main- 
tained without the test insect, the leaf discs, the filter paper and the test insects were 

207 



208 R S Annadurai 

removed and reweighed. Before reweighing the filter paper and the sides of the petri- 
dishes, the left over leaf discs were carefully wiped off with a camel hair brush to 
collect excrements, if any. After correcting for the weight loss due to transpiration by 
the leaf, the comparative food utilisation on different host plants was assessed by 
determining the quantity of food ingested/24 h and the consumption index, growth 
rate, approximate digestibility, efficiency of conversion etc were calculated 
(Waldbauer 1968). Three replicates were made. 

2.2 Fecundity studies 

To study the fecundity of R. atripennis on different cucurbitaceous host leaves of 
different ages (young 3 day old, mature 5-10 day old, senescent more than 10 days 
and flowers 3 day old) freshly hatched adult (imago) male and female pairs, whose 
larvae were formerly reared on their respective host roots were confined in plastic 
vials. Leaves of the respective host plants were provided inside the vials. The fecun- 
dity was calculated as the total number of eggs laid during the life time of an indi- 
vidual. 



2.3 Biochemical analysis 

Total nitrogen (Humphries 1956); total carbohydrates (Dubois et al 1956); total 
phenols (Bray and Thrope 1954); total chlorophyll (Yoshida et al 1976); total ammo- 
acids (Moore and Stein 1948); and total lipids (Folch et al 1957) of the various cucur- 
bitaceous host plants as well as the different leaf stages were analysed. 

In order to analyse the qualitative fatty acid profile of the host leaves of different 
ages the total lipids extracted from the leaf tissue was added with 10 ml of ethanol, 
3 ml of 28% ammonium hydroxide, 25 ml of petroleum ether and 25 ml of diethyl 
ether in a separating funnal, shaken for 5 min and allowed to stand for 20 min. The 
bottom layer was drained off, the ether fraction was dried to which 3 ml of 0-5 N 
NaOH in methanol was added and heated in a steam bath for 15 min. To this 5 ml of 
distilled water was then added and 2 N HC1 were added slowly until the pH reached 
approximately two. The fatty acid methyl esters were then extracted into 5 ml of 
petroleum ether and 5 ml of diethyl ether from the acidified methylated lipid extract. 
Fatty acids were analysed by a Hewlett Packard HPLC system at 230 nm using an 
Hypersil ODS 5 /an column with water and acetonitrile as solvents at a flow rate of 
045 ml according to the gradient programme as per Schuster (1985). The retention 
time and area percentage of fatty acid methyl esters were recorded. 

3. Results 



3.1 Quantitative food utilization 

The amount of food ingested by R. atripennis varied among the different cucurbita- 
ceous hosts. Data on the comparative food utilization of R. atripennis is provided in 
table 1. Among the 3 host plants tested the maximum amount of food ingested was 
on L acutangula (0-050 1 0-003 mg), followed by L. cylmdrwa (0-0474 0-003 mg) 



Influence of leaf age on feeding and reproduction 



Table 1. Quantitative food utilization in R. atripennis. 



209 



Host 


Amount of 
food ingested 
(mg) 


CI 


GR 


AD (%) 


ECD (%) 


ECI(%) 


L. acutangula 














Young 


0-0489 0002 


2-445 0-10 


1-22 0-35 


77-50 6-86 


50-62 4-89 


49-89 3-5 


Mature 


0-0501 0-003 


2-505 0-0 15 


1-34 0-20 


97-80 7-24 


54-69 0-57 


53-49 1-33 


Senescent 


0-0298 0-002 


1-490 0-10 


0-68 0-10 


66-44 3-86 


47-22 3- 10 


45-63 1-00 


Flowers 


0-0492 0-003 


2-46 0-0 15 


1-170-05 


85-77 5-08 


55-45 3-25 


47-563-33 


L. cylindrica 














Young 


0-0398 0-000 


1-990 0-00 


0-82 0-05 


94-48 8-29 


86-70 0-1 6 


40-95 i 0-00 


Mature 


0-0474 0-003 


2-37 0-1 5 


1-130-10 


97-89 11-23 


48-70 0-65 


47-60 9-52 


Senescent 


0-0 197 0-008 


0-985 0-40 


0-43 0-20 


78-68 7-24 


55-48 0-1 8 


45-1 8 5-00 


Flowers 


0-0419 0-012 


2-095 0-60 


1-120-10 


83-29 8-21 


68-78 1-10 


53-46 16-66 


M. scabrella 














Young 


0-01 87 0-01 9 


0-935 0-50 


0-49 0-1 2 


57-21 4-36 


91-584-21 


52-41 5-20 , 


Mature 


0-0202 0-002 


1-0100-10 


1-50 0-26 


90-09 6-02 


55-49 1-02 


50-00 1-00 


Senescent 


0-0 109 0-004 


0-545 0-20 


0-29 0-24 


82-56 7-24 


64-44 1-08 


53-21 1-50 


Flowers 


0-0 192 0-008 


0-960 0-40 


049 1-02 


85-41 3-14 


59-75 0-86 


59-75 3-75 



Standard deviation 

wherein the highest consumption was on the mature leaves. The food ingested was 
least on M. scabrella (0-0202 0-002), the order preference being L. acutangula> 
L. cylindrica > M. scabrella. The various growth parameters computed viz CI, GR, AD, 
ECD and ECI were also in accordance with food preference but with some 
exceptions. The consumption index, growth rate and the approximate digestibility 
were relatively higher in the case of mature leaves and flowers compared to young 
and senescent leaves of all the hosts. The ECD was maximum only in the young 
leaves of L. cylindrica (86-70 0-16) and M. scabrella (91-58 4-21) than the mature 
leaves. With regard to ECI of flowers, L. cylindrica (53-46 16-66) and M. scabrella 
(59-75 3-75) showed a higher percentage than any other plant part. 



3.2 Feeding preferences 

The feeding preferences of R. atripennis was tested in a no-choice as well as free- 
choice situation. Among the 3 host plants offered, R. atripennis showed a selective 
preference towards the mature leaves of L. acutangula in a free-choice situation. But 
in a no-choice situation, when only M. scabrella was offered the beetles readily fed on 
them. In a condition where L. cylindrica and L. acutangula were offered the prefe- 
rence was towards L. acutangula, but when L. cylindrica and M. scabrella were 
offered the preference was towards L. cylindrica. 

R. atripennis utilizes the weed plant Mukia scabrella only when no other cucurbit 
crop is available in that vicinity. If all the host plants are present in the same area 
only L. acutangula is utilised. The random search of an adult beetle guided by 
olfactory and visual cues result in the arrival on a suitable cucurbit host plant, 
following which the insect searches for a proper feeding site, depending on the age of 
the leaf. It is followed by antennal palpation and labial exploration where the sen- 
sillae on these areas play a vital role in the host selection process. Having located a 



210 R S Annadurai 

suitable feeding site, the beetle makes a test bite which results either in a continuous 
feeding till satiation or in a change of feeding site. 

3.3 Host plant influence on the longevity and survival 

The longevity of adults of R. atripennis as well as the survival percentage was also 
influenced by the host plant quality. Adults reared on the mature leaves of L. acutangula 
had a longevity of 114 2-2 days followed by young leaves (106 1-2 days), flowers 
(92 3*8) and senescent leaves (80 24 days). L cylindrica ranked second followed 
by M. scabrella. With regard to the order of superiority among the different ages the 
same trend was followed as in the case of L. acutangula. The percentage survival 
was also maximum in the case of mature leaves of L. acutangula (98-6%), the 
minimum being 72-14% in the case of senescent leaves of M. scabrella (table 2). 

3.4 Host plant influence on the fecundity ofR. atripennis 

Figure 1 provides the comparative fecundity of R. atripennis when fed on the leaves 
of varying ages and flowers of the 3 host plants mentioned above. The fecundity of 
R. atripennis and the total number of egg clutches showed a marked variation among 
the different cucurbit plants and among the leaves of various ages. The maximum 
fecundity was observed in the case of L. acutangula (218) when fed on the mature 
leaves. Individuals fed on young leaves and flowers had a comparatively lesser 
fecundity of 175 and 78 respectively than the senescent leaves. The number of egg 
clutches was maximum when fed on the mature leaves while the senescent leaves of 
all the hosts had a marked influence on the beetle resulting in a very low fecundity. 
As regards the fecundity of R. atripennis the maximum egg output was on 
L. acutangula followed by L. cylindrica and M. scabrella. 

Table 2. Impact of host plant age on the longevity and 
survival of jR. atripennis. 



Longevity of 
Host plant adults Survival (%) 



L. acutangula 






Young 


106 1-2 


96-00 


Mature 


1142-2 


98-60 


Flowers 


92 3-8 . 


83-20 


Senescent 


80 24 


76-80 


M. scabrella 






Young 


86 1-3 


82-28 


Mature 


96 0-0 


86-02 


Flowers 


79 3-1 


76-32 


Senescent 


69 2-8 


72-14 


L. cylindrica 






Young 


98 3-2 


91-22 


Mature 


112 2-8 


96-00 


Flowers 


88 1-3 


81-32 


Senescent 


76 2-7 


75-00 



Mean of 5 replicates. 
Standard deviation. 



Influence of leaf age on feeding and reproduction 



211 



200 - 



150 



100 



50 




4 = 



M Y F 
LA 



M Y F S 

LC 
Host plants 



M Y F S 
MS 



Figure 1. Impact of different cucurbitaceous host plants as well as different ages of leaves 
on the fecundity of R. atripennis. LC, L. cylindrical LA, L. acutangula; MS, M. scabrella; M, 
Mature; Y, Young; S, Senescent; F, Flowers. 



3.5 Biochemical composition 

The chlorophyll content varied considerably among the cucurbit hosts as well as 
among the different leaf tissues. It was higher in the case of mature leaves of M. scabrella 
(3-60 mg/g), followed by L. cylindrica (2-55-mg/g) and L. acutangula (1-56 mg/g). The 
quantitative analysis of nitrogen, protein and carbohydrates showed a uniform 
picture in all the hosts as well as on different stages, the trend being L. cylindrica > 
L. acutangula > M. scabrella. The highest phenolic content was estimated in the case 
of L cylindrica and the lowest in the case of M. scabrella, the moderate being in 
L. acutangula. An increasing trend in the total amino acid content and total phenolic 
content was observed in the mature leaves of all the hosts as compared to other 
stages. However, it was very high in L. acutangula than the other two hosts (table 3). 
The HPLC analysis of the fatty acid composition also indicated some interesting 
results. The triglycerides of varying retention time with varied area percentage of all 
the host plants with 3 different leaf stages were provided as given in table 4. Mature 
leaves of L. acutangula showed the presence of linolenic, linoleic, palmitic and oleic 
acids whereas young and mature leaves showed only the presence of palmetic acid. 
In the case of L. cylindrica young leaves had palmitic and oleic acid. The pattern was 
more or less similar in the case of M. scabrella (figure 2). 

4. Discussion 



Phytophagous insects can be highly selective with respect to the plant they consume 
and that both the degree of selectivity and the identity of preferred plants can change 



212 



R S Annadurai 



Table 3. Chemical composition of some cucurbitaceous hosts. 







Mois- Nit- 


Chloro- 


Pro- Carbo- 


Phe- Amino 


ture rogen phyll 


teins hydrates nols acid 


Lipid C/N 


Host 


Plant part 


(%) (%) 


(mg/g) 


(%) (mg/g) 


(mg/g) (mg/g) 


(mg/g) ratio 


L. acutangula 


Young 


78-14 2-17 


1-73 


13-56 1-63 


2-00 1-80 


12-5 0-75 




Mature 


73-75 2-94 


1-56 


18-38 M3 


3-70 10-00 


15-0 0-38 




Senescent 


66-00 2-01 


1-91 


18-81 0-85 


3-40 5-5 


8-6 0-28 




Flowers 


79-75 4-69 


0-98 


29-31 1-73 


1-56 4-32 


7-8 0-37 


L. cylindrica 


Young 


83-28 5-32 


1-73 


33-25 2-38 


3-00 0-6 


8-2 0-45 




Mature 


77-76 6-44 


2-55 


40-25 1-25 


3-00 2-8 


9-2 0-19 




Senescent 


62-73 4-79 


1-54 


29-94 1-08 


4-30 2-2 


4-3 . 0-23 




Flowers 


8Q-43 5-95 


1-02 


37-19 2-13 


2-60 1-8 


5-2 0-36 


M. scabrella 


Young 


84-00 1-68 


2-56 


10-50 1-25 


1-50 MO 


6-8 0-74 




Mature 


78-77. 1-75 


3-60 


10-94 0-95 


1-60 1-50 


7-3 0-54 




Senescent 


76-63 1-96 


2-63 


12-25 0-93 


1-55 1-00 


5-5 0-47 




Flowers 


82-50 M2 


0-42 


7-00 MO 


2-15 0-82 


4-2 0-98 




Table 4. Showing 


the retention 


time and area (%) of the fatty acid methyl 


esters. 




Retention 


Area 


Retention Area 


Retention 


Area 


Host plant 


time 


(%) 


time 


(%) 


time 


(%) 


L. acutangula 


Young 


Mature 


Senescent 




2-76 


0-342 


2-65 


0-454 


2-81 


8-884 




2-94 


3-496 


2-84 


24-947 


3-54 


23-174 




3-31 


10-837 


6-42 


21-926 


3-75 


65-014 




3-55 


82-227 


7-28 


19-312 


5-16 


0-944 




4-61 


0-033 


9-59 


24-209 


5-80 


1-065 




4-77 


0-066 


10-90 


3-280 


7-91 


0-628 




5-09 


0-280 


11-54 


5-568 


15-63 


0-293 




5-26 


0-160 


15-84 


0-144 








6-72 


0-309 


16-15 


0-160 








7-38 


1-029 












15-66 


0-897 












15-81 


0-324 










L. cylindrica 


2-82 


0-675 


2-84 


1-524 


2-93 


6-180 




3-00 


11-324 


' 3-00 


15-048 


3-32 


16-779 




4-74 


16-603 


4-39 


6-854 


3-44 


76-440 




4-89 


70-951 


4-58 


29-379 








15-10 


0-065 


4-77 


46-843 








15-82 


0-251 


15-55 


0-221 








16-15 


0-132 


15-87 


0-131 






M. scabrella 


2-62 


2-573 


2-85 


22-512 


2-94 


19-408 




2-71 


7-285 


4-25 


27-217 


4-21 


25-004 




3-14 


15-247 


4-43 


47-200 


4-47 


51-932 




3-23 


74-644 


5-61 


0-166 


6-62 


0-163 




15-89 


0-251 


8-36 


1-466 


7-76 


0-124 








15-86 


1-060 


8-10 


0-053 








16-02 


0-379 


15-62 


1-347 












15-79 


0-678 












15-92 


0-791 



Influence of leaf age on feeding and reproduction 



213 



I Fatty acid methyl esters; 



Senescent 




25 25 O 25 

Retention time (min) 

Figure 2. HPLC analyses of fatty acid methyl esters of different cucurbitaceous hosts. 



during an individual's life time. It is widely recognised that plant species and indivi- 
duals can differ in quality or suitability for insect growth, survival and reproduction. 
Hence, the mere establishment of an insect on a particular plant will not provide a 
clear picture with regard to the suitability of the plant. But an understanding of the 
efficiency of utilisation of plant resources in terms of quantitative aspects, evidenced 
by the biochemical composition of the hosts and the reproductive success provide an 
incisive picture in any investigation relating to insect-plant interactions. Hsiao and 
Fraenkel (1968) indicated that the host selection of the potato beetle, L. decemlineata 
is determined not only by the availability of adequate feeding stimuli and nutrient of 
the plant, but also by the presence of host specific substances which releases the 
initial feeding behaviour. While the relative preference of R. atripennis to the mature 
leaves of L. acutangula and a relatively higher CI, GR and AD could be attributed to 
the presence of such host specific substances apart from other nutrient substances, 
the possibility of the presence of such a feeding stimulant in this host was ruled out at 
least for the beetle, Henosepilachna septima (Mary Saroja 1982). However, higher 
food intake of H. septima on the host L. acutangula was suspected to be due to 
nutritional deficiency in the host plant which forced the beetles to consume more 
food to fulfil the nutritional requirements (Ganga et al 1985). But the present 
investigation revealed higher consumption rate and fecundity in R. atripennis when 
fed with L. acutangula which indicate the fact that this host plant is not nutritionally 
deficient as envisaged by Ganga et al (1985). 




214 R S Annadurai 

The growth rate, survival and fecundity of insects are influenced by the concentra- 
tion of nutrients, particularly nitrogen and proteins in their food (Watson 1970; 
Mattson 1980; Scriber and Slansky 1981). In the present study biochemical analysis 
of the different plant parts of cucurbitaceous hosts revealed mature leaves of 
L. acutangula to contain increased amounts of nitrogen, proteins, amino acids, 
carbohydrates, phenols and lipids. In addition, the analysis of the fatty acid methyl 
ester profile of leaves of different age groups revealed the presence of linolenic, 
linoleic, palmitic and oleic acids in mature leaves. While senescent leaves of L. cylindrica 
did not show the presence of these fatty acids, M. scabrella and L. acutangula 
possessed only palmitic acid on which the food utilization as well as egg output were 
comparatively lesser. For instance, in the red pumpkin beetle, Raphidopalpa 
foveicollis, a definite correlation was found to exist between the utilization of different 
plant parts of various cucurbitaceous hosts and the level of nitrogen in those parts 
(Raman and Annadurai 1985). The differential feeding behaviour and increased re- 
productive rate of R. atripennis on different parts of the cucurbitaceous hosts appear 
not only to the variation of several nutrient substances but also due to the presence 
of certain secondary plant substances, notably cucurbitacin E which is presumed to 
be acting as a feeding stimulant (Sinha and Krishna 1970). The age of leaf is also 
known to influence the feeding pattern in several other insects. In Plagiodera versi- 
colora higher fecundity was evident when fed with young leaves than old leaves 
though the females tend to lay their eggs on older leaves (Raupp and Denno 1980). 
On the contrary, the Mexican bean beetle, Epilachna varivestis preferred mature 
leaves wherein the fecundity was also higher compared to young or senescent leaves 
(Kitayama et al 1979). Similarly, higher concentrations of soluble nutrients in the 
mature leaves of eucalyptus increased the feeding preference of the psyllid, Cardia- 
spina densidextra (White 1970). Thus, the results from the present study indicate that 
the differential preference of the beetle, R. artipennis vary on leaves of different age 
groups which by and large appear to be due to the variations in the nutritional 
quality of the concerned plant part. 

Acknowledgements 

The author is grateful to Prof. T N Ananthakrishnan for valuable suggestions and 
critical perusal of the manuscript and to Dr G Suresh for his help in the biochemical 
analysis. The work was possible through COSIST assistance. 

References 

Bray H C and Thrope W V 1954 Analysis of phenolic compounds of interest in metabolism; Methods 

Biochem. 1 27-52 
de Wilde J, Bonger W and Schooneweld H 1969 Effect of hostplant age on phytophagous insects; Entomoi 

Exp. Appl 12 714-720 
Dubois M, Gilles K A, Hamilton J K, Robers P A and Smith F 1956 Colorimetric determination of sugars 

and related substances; Anal Chem. 28 351-356 
Folch J, Less M and Sloane-Stanely-Stanley G H 1957 A simple method for the isolation and purification 

of total lipids from animal tissues; J. Biol Chem. 226 497-506 
Ganga G, Sulochana Chetty J, Senthamil Selvi R and Manoharan T 1985 Influence of food plants on the 

food utilization and chemical composition of Henosepilachna septima (Coleoptera: Coccinellidae); 

Proc. Indian Acad. Sci, (Anim. SCL) 94 161-167 



Influence of leaf age on feeding and reproduction 215 

Orison P 1957 Les facteurs alimentaries de la focondite chez le doryphire (Leptinotarsa decemlineata Say) 

(Col: Chrysomelidae); Ann. Epiphyt. 8 304-381 
Orison P 1958 L' influence de la plant note sur; la fecondite de 1'insecte phytophage; Entomol. Exp. Appl. I 

73-93 
Hsiao T H and Fraenkel G 1968 The influence of nutrient chemicals on the feeding behaviour of the 

Colorado Potato Beetle Leptinotarsa decemlineata Say; Ann. Entomol. Soc. Am. 61 44-54 
Humphries E C 1956 Mineral compounds and ash analysis; in Modern methods of plant analysis (eds) 

K Peach and M V Tracey (Berlin: Springer Verlag) pp 237-248 
Kitayama K, Stinner R E and Rabb R L 1979 Effects of temperature, humidity and Soyabean maturity on 

longevity and fecundity of the adult Mexican bean beetle Epilachna varivestis; Environ. Entomol. 8 

458- 464 

Mattson W J 1980 Herbivory in relation to plant nitrogen contents; Annu. Rev. Ecol. Syst. 11 119-161 
Mary Saroja Y 1982 Studies on feeding behaviour of two closely related pests of cucurbit ace >ae, M.Phil. 

dissertation, Madurai Kamaraj University, Madurai 
Moore S and Stein W H 1948 Photometric ninhydrin method for use in the chromatography of amino 

acids; J. BioL Chem. 176 367-388 
Phillips W M 1976 Effect of leafage on feeding preference and egg laying in the chrysomelid beetle Haltica 

lythri\ Physiol: Entomol. 1 223-226 
Raman K and Annadurai R S 1985 Host selection and food utilization of the red pumpkin beetle 

Raphidopalpa foveicollis (Lucas) (Chrysomelidae; Coleoptera); Proc. Indian Acad. Sci. (Anim. Sci.) 94 

547-556 
Raupp M J and Denno R F 1980 Interactions among willow herbivores as a determinant of feeding and 

oviposition of the imported willow leaf beetle, Plagiodera versicolora Laich. (Coleoptera: 

Chrysomelidae); J.N.Y. Entomol. Soc. 88 67 
Scriber J M and Slansky F Jr. 1981 The nutritional ecology of immature insects; Ann. Rev. Entomol. 26 

183-211 
Schuster R 1985 Determination of fatty acids in mangarine and butter by on-column derivatization; HPLC 

Application Helwett Packard publ. No. 12-5954-0826 
Sinha.A K and Krishna S S 1970 Further studies on the feeding behaviour of Aulacophora foveicollis on 

cucurbitacin; J. Econ. Entomol. 30 333-334 

Waldbauer G P 1968 The consumption and utilization of food by insects; Adv. Insect. Physiol. 5 229-288 
Watson A (ed) 1970 Animal Populations in Relation to their Food Resources (Oxford: Black well) 
White T C R 1970 Some aspects of the life history, host selection dispersal and oviposition of adult 

Cardiaspina densidextra; Aust. J. Zool. 18 105-117 
Yoshida S D, Farno A, Cock J H and Comez K A 1976 Laboratory manual for physiological studies of rice, 

3rd edition, International Rice Research Institute, Los Banos, Philippines 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 217-220. 
(Q Printed in India. 



Effect of food quality on fecundity of Mylabris pustulata (Coleoptera: 
Meloidae) 

T MANOHARAN, S CHOCKALINGAM and 
K P S JEYACHANDRAN 

Zoological Research Laboratory, Thiagarajar College, Madurai 625 009, India 

Abstract. Observations on the fecundity of Mylabris pustulata maintained on two different 
food plants Ipomoea cornea and Ipomoea tuberosa, with males and females reared separately 
on the flowers of these two host plants are discussed. Mylabris pustulata reared on Ipomoea 
cornea laid 144 30-80 eggs while that on Ipomoea tuberosa only 93 38-87 eggs. The chemical 
composition of the flowers of L cornea and /. tuberosa revealed that protein concentration is 
greater in Ipomoea cornea (4-88%) than Ipomoea tuberosa (4-64%). The quantity of food 
ingested is also greater when reared on Ipomoea cornea (1055 mg). In the light of these 
observations it is presumed that the food rich in protein concentrations is among the 
principal factors influencing fecundity. 

Keywords. Mylabris pustulata; food quality; fecundity. 



1. Introduction 

Polyphagous insects feed on a variety of food plants which differ in the possession of 
organic and inorganic constituents. The egg production in phytophagous insects is 
related with the quality and quantity of food ingested (Mulkern and Brusven 1962; 
McCaffery 1975). Wigglesworth (1965) has stated that nutritionally poor plants will 
have an impact on the fecundity in phytophagous insects. Water and nitrogen 
contents in the food plants influence the egg production in insects (Kehat and 
Wyndham 1972; Van Emden 1973). Mylabris pustulata exclusively feeds on the petals 
of flowers of plants and never feeds on leaves. The results presented in this paper 
indicate the nutritive relationship between two different flowers and fecundity of 
M. pustulata. 



2. Materials and methods 

M. pustulata collected from local gardens in the vicinity of Madurai were reared in 
the laboratory on flowers of Ipomoea cornea and Ipomoea tuberosa at 30 1C and 
70 RH. Beetles were fed ad libitum every day. 

2.1 Fecundity studies 

A pair of male and female (400-450 mg) insects were selected from the culture and 
introduced in a plastic container (10 x 6 x 6 cm) and fed ad libitum with concerned 
flower. Six replicates were maintained for $ach flower. The eggs laid by each female 
insect were recorded. 



218 T Manoharan, S Chockalingam and K P S Jeyachandran 

2.2 Biochemical studies 

The protein, carbohydrate and lipid contents in the flowers of food plants were 
estimated following the methods of Lowry et al (1951), Seifter et al (1950) and 
Bragdon (1951) respectively. Water content in the leaves of each food plant was 
measured by drying the fresh flowers in hot air oven at 60C to a constant weight. 

2.3 Food consumption studies 

The female beetles were fed ad libitum on weighed quantities of their respective food 
for 1 5 days and 6 replicates for each food were maintained. Unfed leaves and faeces 
were collected daily and dried at 60C to find out the percentage of water content. 
The amounts of food consumed, defecated, assimilated and converted were estimated 
directly following IBP formula (Petrusewicz and Macfadyen 1970) represented as 
C = P + R + (F-i- 17) where C is the total food consumed, P the growth, R the energy 
spent on metabolism and F+ U the energy loss via faeces including nitrogenous 
excretory products. 

3. Results and discussion 

3.1 Fecundity of M. pustulata 

Data presented in table 1 indicate that the maximum number of eggs were laid when 
M. pustulata was reared on /. cornea (144 30-80) and minimum on /. tuberosa 

(93-33 38-87). 

3.2 Relation between the composition of flowers and fecundity 

The nutritive value of the flowers was assessed by estimating the protein, lipid, 
carbohydrate and water in them. The concentration of protein, lipid and water was 
comparatively greater in /. cornea than /. tuberosa (table 2). 

Table 1. Eggs laid per female of M. pustulata 
reared on flowers of /. cornea and /. tuberosa. 

Food plants Fecundity 



/. cornea 144-00 30-80 

/. tuberosa 93-33 38-87 



Each value represents the average performance of 
6 individuals (meaniSD) maintained at 
301C. 

Table 2. Composition of the flowers of L cornea and /. tuberosa expressed in 

percentage. 

Food plants Carbohydrate Protein Lipid Water 

/. cornea 5-3 0-16 4-880-04 6-0l-6 89-95dbl-20 

L tuberosa 5-8 0-04 4-64 0-09 1-9 88-62 0-90 



Food quality effect on fecundity of Mylabris pustulata 



219 



The high fecundity observed on /. cornea may be due to its high protein content 
(4-88%) than /. tuber 'osa (4-64%), High protein and nitrogen content and low sugar in 
Luffa cylindrica were said to be the causative factors for the increased fecundity in 
the pest Cyrtopeltis tenuis (Raman and Sanjayan 1984). In the case of the seed bugs, 
Oxycarenus hyalinipennis and Spilostethus hospes maximum egg production was 
achieved on the host plant seed which contained higher proteins, nitrogen and a 
narrow C/N ratio (Ananthakrishnan et al 1982a, b). 

Data were statistically analysed and a high correlation was found between protein 
content and fecundity (r = 0-9748) which showed a significant fit at 0-05 level (figure 1). 
Slansky (1982) found that protein is required not only for adult maintenance but also 
to supply the energy and nutrients for provisioning the eggs and egg production in 
insects. 

3.3 Feeding budget of M. pustulata 

Recent studies have indicated that not only the quality but the quantity of food 
consumed by the insect may also have an effect on fecundity. Data given in table 3 
indicate that all the feeding parameters viz food consumption, assimilation, 
conversion and feeding rate were greater in M. pustulata maintained on /. cornea. 
Statistical analysis showed that fecundity of M. pustulata depended on the 




6 160- 
120- 



r 0-9066 



4-5 4-6 4-7 
r= 0-9743 



r =0-8010 



1-8 1-9 2-0 



r- 0-8623 



700 780 860 940 
r = 0-9044 




4-8 4-9 5-0 
protein 



4 6 8 10 
Lipid 



IOOO IO50 IIOO 
Food consumed 



Figure I. Regression' to describe the relationship of protein and lipid content of food plants 
and food consumption by M. pustulata against fecundity. 



Table 3. Feeding budget of M. pustulata ($) fed ad libitum on flowers of /. cornea and /. tuberosa. 



Food 
plants 


Food 
consumed 


Faeces 
produced 


Food 
assimilated 


Food 
converted 


Food 
metabolized 


Feeding 
rate 
(mg/glivewt/day.) 


/. cornea 
L tuberosa 


1055 32-58 
784 55-93 


447 26-67 
3 12 35-07 


608 23-53 
472 34-82 


83 14-66 
37 9-40 


525 36-23 
435 32-27 


330 10 
110 09 



Each value represents the average performance of 6 individuals (meanSD) (mg/dry wt) maintained for a 
period of 15 daysat30lC 



220 T Manoharan, S Chockalingam and K P S Jeyachandran 

consumption of /. cornea (r = 0*9044; P<001). Mukerji and Guppy (1970) also 
reported that the fecundity increased with the rate of food intake in Pseudaletia 
unipuncta. 

This study highlights that the protein content of the food plants and the quantity 
of food consumed essentially determine the fecundity in phytophagous insects. 

Acknowledgements 

Our sincere thanks are due to Dr (Mrs) Radha Thiagarajan, Mr T Kannan and 
Dr S P Annamalai for constant encouragement. One of us (KPSJ) is thankful to the 
Council of Scientific and Industrial Research, New Delhi for the award of a fellowship. 

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Proc. Indian Natl. ScL Acad. B48 577-584 
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unipuncta; Can. EntomoL 102 1179-1188 
Mulkern G B and Brusven M A 1962 Biology and ecology of North Dakota grass hoppers. I. Food habits 

and preference of grass hoppers associated with alfalfa fields; N. D. Agric. Exp. Stn. Res. Rep. No. 7 
Petrusewicz K and Macfadyen A 1970 Productivity of terrestrial animals, principles and methods; in IBP 

Handbook No. 13. (Oxford: Black Well Publ.) pp 190 
Raman K and Sanjayan K P 1984 Host plant relationships *and population dynamics of the mirid, 

Cyrtopeltis tennis Reut; Proc. Indian Natl. ScL Acad. B50 355-361 
Seifter S, Dayton S, Novic B and Muntwyler E 1950 The estimation of glycogen with the an throne 

reagent; Arch. Biochem. Biophys. 25 190-200 

Slansky F 1982 Insect nutrition: An adaptationist's perspective; Fla. EntomoL 65 45-71 
Van Emden H F 1973 Aphid host plant relationships: Some recent studies in perspectives in aphid 

biology; in EntomoL Soc. N. Z. Bull, (ed) A D Lowe p 54 
Wigglesworth V B 1965 The principles of Insect physiology (London: Methuen and Co., Ltd.) p 741 



Proc. Indian Acad. Sci. (Anim, Sci.), Vol. 96, No. 3, May 1987, pp. 221-227. 
Printed in India. 



Effect of temperature and host seed species on the fecundity of 
Callosobruchus maculatus (F.) 

J CHANDRAKANTHA*, J MUTHUKRISHNAN** and 

S MATHAVAN 

School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India 
^Department of Zoology, APA College for Women, Palani 624601, India 
"'Department of Zoology, APA College, Palani 624 602, India 

Abstract. Callosobruchus maculatus was reared on Vigna unguiculata, Phaseolus radiatus 
and Dolichos lab lab from hatching to death at 20, 25, 30 and 35C. Food consumption, 
growth and egg production were estimated in terms of energy. At the tested temperatures, 
host seed species significantly influenced feeding and energy allocated to egg production. 
Irrespective of the seed species, food consumption decreased with increasing temperature. 
Energy density of an egg did not vary with seed species but significantly decreased with 
increasing temperature. Fecundity (egg/females) of Callosobruchus maculatus ranged from 
73 on D. lab lab at 35C to 118 on Vigna unguiculata at 30C Energy allocated to egg 
production at 20C was more than at other temperatures. A statistically significant inverse 
relationship exists between overall feeding rate of Callosobruchus maculatus and energy 
allocated to egg production. 

Keywords. Feeding; fecundity; egg production; efficiency; temperature; seed species effect; 
Callosobruchus maculatus. 



1. Introduction 

Callosobruchus maculatus (Coleoptera : Bruchidae) infests a variety of pulses such as 
bengal gram, green gram, black gram and pigeon pea and inflicts extensive 
deterioration of the quantity and quality of the seeds. Considerable information is 
available on the oviposition behaviour and fecundity of stored grain pests. Srivastava 
and Bhatia (1959) and Gokhale and Srivastava (1976) have reported ovipositional 
preference of C. chinensis and C. maculatus on several leguminous seeds (see also 
Saxena et al 1971; Bhattacharya et al 1977). These authors have related number of 
eggs oviposited to nature (smooth and rough) or area of seed surface available for 
oviposition. They have totally ignored the nutritional adequacy of the tested seeds as 
a factor in determining the fecundity of the pests. Leelavathy (1982) and Mark (1982) 
have stressed the importance of nutritional quality of seeds in determining 
ovipositional preference and fecundity (see also Fox and Morrow 1981). Presence of 
oviposition and/or feeding deterrents in the seed is also an important factor in 
determining fecundity (see Applebaum and Guez 1972; Janzen et al 1976, 1977). 
Klekowski et al (1967) and Campbell et al (1976) have studied the pattern of 
transformation of food in Tribolium castaneum and Sitophilus granarius., respectively. 
The species of bruchids studied by them do not bore into the seeds but feed from 
outside throughout their life time (except during the pupal period). C. maculatus and 
C. ' analis bore into the seeds and feed till the completion of larval development 
Therefore, the pattern of utilization of food and energy allocation to egg production 
in C. maculatus is likely to be different from T. castaneum and S. granarius. The 

221 



222 J Chandrakaritha, J Muthukrishnan and S Mathavan 

present paper reports effects of seed species and temperature on feeding and energy 
allocation to egg production in C. maculatus. 

2. Materials and methods 

Freshly emerged C. maculatus (5?$ and 5(Jc?) were allowed to oviposit on 100 
seeds each of Vigna unguiculata, Phaseolus radiatus and Dolichos lab lab at 20, 25, 30 
and 35C and 75% RH. Seeds of similar size were taken in 5 cm dia petridishes and 
exposed to C. maculatus females for oviposition. Subsequent to oviposition, seeds 
with single egg were separated, weighed individually, numbered and transferred to 
glass vials. Ten replicates of 200 seeds of each species with one egg on each seed were 
maintained at the desired temperatures in BOD incubators. Simultaneously, 100 seeds 
of more or less the same weight but without any eggs were maintained at the tested 
temperatures as control. To ascertain hatching and moulting to higher instar, a 
minimum of 5 infested seeds from each replicate were dissected every day and head 
capsule width of the larva inside was measured. Using the data on head capsule 
width for the different larval instars reported by Sharifi and Mills (1971), the larval 
instars were determined. On the day of moulting to the next instar, unfed seed 
remains, egesta and exuvia were separated and dried. Subtracting the dry weight of 
the unfed remains at the end of an instar from the dry weight of the seed at the 
commencement of the instar, food consumption for the instar was determined. Total 
consumption for the entire larval period was obtained by adding the food 
consumption for the different instars. Overall feeding rate (KJ g' 1 live insect d" 1 ) 
was calculated as follows 

Food consumed (KJ/instar) 
Feeding rate (Cr) = 



Overall Cr = 



Midbody weight (g) x duration of the instar (day) ' 

Cr of instar I x duration + Cr of instar' II x duration . . . instar IV 
Total larval period (day) 



Somatic growth was estimated as the difference between the dry weight of the freshly 
hatched larva and that of the terminal larva. Freshly mated females were exposed to 
the respective seed species on which they were reared for oviposition. Number of 
eggs oviposited by a female and the egg weight were estimated. Samples of food, test 
insects and eggs were dried to constant weight at 75C and energy content of the 
samples was estimated in a Parr 1421 bomb calorimeter. Egg production efficiency 
(%) was calculated as follows 

_ , , . /0/ , Energy allocated to egg production (J/insect) 

Egg production efficiency (%) = - 



Food energy consumed (J/insect) 



3. Results and discussion 



Data on food consumption and energy allocated to somatic growth and reproductive 
growth by C. maculatus developing in the different species of seeds tested are 
provided in table 1. Irrespective of the seed species, food consumption decreased with 
increasing temperature. However, due to shortening of larval period at higher 



E/Jecf o/ temperature and host seed species on C. maculatus 



223 



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224 J Chandrakantha, J Muthukrishnan and S Mathavan 

temperatures, feeding rate increased with temperature. For instance, C. maculatus 
developing in V. unguiculata consumed 444, 419, 386 and 367 J/insect at the rate of 
11-53, 15-93, 22-34 and 39-25 KJg" 1 live weight d" 1 at 20, 25, 30 and 35C 
respectively (table 1). Of the 3 species tested, C. maculatus developing in D. lab lab 
ingested the maximum food energy at all the temperatures. For instance, at 30C 
consumption in D. lab lab was 1040 J/insect compared with 386J/irisect for that 
developing in V. unguiculata. Prolongation of larval development in D. lab lab is 
responsible for more than two fold increase in food consumption by C. maculatus 
developing in D. lab lab over that developing in the other two tested seed species. 
The pattern of energy allocation to somatic growth at the tested temperatures was 
similar to that observed for food consumption. C. maculatus realised maximum 
somatic growth in D. lab lab. It ranged from 73-42 J at 35C to 110-06 J at 20C 
(table 1). P. radiatus supported the lowest growth at all the tested temperatures. 
Weight and energy content of a single egg oviposited by C. maculatus did not vary 
with the host seed species but decreased with increasing temperature. The mass of an 
egg averaged to 30-1, 22-2, 19-0 and 15-0 ug at 20, 25, 30 and 35C respectively; 
corresponding values for the energy content were 0-286, 0.210, 0.181 and 0-143 J/egg 
(table 1). The number of eggs oviposited by C. maculatus significantly varied with 
seed species and temperature. Maximum number of eggs were oviposited by C. 
maculatus reared on V. unguiculata at 30C (table 1). 

Of the 3 species of seeds tested, fecundity of C. maculatus developing in P. radiatus 
was low (table 1). Consequent to the difference in fecundity of C. maculatus in the 
different seed species, energy allocated to egg production and hence egg production 
efficiency varied. For instance, at 20C C. maculatus developing in V. unguiculata 
allocated 29-24 J for egg production compared with 25-8 J and 24-36 J by that developing 
in D. lab lab and P. radiatus. Egg production efficiency expressed as percentage of 
ingested food energy allocated to egg production decreased with increasing 
temperature. The decrease was from 6-6% to 20C to 3-4% at 35C for the insect 
developing in V. unguiculata (table 1). Egg production efficiency of the insect in D. lab 
lab was less than that in the other seed species. For instance, the efficiency at 20C 
was 6-6% in V, unguiculata compared with 5-2% and 2-1% in P. radiatus and D, lab 
lab (table 1). 

The results presented above clearly indicate that C. maculatus maximises egg 
production in V. unguiculata at 30C. On the basis of egg production efficiency also, 
V. unguiculata and P. radiatus appear to be more suitable for feeding and energy 
allocation to egg production. Although food consumption in D. lab lab at the tested 
temperatures is twice more than that on the other seed species, prolonged larval 
development and the consequent increase in the expenditure of energy on 
maintenance metabolism resulted in the restriction of energy available for egg 
production. Chandrakantha (1985) found that V. unguiculata seeds contain more 
nitrogen, lipids, energy and water than P. radiatus and D. lab lab (table 2) and that C. 
maculatus utilizes V. unguiculata more efficiently than P. radiatus and D. lab lab. 
Accumulation of sufficient nitrogen during the feeding larval period is essential to 
maximise egg production during the non-feeding adult period. Gross nitrogen 
accumulation efficiency of C. maculatus developing in V. unguiculata was 71-6% 
compared with 68-8% and 62-8% for that developing in P. radiatus and D. lab lab 
(Chandrakantha 1985). Therefore to maximise egg production C. maculatus prefers 
nitrogen rich V. unguiculata, which affords faster development. The presence of 



Effect of temperature and host seed species on C. maculatus 



225 



Table 2. Chemical constituents, energy and water content of the host seeds provided to 
C. maculatus. 



Seed species 


Nitrogen 


Lipid 


Carbohydrate 


Ash 


Energy 


Water 


V. unguiculata 
P. radiatus 
D. lab lab 


2-7 0-01 
2-5 0-01 
1-9 0-02 


1-3 0-04 
0-9 0-03 
0-7 0-04 


55-7 0-07 
56-6 0-04 
59-3 0-09 


2-9 0-16 
4-3 0-23 
4-4 0-20 


19-70-16 
18-90-12 
17-2011 


27-3 0-97 
25-6 0-94 
23-2 0-86 



Values of chemical constituents and water are expressed in percentage dry weight and energy content in 
terms of J nig" 1 dry matter. 



saponins and protease inhibitors in D. lab lab (see Gokhale 1976; Applebaum and 
Guez 1972) is likely to interfere with digestion and assimilation of nutrients 
rendering D. lab lab less sizable for food consumption and utilization. 

The finding in the present study that C. maculatus oviposited more eggs at 30C in 
the tested species of seeds conforms with the conclusion of Mookherjee and Chawla 
(1964) and Giga and Smith (1983). However the obvious increase in the 'metabolic 
cost at 30C resulted in decrease in egg production efficiency and allocation of less 
energy for egg production than that at 20C and 25C. The adult life span of 
C. maculatus at the tested temperatures ranged from 8-13 days. Granivorous insects 
like'S. granarius (Campbell et al 1976), Cryptolestes ferrugenius (Campbell and Sinha 
1978) and Oryzaephilus surinamensis pass through a prolonged adult life span of over 
30 days. These insects feed throughout their life time as against C. maculatus which 
feeds only during the larval period. Whereas C. maculatus resorts to shorten the 
adult duration and maximise egg production, the other granivores shorten the larval 
duration and allocate most part of the food energy consumed during the adult period 
for egg production. 

Figure 1 shows the inverse relationship between feeding rate and energy allocated 



29 



L 25 





T3 

. 21 



en 
en 
UJ 



17 



13 



Y= 33-41 + 1-0-637) X 
r = 0-907 




10 20 30 

Cr (KJ g~ 1 day~ 1 ) 



40 



Figure 1. Energy allocated to egg production of C. maculatus as a function of overall 
feeding rate. 



226 J Chandrakantha, J Muthukrishnan and S Mathavan 

Table 3. Observed and predicted values of -energy allocated to egg production by 
C. maculatus. 



Host seed species 


Temp. 
(C) 


Observed 
egg 
energy (J) 


Predicted 
from TL 


X 2 


Predicted 
from pupa 


Predicted 
X 2 from imago X 2 


V. unguiculata 


20 


29-24 


25-93 


0-374 


27-45 


0-109 


29-89 


0-014 




25 


22-50 


22-97 


0-009 


23-67 


0-060 


22-21 


0-003 




30 


21-24 


18-93 


0-251 


20-17 


0-053 


20-32 


0-039 




35 


12-47 


15-47 


0-721 


15-78 


0-879 


17-17 


1-810 


P. radiatus 


20 


24-36 


20-79 


0-523 


21-12 


0-043 


22-16 


0-198 




25 


18-71 


19-34 


0-021 


19-30 


0-018 


18-43 


0-004 




30 


17-25 


17-65 


0-009 


17-19 


0-0002 


16-85 


0-009 




35 


10-32 


14-24 


1-488 


12-87 


0-630 


13-54 


1-004 


D. lab lab 


20 


25-80 


26-22 


0-006 


27-39 


0-097 


25-77 


0-0003 




25 


19-97 


23-47 


0-613 


23-02 


0-465 


22-44 


0-305 




30 


18-51 


19-85 


0-097 


17-95 


0-016 


17-20 


0-092 




35 


10-31 


16-77 


4-047 


12-99 


0-696 


11-34 


0-102 



to egg production in C. maculatus. The statistically significant correlation between 
the two variables may be made use of to predict the energy allocated to egg 
production from feeding rate. The observed and predicted values of energy allocated 
to egg production by C. maculatus developing in the different seed species are 
provided in table 3. The observed and predicted data do not differ significantly and 
hence the proposal of prediction of energy allocation to egg production from feeding 
rate stands valid. 

References 

Applebaum S W and Guez M 1972 Comparative resistance of Phaseolus vulgaris beans to C. chinensis and 

Acanthoscelides obtectus (Coleoptera : Bruchidae): The differential digestion of soluble hetero- 

polysaccharide; Entomol Exp. Appl 15 203-207 
Bhattacharya A K, Pathak P K and Shah S P 1977 The oviposition and development of Callosobruchus 

chinensis on several host species; Bull. Grain Technol. 15 38-41 
Campbell A, Singh N B and Sinha R N 1976 Bioenergetics of the granary weevil, Sitophilus granarius (L.) 

(Coleoptera : Curculionidae); Can. J. ZooL 54 786-798 
Campbell A and Sinha R N 1978 Bioenergetics of granivorous beetles, Cryptolestes ferrugineus and 

Rhyzopertha dominica (Coleoptera : Bostrichidae); Can. J. Zool. 56 624-633 
Chandrakantha J 1985 Studies on seed-insect interactions (Bioenergetics and reproduction of Callosobruchus 

maculatus Fab), Ph.D. thesis, Madurai Kamaraj University, Madurai 

Fox L R and Morrow P A 1981 Specialization: Species property or local phenomenon; Science 211 887-893 
Giga D P and Smith R H 1983 Comparative life history studies of four Callosobruchus species infesting 

cowpeas with special reference to Callosobruchus rhodesianus (PIC) (Coleoptera : Bruchidae); J. Stored 

Prod. Res. 19 189-198 
Gokhale V G 1976 Developmental compatibility of several pulses in the bruchidae. II. Hyacinth bean 

(Dolichos lab lab L.) seed coat as a factor in the development of the pulse beetle, Callosobruchus 

maculatus; Indian J. Entomol 38 121-124 
Gokhale V G and Srivastava B K 1976 Ovipositional behaviour of Callosobruchus maculatus (F.) 

(Coleoptera : Bruchidae) II. Distribution of eggs and relative ovipositional preference on several 

leguminous seeds; Indian J. Entomol 38 121-124 
Janzen D H, luster H B and Bell E A 1977 Toxicity of secondary compounds to the seed eating larvae of 

the bruchid beetle Callosobruchus maculatus; Phytochemistry 16 223-227 
Janzen D H, luster H B and Liener I E 1976 Insecticidal action of the phytohaemagglutinin in black beans 

on a bruchid beetle; Science 192 795-796 



Effect of temperature and host seed species on C. maculatus 227 

Klekowski R Z, Prus T and Zyromska-Rudzka H 1967 Elements of energy budget of Tribolium castaneun 

(Hbst) in its developmental cycle; in Secondary productivity of terrestrial ecosystems (Principles and 

methods) (ed) K Petrusewicz (Warzawa : Krakow) pp 859-879 
Leelavathy S 1982 Studies on a stored grain pest: Callosobruchus analis (F.), Ph.D. thesis, Madurai 

Kamaraj University, Madurai 
Mark G A 1982 Induced oviposition preference, periodic environments and demographic cycles in the 

bruchid beetle, Callosobruchus maculatus; EntomoL Exp. Appl. 32 155-160 
Mookherjee P B and Chawla M L 1964 Effect of temperature and humidity on the development of 

Callosobruchus maculatus (F.), a serious pest of stored pulses; Indian J. EntomoL 26 345-351 
Saxena H P, Amrit Phokela and Yeshbir Singh 1971 Report of plant prot. workshop, Madhya Pradesh 
Sharifi S and Mills R B 1971 Radiographic studies of Sitophilus zeamais (mots) in wheat kernel; J. Stored 

Prod. Res. 7 195-206 
Srivastava B K and Bhatia S K 1959 The effect of host species on the oviposition of Callosobruchus 

chinensis (F.), (Coleoptera : Bruchidae); Ann. Zool. Agra 3 37-42 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 229-237. 
Printed in India. 



Nutritional influence on the growth and reproduction in two species of 
acridids (Orthoptera: Insecta) 

K P SANJAYAN and K MURUGAN 

Entomology Research Institute, Loyola College, Madras 600 034, India 

Abstract. Comparative analysis of the effect of different natural host plants on the 
nymphal duration, quantitative food utilization and fecundity of two species of acridids, 
Aiolopus thalassinus (Fabricius) and Gesonula .punctifrons Stal revealed significant 
differences that were attributed to the varied nutritive value of the host plants. Aiolopus 
thalassinus appeared to show greater preference for Panicum maximum and Cyperus 
rotundus as against Cynadon dactylon and Coix lachryma. Similarly Gesonula punctifrons 
showed greater food intake and fecundity on Eichhornia crassipes and Colocasia sp than 
Musa paradisiaca and Ipomoea sp. Various growth parameters such as consumption index, 
approximate digestibility, growth rate, efficiency of conversion of ingested food and digested 
food were also analysed for the study of the nutritional impact in addition to the biochemi- 
cal profile of the host plants. 

Keywords. Aiolopus thalassinus; Gesonula punctifrons; nymphal duration; quantitative 
food utilization; fecundity; consumption index; growth rate. 



1. Introduction 

Earlier studies on insect nutrition were mainly focussed on the qualitative food 
requirements of insects, the basic criteria used in evaluating a diet being based on the 
time taken for the first instar nymph to reach the adult, gain in body weight and sex 
ratio. The data present strictly the gross comparative value of food plants and do not 
make distinction between the nature of food and the basic causes for the failure of 
development of insects on certain plant species. For a better understanding of the 
nature of food material, it is obligatory to collect information on the rate of feeding 
and its effect on growth and development, the amount of food digested, and the 
quantity of food converted into body mass. Ananthakrishnan et al (1986a,b) used such 
parameters for analysing the host preference of the cotton grasshopper 
Cyrtacanthacris ranacea and also stressed the importance of sensillar density and 
diversity in the feeding range. The role of physicochemical factors in host selection of 
grasshoppers have been well recognised (Mulkern 1967; Bernays and Chapman 1977; 
Sanjayan and Ananthakrishnan 1987). The nutritional influence of different host 
plants discussed here is based on two criteria: (i) Long term physiological 
components involving the effect of different hosts on the duration of post embryonic 
development and fecundity of the acridids and (ii) short term behavioural 
component in terms of the effect of different host plants on the quantitative food 
intake and subsequent analysis of various growth parameters. Two species of acridids 
were chosen for this study namely Aiolopus thalassinus (Fabricius) and Gesonula 
punctifrons Stal, both being restricted oligophagous grasshoppers feeding mainly on 
monocots and completely avoiding the dicots. 

229 



230 K P Sanjayan and K Murugan 

2. Materials and methods 

A. thalassinus and G. punctifrons were field collected from their respective host plants 
and reared in cages measuring 25 x 25 x 30 cm. Newly hatched first instar nymphs 
were segregated in groups and each group was fed with any one of the host plants. 
Observations were made on the duration spent by each instar on the different host 
plants. The total nymphal duration on each test plant was taken as an index for 
comparison of the rate of development. 

2.1 Quantitative food utilization 

The gravimetric method was adopted for assessing the quantity of food utilized. 
Experimental insects were separated from the stock culture, confined individually 
and provided with water to clear off their guts for a period of 10 h. After measuring 
the initial weight, the grasshoppers were introduced one per cage and allowed to feed 
on weighed quantity of the host plant leaves for a period of 24 h. At the end of the 
experiment the left over food, excreta and the insects were weighed. The difference in 
the weight of the insect gives the weight gained during the period of study. 
Comparison of the various food utilization was carried out by studying the growth 
parameters of Waldbauer (1968). 

2.2 Biochemical estimations 

The host leaves were subjected to biochemical estimations for their total proteins 
(Lowry et al 1951), carbohydrates (Dubois et al 1956), nitrogen (Vogel 1963) and 
phenol (Hori 1974). For analysis of the qualitative profiles of amino acids in the 
different host leaves, Hewlett Packard High Performance Liquid Chromatography 
(HPLC) was used. O-phthalaldehyde derivatives of the amino acids in the alcoholic 
extract was chromatographed (Lindroth and Mopper 1979) using Hypersil ODS 
5 /mi column. 0*1 M phosphate buffer pH 7-7 and methanol were used as mobile phase 
and fluorescent derivatives were detected at 340 nm. The amino acids were identified 
based on standard chromatograms of individual amino acids and the area per cent 
individual amino acid peaks were calculated. 

3. Observations 

3.1 Duration of post embryonic development 

Data on the duration of nymphal development and fecundity of the two acridid 
species is provided in table 1. Maximum fecundity and fastest rate of development of 
A. thalassinus were observed on Cyperus rotundus followed by Panicum maximum, 
Cynadon dactylon and Coix lachryma. For G. punctifrons, Eichhornia crassipes 
appeared to facilitate the highest fecundity as well as the shortest nymphal duration. 
No eggs were laid for acridids fed on Musa paradisiaca. Based on the data on the 
fecundity and duration of post embryonic development, the host plants could be 
arranged in the order of their preference as follows: (i) A. thalassinus: C. rotundus > 



Nutritional impact on growth and reproduction of acridids 231 

Table 1. Impact of host plants on duration of post embryonic development 
and fecundity. 





Total nymphal duration 


Fecundity 


A. thalassinus 






P. maximum 


43 0-53 


61-2 3-2 


C. rotundus 


40 045 


72-3 2-5 


C. dactylon 


49 0-63 


33-5 6-8 


C. lachryma 


51 0-98 


14-1 4-4 


G. punctifrons 






E. crassipes 


40-5 3-32 


39-0 1-09 


Colocasict sp 


42-8 2-68 


264 0-89 


M. paradisiaca 


41 -Oil -41 






Values represent mean SD of 6 replications. 

P. maximum > C. dactylon>C. lachryma; (ii) G. punctifrons: E. crassipes > Colo- 
casia sp > M. paradisiaca. 

3.2 Quantitative food intake 

Tables 2 and 3 provide data on the consumption and utilization of different host 
leaves by the various instars as well as adults of A. thalassinus and G. punctifrons 
respectively. Analysis of the amount of food ingested showed variations not only 
with the host plants but also among the developmental stages of the acridids. The data 
of the fifth instar nymphs have been taken for comparison of the host plants with 
regard to their utilization. The maximum weight of food ingested by A. thalassinus 
was from C. rotundus (248-10 mg/g) in comparison to the other hosts. G. punctifrons 
ingested more of E. crassipes (188-0 mg/g) followed by M. paradisiaca (172-0 ing/g) 
and Colocasia sp (121-5 mg/g). The weight gained was also the highest on the most 
preferred host. Various growth parameters were computed for an understanding of 
the nutritive value of the host plants. In general the acridids showed a decrease in the 
consumption index with the advancement of the nymphal stages. On all the host 
plants the consumption index (CI) of the first and second instars of A. thalassinus was 
comparatively higher than the other nymphal stages. The growth rate was also 
significantly higher in the early nymphal instar. A comparison of the data on the 
approximate digestibility (AD) indicate that the digestibility of all the plants tested to 
be fairly high and this could explain the acridid completing a number of generations 
even on a host plant of lesser nutritive value. Comparison of the efficiency of 
conversion of ingested food and digested food values of the fifth instars fed with 
various host plants indicates the efficiency of ingested and digested food to be high 
on the lesser preferred host. 

3.3 Biochemical correlates 

Table 4 provides data on the chemical analysis of the various host plants of the two 
acridids in terms of their total protein, carbohydrates, phenols, nitrogen and water. A 
comparison of the hosts of A. thalassinus indicate the preferred host plant, 
C. rotundus, to have lesser amount of proteins, carbohydrates, phenols and nitrogen. 



212 



K P Sanjayan and K Murugan 





8 


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Nutritional impact on growth and reproduction of acridids 233 





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234 



K P Sanjayan and K Murugan 



However, the water-nitrogen index computed was significantly high. A reverse trend 
was observed for these parameters on the preferred host of G. punctifrons namely 
E. crassipes. E. crassipes was comparatively rich in proteins, carbohydrates and 
nitrogen but had a very low water-nitrogen index. Figures 1 and 2 provide the 
HPLC analysis for amino acids of the various host plants. It is interesting to note 
that histidine and lysine were present in all the host plants of A. thalassinus. 
Aspergine, arginine, alanine, tyrosine, cysteine, tryptophan, methonine and leucine 



Table 4. Biochemical analysis of host plants of the two acridids. 



Host plants 


Proteins 

(mg/g) 


Carbohydrates 

(nig/g) 


Phenols 
(mg/g) 


Nitrogen 
(%) 


Water 
(%) 


Water/ nitrogen 


C. rotundus 


42-0 


193 


73-5 


6-72 


71-74 


10-67 


C. dactylon 


55-5 


583 


102-0 


8-88 


61-97 


6-97 


P. maximum 


159-0 


586 


160-5 


9-28 


77-29 


8-32 


E. crassipes 


130-0 


66 


300-0 


7-80 


82-25 


10-54 


M. paradisiaca 


10-0 


36 


460-0 


1'60 


68-92 


43-08 


Colocasia sp 


8-0 


30 


250-0 


1-28 


77-35 


60-43 



Mean of 3 replicates. 



P. maximum 



C. rotundus 

Column- hypersil CDS 5pm 5 

Mobile phase- O-1 m Phosphate buffer 

-Methonol 

Flow rate 0-4ml/min 
Detection W-L- - 34Onm 




C. dactylon 





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4 



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8 12 16 22 4 

Retention time (min) 



r- 
12 



T- 
16 



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22 



Figure I. HPLC analysis of amino acids. 



Nutritional impact on growth and reproduction of acridids 235 



M. paradisiaca 




12 



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Figure 2. Same as in figure 1. 

were present in all the hosts of G. punctifrons. The presence of glutamine at a high 
percentage only on the preferred host, C rotundus, may play an important role in 
the greater performance of the acridids reared on C. rotundus. 



4. Discussion 

The interactions between insects and plants is a complex process involving analysis 
of the responses of insects to plants as well as those plant characteristics which 
evoke these responses. Although it is known that plants contain all the essential 
nutrients required by insects, in nature an insect does not feed on all the plants. At 
the same time a plant in a particular habitat is not attacked by all the insect species 
in that habitat. This shows that there is some degree of association between the 
insects and plants such that the insect feeds on certain species of plants or certain 
groups of plants. The relationship between the acridid and its host plant is studied 
here based on such responses of the acridids as the quantitative intake of different 
host leaves, growth and fecundity, while the characteristics of the plants which 
evoke the responses of acridids relates to the biochemical composition of the plants 
in addition to the physical nature of the leaves. 

After an insect has located its host plant and has begun to feed, the two variables 
that interact are the nutritional requirements of the insects in terms of the qualitative 



236 K P Sanjayan and K Murugan 

and quantitative aspects and the digestibility and nutritive quality of the host plant 
(House 1969). The digestibility and nutritive quality of plants vary with insects as 
also the nutritive requirements of insects. However, all insects are found to have 
quite a similar qualitative requirements, and it is very likely that all the plants contain 
25 essential nutrients required by insects; it appears that qualitative factors may not 
play a decisive role in the relationship between insects and plants. More likely, the 
quantitative factors are most important. The quantity of food utilized by the two 
acridids studied here varied with the host plants. While the food ingested by 
A. thalassinus was greatest on C. rotundus, followed by P. maximum, C. dactylon and 
C. lachryma, the food ingested by G. punctifrons was highest on E. crassipes in 
comparison to M. paradisiaca and Colocasia sp. The weight gained by the insect as a 
result of feeding on a particular host plant would give a measure of the nutritive 
value of the host plant. It gives a direct correlation of how much the ingested food 
have been converted into the body mass. The weight gained by the two acridids was 
also the highest on the most preferred host indicating that for A. thalassinus and 
G. punctifrons, C. rotundus and E. crassipes respectively had a high nutritive value. 
Various growth parameters were computed for an understanding of the nutritive 
value of the host plant. In general the acridids showed a decrease in the consumption 
index (CI) with the advancement of the nymphal stages. A general trend in the 
decrease in CI in succeeding instars was also recorded for S. gregaria and 
L. migratoria fed with cabbage (Mehrotra et al 1972). On all the host plants the CI of 
the first and second instars of A, thalassinus was high compared to the other 
developmental stages. Comparison of the CI of the fifth instars on various host plants 
showed the value to be highest on C. rotundus and the least on C. lachryma. On good 
host plants the CI was very high while it was too low on poor host plants. Therefore 
it can be stated that the CI increased with increase in nutrient levels. The early 
nymphal stages showed the highest growth rate on all the host plants. The 
approximate digestibility computed indicated all the host plants provided were 
highly digested by the acridids. The efficiency of conversion of the ingested and 
digested food was significantly high in the less preferred host thereby enabling the 
insect to complete its development and reproduce when fed on the lesser preferred 
hosts. 

Faster post-embryonic development of A. thalassinus and G. punctifrons when fed 
on C. rotundus and E. crassipes respectively holds good with the observations of 
Ananthakrishnan et al (1986a) where they mentioned nymphs of Cyrtacanthacris 
ranacea had the shortest duration in the most preferred host. This effect of food on 
the insect may be either due to the differences in the amount of food ingested or due 
to the nutritive value of the host plant (Johansson 1964). 

To further understand the nutritive differences that exists among the host plants, 
biochemical estimation of the plants for the total proteins, carbohydrates, nitrogen, 
phenols and aminoacids yielded significant results: C. rotundus, the most preferred 
host of A. thalassinus had a lower quantity of proteins, carbohydrates and phenols in 
their leaves in comparison to the other hosts. The percentage of nitrogen was also 
less in C. rotundus but the water-nitrogen index computed was the highest. Of 
particular significance was the HPLC analysis of host plants for the individual 
amino acid composition. Of significance was also the detection of glutamine only in 
C. rotundus which probably would explain its greater preference by A. thalassinus. 
Schistocerca gregaria and Locusta migratoria respond differently to protein and 



Nutritional impact on growth and reproduction of acridids 237 

amino acid additions to their diet. Casein provides an adequate source of amino acids 
for S. gregaria but L. migratoria fails to develop beyond the third instar (Dadd 1960). 
Good growth occurs in a diet when protein constitutes 20-40% of dry weight (Dadd 
1960). Carbohydrates are essential as a source of energy to acridids and cannot be 
replaced efficiently by addition of protein or lipid (Dadd 1960). Acridids have no 
absolute requirement for lipids except for a sterol and some amount of fatty acid 
namely linoleic or linolenic acid. 

Acknowledgements 

The authors are thankful to Prof. T N Ananthakrishnan for his encouragement and 
critical perusal of the manuscript. The help rendered by Dr G Suresh for HPLC 
analysis of samples is also gratefully acknowledged. 

References 

Ananthakrishnan T N, Sanjayan K P and Suresh Kumar N 1986a Host plant preferences in Cyrta- 

cunthacris ranacea Stoll in some malvaceous hosts in terms of food utilization; Proc. Indian Natn. Sci. 

Acaci B51 351-357 
Ananthakrishnan T N, Suresh Kurnar N and Sanjayan K P 1986b Sensillar diversity, density and 

distribution during post-embryonic development of Cyrtacanthacris ranacea Stoll and their role in 

feeding; Proc. Indian Acad. Sci. (Anim. Sci.) 95 117-124 
Bernays E A and Chapman R F 1977 Deterrent chemicals as a basis of oligophagy in Locusta miyratoria 

(L); Ecol Entomol. 2 1-18 
Dadd R H 1960 Observation on the palatability and utilization of food by locusts with particular 

reference to the interpretation of preference in growth trials using synthetic diets; Entomol. Exp. Appi 3 

282-304 
Dubois M, Giller K A, Hamilton J K, Rebers P A and Smith F 1956 Calorimetric determination of sugars 

and related substances; Anal Chem. 28 351-356 
Hori K 1974 Studies on the feeding habits of Lygus disponsi Linn. (Hemiptera: Miridae) and the injury to 

its host plant. V. Phenolic compounds, acid phosphatase and oxidative enzymes in artificially infested 

tissue of the sugarbeet leaf; Appl. Entomol. Zool. 9 225-230 

House H L 1969 Effects of different proportions of nutrients on insects; Entomol. Exp. Appl. 12 651-669 
Johansson A S 1964 Feeding and nutrition in reproductive processes in insects; R. Entomol. Soc. London 

Symp. 2 43-55 
Lindroth P and Mopper K 1979 High performance liquid chromatographic determination of subpicomole 

amounts of amino acids by precolumn fluorescence derivatization with O-phthalaldehyde; Anal. Chem. 

51 1667-1674 
Lowry O H, Rosebrough N G, Farr A L and Randall R G 1951 Protein measurements with folin phenol 

reagents; J. Biol Chem. 193 265-275 
Mehrotra K N, Rao P J and Faroqui T N A 1972 The consumption, digestion and utilization of food by 

locusts; Entomol. Exp. Appl. 15 90-96 

Mulkern G B 1967 Food selection by grasshoppers; Ann. Rev. Entomol. 12 59-78 
Sanjayan K P and Ananthakrishnan T N 1987 Host preferences of some acridids (Insecta: Orthoptera) in 

relation to some biochemical parameters; Proc. Indian Acad. Sci. (Anim. Sci.) 96 15-21 
Vogel I A 1963 Determination of nitrogen by Kjeldahl's method; in A Text Book of quantitative elementary 

Instrumental Analysis pp 256-257 
Waldbauer G P 1968 The consumption and utilization of food by insects; Adv. Insect. Physiol. 5 229-288 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 239-244. 
Printed in India. 



Correlation of nutritional changes with the reproductive potential of 
Aphis gossypii Glover on egg plant 

T K BANERJEE and D RAYCHAUDHURI 

Aphid Research Unit, Entomology Laboratory, Department of Zoology, University of 
Calcutta, 35 Ballygunge Circular Road, Calcutta 700019, India 

Abstract. Brinjal (Solanum melonyena Linn.) leaves of varied maturity show a remarkable 
variation in respect to the retention of nutrient contents therein. The contents also vary 
along with the increase of plant age. Aphis gossypii Glover, an important pest of the crop, 
shows high reproductive potential on old and young leaves when the plants are young (2-4 
months old) and mature (4-6 months old) respectively. Amongst the nutrient content of the 
leaves, nitrogen level shows a significant correlation with the reproductive potential of the 
aphid species leading to its population outbreak and niche selection. 

Keywords. Brinjal leaves; Aphis gossypii', nutrient contents; reproductive potential; niche 
selection. 



1. Introduction 

Nutritional composition of the host plant appears to be a dominating factor in the 
dynamics of insect-plant-interactions ensuring feeding and oviposition of the insects. 
Host tissues are related with the nutritional (Ananthakrishnan et al 1982; Raman 
and*Sanjayan 1984) arid non nutritional (Reese 1929; Rosenthal and Janzen 1979) 
attributes which reflect the said behavioural responses. Of the phytophagous insects, 
aphids are too much sensitive to the nutrient values of the food plants and their 
quantitative availability may also act rapidly on their developmental switch 
mechanism (Lees 1966; White 1972). Thus, host-plant condition appears as a 
complement in nutritional ecology of aphids. High correlation between the 
reproductive activity of aphid species and nitrogen content of the host leaves has 
been established in Brevicoryne brassicae and Myzus persicae (van Emden and 
Bashford 1969) and Drepanosiphum platanoides (Dixon 1966). 

The present study is an attempt to find out the appreciable correlation, if any, 
between the nutrient quality of host leaves and reproductive activity of egg plant- 
aphid, Aphis gossypii Glover during kharif season. 

2. Materials and methods 

In a plot (5 m x 4 m) in Hooghly district (West Bengal) 20 plants of egg plant (brinjal) 
(Pusa purple cluster variety) were planted during July 1985-86 (kharif season) when 
the plants were one month old. Observations on population incidence of aphid, 
A. gossypii were made on 3 randomly selected leaves (old, young (mature) and 
tender) per plant at weekly interval. The plants were uprooted at the end of 
December. Population distribution of the aphid (%) and its reproductive potential 
were analysed. 

__ 



240 



T K Banerjee and D Raychaudhuri 



Estimation on reproductive potential (index r) of the aphid species was adopted after 
the methods of Chapman (1928), Birsh (1948) and Odum (1971). 

To analyse the major nutrient contents (carbohydrate, total nitrogen, fat, sterols 
and inorganic salts) dried and powdered leaves (200 g) of egg plant was thoroughly 
extracted with chloroform in Soxhlet apparatus. The crude extract (2 g) after removal 
of the solvent was chromatographed on a silica gel column using solvents of 
increasing polarity. Residues from eluents of different solvents were analysed 
(Ananthakrishnan 1986) and their approximate quantities determined. The said 
experiments were repeated in favour of old, young and tender leaves taking from the 
plants of 2, 4 and 6 months old during the experimental period. 

3. Results 

3.1 Aphid incidence 

There was a gradual decrease in the population incidence of aphid (figure 1) in the 
old leaves while in young and tender leaves it was found to increase gradually 
towards harvest. Again the greater population volume of the aphid appeared in old, 
young and tender leaves during premature (2 months old), mature (4 months old) 
and post mature (6 months old) periods of the plant respectively. 

3.2 Reproductive potential 

Data on the reproductive potential or natality rate (figure 2) reveals that the aphid 
showed variations in the reproductive rate at different age segments of the brinjal 
crop. Same was also seen in old, young and tender leaves at different ages of the crop. 
Again, the aphid appeared with high reproductive potential in qld and young leaves 
when the crop was 2-4 and 4-6 months old respectively. Furthermore, the high 
reproductive potential of the aphid was found to be associated with the incidence 



5-0 




1-0 



2 4 

Age of the plant (months) 



Figure 1. Population incidence of A. gossypii on old, young and tender leaves of brinjal at 
different age of the plant in kharif season during 1985-86. 



Reproductive potential of A. gossypii 



241 



.2 


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Figure 2. Reproductive potential of A. gossypii on old, young and tender leaves of brinjal 
plant during kharif season (1985-86). 



5-0 



Is 

||4-0 



3-0 



- Old leaves 



4-0 



3-0- 




3-0 



Tender leaves 



i-oL 



0-10 0-15 0.2O 0-10 0-20 0-3O 0-05 O-lO O-15 

Log number of reproductive potential /week 



Figure 3. The relationship between the population incidence and reproductive potential of 
A. gossypii on old, young and tender leaves of brinjal plant in kharif season (1985-86). 

on the mentioned leaf niches (figure 3). The slopes of the regression lines relating to the 
reproductive activity and population volume (%) of the aphid showed a significant 
correlation (P>0-05), when the regression equations were recalculated using the 
common coefficients, the relationship between the aphid population (x) and 
reproductive potentials (y) appeared in the following forms 

(i) In old leaves : -4- 59+1.19* 

(ii) In young leaves : -0-12 + 0-07x 
(iii) In tender leaves : -0-01 + 0-03x 



3.3 Nutrient status of the leaves 

There was a marked difference in the major chemical components of the leaves (old, 
young and tender) at a particular age of the plant (figure 4). Of these nutrients 
carbohydrate, nitrogen content and fat appeared in greater proportion in young 
leaves while the sterols and inorganic salts abundantly appeared in old leaves. Again, 
most of these nutrients were in maximum in old leaves during the young age (2-4 
months) of the plant while the same was seen in young and tender leaves at the old 
age (4-6 months) of the plant. Furthermore, studies on the relationships of the 
said leaf nutrients with the reproductive potentials of A. gossypii (table 1) reveal that 



242 



K Banerjee and D Raychaudhuri 



Nutrients 

20-0- 



10-0-nr 





"o 

L. 
~O 





O 
O 



e 



o 

CD 



O 

P 
O 

c 



1-0- 



2-0- 



ITTlotel 




^Tender leaf 
Young leaf 
[HH Old leaf 



Age of the plant (months) 

Figure 4. Distribution of nutrient content of leaves of brinjal at different age of the plan 

kharif season (1985-86). 



Table 1. Correlation coefficients between the reproductive potentials of A. gossypii \ 
nutrient contents of the leaves. 



Different 
leaves 


Nutrients of the leaves 


Carbohydrate 


Nitrogen 


Fat 


Sterol 


Inorganic sal 


Old 

Young 
Tender 


-0-280 
-0-079 
0-435 


0-792* 
0-889* 
0-910* 


-0-333 
-0-812 
-0-235 


-0-765 
-0-111 
-0-718 


-0-553 
-0-917 
-0-089 



*Significance at 5% level. 

the nitrogen content of the leaves appear to show significant correlation and that v 
true for all the 3 types of leaves. 



4 Discussion 

It is evident from above that population density and reproductive potential of apl 
are extremely varied, as varied as the nutrient content of the leaves along with i 
increase of crop age. Thus, variation in the nutrient content of the leaf niches (c 
young and tender) of egg plant indicate an impending condition of host plant 
parts therein. Positive correlation between the nitrogen content of the leaves and 
reproductive activity of the aphid species appear to show a preferential response 
aphid to nitrogen content of the leaves on which the aphid feeds. Again, the variati 
in the natality of the said aphid species in accordance with the natality of the h 



Reproductive potential of A. gossypii 243 

plant deserves a considerable importance. However, it is apparent that the survival 
and reproduction of insects are bound to be influenced by the quantity and quality of 
nitrogen present in the tissues which are subjected to variation with the host plant 
age (Raman and Ananthakrishnan 1986). Again, the reproductive activity of different 
aphid species has been found to vary at different age of the host due to the changes of 
amino acid contents of the tissue concerned (van Emden and Bashford 1969; Daiber 
1970; Harrewijn 1970; El Ibrashy et al 1972). Therefore, it appears to be possible for 
A. gossypii to present a high reproductive activity by changing the niches from old to 
young to have greater amount of nitrogen content during the increase of crop age. 
This study though does not conclusively demonstrate the key factor responsible for 
reproductive response of A. gossypii, but the present observation is in well 
conformity with the findings of Kennedy and Booth (1951), Dixon (1971), Sogawa 
(1971), Cheng and Pathak (1972), Me Neill (1973) and Me Neill and Southwood 
(1978) about the nutrient discrimination of insects in host selection to exert greater 
reproductive performance. 

Acknowledgements 

Authors remain indebted to Prof. T N Ananthakrishnan, Director, Entomology 
Research Institute, Loyola College, Madras, who kindled the light of inspiration for 
such an investigation. Thanks are also due to Dr S Sengupta, Raja Peary Mohan 
College, Uttarpara, West Bengal for biochemical analysis. 

References 

Ananthakrishnan T N 1986 Instructional Manual on Insect-Plant Interactions (eds) V Subramanian and 

S Viswanathan, pp 1-1 14 
Ananthakrishnan T N, Daniel A M and Suresh Kumar N 1982 Spatial and seasonal distributional 

patterns of some phytophagous thrips infesting Ricinus communis L. (Euphorbiaceae) and Achyranthes 

aspera L. (Amaranthaceae); Proc. Indian Natl ScL Acad. B48 183-189 

Birsh L C 1948 The intrin rate of natural increase of an insect population; J. Anim. Ecol. 17 15-26 
Chapman R N 1928 The quantitative analysis of environmental factors; Ecology 9 111-122 
Cheng C H and Pathak M D 1972 Resistance to Nephotettix virescens in rice varieties; J. Econ. Entomol 

651148-1153 

Dabier C C 1970 The influence of host plant on the biology of cabbage aphids; Phytophylactica 2 149-156 
Dixon A F G 1966 The effect of population density and nutritive status of the host on summer 

reproductive activity of the sycamore aphid Drepanosiphum platanoides (Schr.); J- Anim. Ecol. 35 105- 

112 
El Ibrashy M T, El-Ziady S and Riad A A 1972 Laboratory studies on the biology of corn leaf aphid, 

Rhopalosiphum maidis (Homoptera: Aphididae); Entomol. Exp. Appl. 15 166-174 
Harrewijn P 1970 Reproduction of aphid, Myzus persicae related to mineral nutrition of potato plants; 

Entomol. Exp. Appl. 13 307-3.19 
Kennedy J S and Booth C O 1951 Host alternation in Aphis fabae Scop. I feeding preferences and 

fecundity in relation to the age and kind of leaves; Ann. Appl Biol 38 25-64 
Lees A D 1966 The control of polymorphism in aphids; Adv. Insect Physiol 3 207-277 
Me Neill S 1973 The dynamics of a population of Leptopterna dolabrata (Heteroptera : Miridae) in relation 

to its food resources; J. Anim. Ecol. 42 495-507 
Me Neill S and Southwood T R E 1978 The role of nitrogen in the development of insect/plant 

relationships; in Biochemical aspects of plant and animal coevolution (ed) J Hasborne (London: 

Academic Press) pp 77-98 

Odum E P 1971 Fundamentals of Ecology; (London: W B Saunders Company) pp 179-183 
Raman A and Ananthakrishnan T N 1986 Mechanisms of host plant selection in phytophagous insects; in 



244 T K Banerjee and D Raychaudhuri 

Dynamics of Insect Plant Interactions (eds) T N Ananthakrishnan and S Viswanathan (P & P Pvt 

Ltd.) pp 16-36 
Raman K and Sanjayan K P 1984 Host plant relationships and population dynamics of the mirid, 

Cyrtopeltis tennis Reut (Hemiptera : Miridae); Proc. Indian Nad. Sci, Acad. B50 351-361 
Reese C J C 1929 Interactions of allelochemics with nutrients in herbivore food; in Herbivores: Their 

interaction with secondary plant metabolites, (eds) G A Rosenthal and D H Janzen (New York: 

Academic Press) 
Rosenthal G A and Janzen D H 1979 Herbivores: Their interaction with secondary plant metabolites 

(New York: Academic Press) pp 718 
Sogawa K J 1971 Effects of feeding of brown plant-hopper on components of leaf blade of rice plants; Jpn. 

J. Appl Entomol. Zool 15 175-179 
van Emden H F and Bashford M A 1969 A comparison of the reproduction of Brevicoryne brassicae and 

Myzus persicae in relation to soluble nitrogen concentration and leaf age (leaf position) in the Brussels 

sprout plant; Entomol Exp. Appl. 12 351-364 
White D 1972 Effect of varying dietary amino acid and sucrose concentrations on production of apterous 

cabbage aphids; J. Insect Physiol. 18 1241-1248 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 245-251. 
Printed in India. 



Influence of nutrition on the reproductive biology of sugarcane pests and 
their natural enemies 

H DAVID, S EASWARAMOORTHY and K SUBADHRA 

Sugarcane Breeding Institute, Coimbatore 641 007, India 

Abstract. The survival and fecundity of the scale insect, Melanaspis glomerata (Green) 
varies according to the sugarcane varieties on which they feed. When the sugarcane setts 
infested with scale insect crawlers were maintained on different nutritional bases, high 
survival and fecundity were observed in distilled water as the medium. The nutrition of 
immature stages and adults of the egg parasite, Trichogramma spp. was found to influence 
the longevity and reproduction. The eggs of Corcyra reared on groundnut kernels had 
appreciable quantities of growth promoting amino acids like glysine, analine and tyrosine 
and this influenced the size of the adult parasites. The higher fecundity of the parasites 
obtained from the eggs of Corcyra reared on greengram was attributed to the maximum 
level of nutrients. The individuals which emerged from superparasitized eggs showed 
reduction in mating and fecundity because of the sharing of the nutrients. The adult 
longevity and fecundity increased when fed with different sugars in the presence of host eggs. 
Increase in the protein content in adult diet decreased the fecundity. 

In the larval parasite, Sturmiopsis inferens the qualitative variation in the nutrient 
contents due to interspecific variation and quantitative variation as influenced by the size of 
the host influenced the reproductive capacity of the parasite. 

Keywords. Sugarcane pests; natural enemies; nutrition; fecundity. 

1. Introduction 

Though investigations on nutritional aspects have been done well in some 
phytophagous and parasitic insects, such basic information is scanty with regard to 
sugarcane pests and their insect natural enemies. The sugarcane entomologists in the 
past were more concerned with biology, ecology and control of sugarcane pests. But, 
it is well understood that basic nutritional information is necessary for species of 
economic importance, like various lepidopteran and hemipteran insects attacking 
sugarcane, which may demand studies involving the use of defined food, 
standardised culture or mass rearings. Attempts already made in this direction has 
resulted in the development of artificial diet for stalk borer, Chilo auricilius Ddgn 
(Varma et al 1975; Bhardwaj et al 1984; Jaipal et al 1986), internode borer, Chilo 
sacchariphagus indicus (Kapur) (Mehta and David 1978), pink borer, Sesamia inferens 
Wlk (Chatterji et al 1969; Qureshi et al 1975; Lingappa 1978; Easwaramoorthy et al 
1986) and Sorghum borer, Chilo partellus Swinhoe (Dang et al 1970; Seshu Reddy 
and Davies 1978) infesting sugarcane. However, not much information has been 
generated on the impact of nutrition on reproductive biology of sugarcane pests and 
their natural enemies. The available information is summarised in this paper. 

2. Sugarcane scale insect, Melanaspis glomerata (Green) 

2.1 Influence of sugarcane varieties 

The sugarcane varieties show quantitative variation in the chemical composition and 

245 



246 H David, S Easwaramoorthy and K Subadhm 

there occurs significant variation in the reducing and non-reducing sugar content. 
This is found to influence the establishment, development and reproduction of scale 
insect, M. glomerata. Under laboratory conditions, the number of crawlers that 
successfully reached reproductive stage varied with the variety. While 72-4% of 
crawlers reached adult stage on variety Co 740, only 17-1% reached adult stage on 
CoC 671, probably due to the variation in the nutrient contents. On the other hand 
the females reared on Co 413 produced significantly more number of crawlers 
compared to females settled on varieties like Co 775, Co A 7602, CoC 671 and Co 975 
(table 1). 

2.2 Influence of media 

The sugarcane setts after the establishment of crawlers, when planted on different 
media affected the development and fecundity of the scale insect. The proportion of 
crawlers reaching adult stage (table 2) was more when the medium used to support 
the sugarcane sett was distilled water, followed by mixed medium (silt, sand and 
farm yard manure in equal proportion) and White's medium (Thorpe 1981). The 
fecundity of females was more, when they were reared on cane setts planted in 
White's medium followed by those that developed on cane setts kept in distilled 
water. The increased fecundity may be attributed to increase in the size of the insect. 
This may be due to the availability of more amount of essential nutrients for the 
insects developing on cane setts planted in these media. 

3. Egg parasite, Trichogramma spp. 

Trichogramma spp. are the most important egg parasites of sugarcane borers in 
India. It is mass reared on the factitious host, Corcyra cephalonica St. The success of 
mass production in the laboratory and also performance in the field depends to a 
greater extent on the nutrition. 

3.1 Nutrition of immature stages 

Nutrition of the host insect significantly influences the reproductivity capacity of the 

Table 1. Survival (%) and fecundity of scale insect on 
different sugarcane cultivars. 



Variety 


Survival (%) 


Mean fecundity 


Co 413 


614(51-62)* 


160-8 


Co 740 


72-1 (58-08) 


125-7 


Co 775 


31-5(34-05) 


115-9 


Co 975 


61-4(51-62) 


102'9 


Co 62175 


44-5 (41-81) 


147-1 


CoA 7602 


44-8 (42-03) 


111-8 


CoC 671 


17-1(24-44) 


107-2 


CoR 8001 


36-6 (33-63) 


159-5 


CD at 5% 


7-16 c 


9-59 c 



"Sugarcane setts planted on mixed medium 
^Figures in parentheses are transformed values 
''Significant at 1% level. 



Biology of pests and their natural enemies of sugarcane 



247 



Table 2. Survival (%), weight, fecundity and sex ratio of scale insect on sugarcane setts 
kept in different media. 



Medium 


S'urvival(%) 


Weight of 25 scale 
insects(mg) 


Mean 
fecundity 


Sex ratio 
(M:F) 


Sand + White's medium 


.19-3 


235-9 


127-1 


1:1-67 


Mixed + White's medium 


31-7 


3014 


191-5 


1:1-38 


White's medium 


55-4 


281-1 


225-9 


1 : 0-77 


Sand 


21-2 


223-9 


99-7 


1:0-81 


Mixed medium 


56-0 


276-2 


147-4 


1:1-33 


Distilled water 


61-4 


309-2 


219-0 


1:0-83 




7-3 




30-57 





Table 3. Amino acid contents in the diets and eggs (/ig/g) 


Amino acid Groundnut Greengram 


Sorghum 


Total free (diet) 2160 5620 
Total bound (diet) 1,03,600 86,000 
Total free (eggs) 3 1 ,800 29,600 


3200 
62,720 
27,400 



Table 4. Effect of larval diets on the parasites, T. chilonis and T. japonicum. 



T. chilonis 



T. japonicum 



Larval diet 



Size offemale(n) 

Length Breadth Fecundity Sex ratio 



Size offemale(fi) 

Length Breadth Fecundity Sex ratio 



Sorghum 


409-2 


171-6 


14-5 


1:1-20 


457-6 


158-4 


13-5 


1:2-78 


Greengram 


466-4 


176-0 


39-3 


1:1-82 


501-6 


180-4 


23-4 


1:2-92 


Groundnut 


448-8 


171-6 


30-8 


1:1-08 


484-0 


180-4 


12-9 


1 :2-84 



parasite (Navarajan Paul 1980). The effect of host nutrition on the longevity, 
fecundity and sex ratio of T. chilonis Ishii (=71 australicum Gir.) was studied by 
Katiyar (1962). He concluded that the parasite was most efficient when bred on eggs 
of C. cephalonica obtained from crushed sorghum mixed with 8% yeast. Later, 
Navarajan Paul et al (1975) made detailed studies on the effect of eggs of C. 
cephalonica bred on sorghum grains, greengram seeds and groundnut kernels on T. 
chilonis and T. japonicum Ashm. The size of the host eggs obtained from the different 
diets varied greatly. Eggs obtained from the adults of C. cephalonica reared on 
groundnut diet were comparatively larger than those from greengram and sorghum 
diets. It was noticed that the development of the parasites was influenced by the size 
and nutrient content of host eggs. Seshagiri Rao (1954) also observed a similar 
phenomenon in the pupae of C. cephalonica reared on different diets. 

The eggs of Corcyra reared on groundnut kernels had appreciable quantities of 
growth promoting amino acids like glysine, alanine and tyrosine, when compared 
with the amino acid contents in the eggs of moths reared on other diets (table 3). This 
has influenced the size of the adult parasites. In both the species, the size of the adult 
parasites emerged from the eggs of Corcyra reared on groundnut kernels was the 
biggest (table 4). 

The fecundity of the parasites obtained from the eggs of Corcyra bred on different 
diets showed remarkable differences. Highest fecundity of the parasites was noticed 



248 H David, S Easwaramoorthy and K Subadhra 

Table 5. Nutrient contents in the eggs of C. cephalonica. 

Nutrient contents (%) 











Total 


Total soluble 




Eggs obtained from 


Calcium 


Potassium 


Phosphorus 


nitrogen 


carbohydrates 


Glycogen 


Sorghum diet 


1-40 


0-90 


0-312 


2-80 


26-67 


12-50 


Greengram diet 


1-65 


2-00 


0450 


3-08 


40-84 


19-17 


Groundnut diet 


1-50 


1-00 


0-300 


2-80 


29-17 


17-50 



in both the species reared on the eggs of Corcyra bred on greengram diet (table 4). 
The higher rate of fecundity was attributed to the maximum level of nutrients viz 
calcium, phosphorus, total nitrogen, glycogen and total soluble carbohydrates 
present in the eggs of moths bred on greengram (table 5). 

Even though the different larval diets of the host had influence on the size and 
nutrient content of the eggs of Corcyra, these had no effect on the sex ratio in both 
the species (table 4). 

Another aspect of nutrition of immature stage is noticed when superparasitism 
occurs. As a result of superparasitism, though 1-3 adults of T. chilonis emerged from 
a single host egg (Corcyra egg), they were defective with poorly developed wings. 
Such individuals were inactive and only a few showed a tendency for mating with 
forms of about their own size. The fecundity of such females that emerged from 
superparasitized eggs was low (Chacko 1953; Narayanan and Chacko 1957). The 
reason for the improper development of the parasites and reduced fecundity is attri- 
buted to the sharing of the limited amount of food in the host eggs between the 
developing parasites. 

3.2 Nutrition of adult parasites 

The adults of T. evanescens West, fed on dilute honey lived significantly longer and 
produced more progeny than starved females (Lund 1938). Later, Narayanan and 
Mookherjee (1955) made detailed studies on adult nutrition. There was a significant 
increase in the longevity and fecundity of T. chilonis adults when fed with 10% 
solutions of glucose, fructose, maltose and sucrose compared to unfed females. 
Longevity and fecundity also increased to a certain extent with the addition of 10% 
solution of yeast extract, Corcyra eggs extract or centrifuged milk, to a 10% solution 
of glucose given as feed in equal proportions (table 6). There was a great increase in 
the longevity of the parasite, when fed merely on sugar than when fed with different 
proteins in combination with glucose. The fecundity of the parasite was highest when 
fed merely on sugar in the presence of host eggs and an addition of yeast extract, 
Corcyra egg extract or skimmed milk to the sugar solution lowered its fecundity. So, 
it is evident that host proteins are of more value than other proteins. The parasite, 
however, seems to need only a limited quantity of the host protein to lay its' normal 
quota of eggs and any increase in the proportion of its protein beyond what the 
parasite normally imbibes from eggs at the time of oviposition has resulted in a 
lowering of its fecundity. It is, therefore, apparent that its protein requirements are 
normally derived from the host itself. 
Ramachandra Rao (1955) also reported that the fecundity and longevity of 



Biology of pests and their natural enemies of sugarcane 

Table 6. Effect of adult nutrition on longevity and fecundity in T. chilonis. 



249 



Longevity of males (days) 


Longevity of females (days) 














Mating in 


Fecundity of 














presence 


females* 




No mating 


Mating 




No mating 




of host 






Host eggs 


Host eggs 


Mating 


Host eggs 


Mating 


eggs and 


Number of 




not 


not 


Host eggs 


not 


Host eggs 


laying 


Corcyra eggs 


Treatments 


provided 


provided 


provided 


provided 


provided 


eggs 


parasitised 


No food 


0-99 


1-15 


1-06 


1-49 


1-21 


1-80 


46-05 


Glucose 


4-00 


4-30 


4-80 


18-40 


10-15 


13-50 


94-90 


Maltose 


3-95 


3-65 


4-75 


9-20 


7-35 


11-45 


94-40 


Fructose 


4-95 


4-95 


5-15 


10-10 


7-90 


14-05 


106-60 


Sucrose 


5-65 


4-15 


4-65 


9-25 


8-70 


10-80 


81-30 


Skimmed milk 
















and glucose 


3-95 


1-60 


1-95 


3-70 


2-20 


3-45 


57-90 


Yeast extract 
















and glucose 


3-30 


2-75 


2-55 


5-70 


4-00 


5-55 


6945 


Corcyra eggs extr- 
















act and glucose 


3-70 


2-80 


2-40 


7-60 


3-45 


7-65 


69-45 


Tap water 


2-25 


1-27 


1-23 


2-57 


1-14 


1-76 


35-1 


Corcyra egg 
















extract 


1-67 








1-27 











CD at 5% 


0-86 


1-07 


1-40 


1-95 


1-57 


0-96 


20-50 



* Females mated and laid eggs in the presence of host eggs. 



Table 7. Effect 


of different hosts on fecundity 


of S. inferens. 






Mean host 


Mean parasite 


Mean fecundity 


Host 


pupal weight (mg) puparial weight (mg) 


(number/female) 


Chilo infuscatellus Snell. 


72-7 


34-0 


196-4 


Chilo partellus Swinhoe 


79-4 


40-8 


252-6 


Scirpophaga excerptalis Wlk. 


96-0 


39-3 


270-0 


Sesamia inferens Wlk. 


123-3 


48-5 


322*0 


Galleria mellonella (L.) 


115-6 


41-3 


357-1 


Corcyra cephalonica Stn. 


23-6 


21-2 


No mating occurred 



T. chilonis increased considerably when the parasites were fed on 10% solution of 
different sugars. Both virgin and mated females fed on honey-water solution lived 
upto 5 days and retained their eggs upto 72 h after emergence without any 
detrimental effect (Sharma 1968). 



4. Larval parasite Sturmiopsis inferens Tns. 

5. inferens is an endo-larval parasite of sugarcane shoot borer, pink borer, stalk borer 
and Gurdaspur borer, Acigona steniellus Hmpsn. Though the exact nutrient 
requirements of the parasite was not worked out, it was found that the size of the 
host is found to influence the size and fecundity of the parasite (table 7). A positive 
correlation was obtained between the weight of the host pupae and parasite puparia 
irrespective of the host. The weight of the puparia, in turn, influenced the fecundity of 



250 H David, S Easwaramoorthy and K Subadhra 



Table 8. Influence of host sex on the pupal and adult weight. 









Weight 


(mg) 






Male 


Female 




Male 


Female 






parasite 


parasite 


Mean 


parasite 


parasite 


Mean 




Male host 






Female host 




Host pupa 
Parasite puparia 
Parasite adult 


44-2 
25-0 
18-1 


43-0 
24-8 
17-1 


43-6 
24-9 
17-8 


86-0 
39-3 
23-8 


87-2 
38-7 
23-9 


86-7 
38-9 
23-8 


Host pupa 


Parasite 


puparium 


Parasite adult 




SE 


CD 


SE 


CD 


SE 


CD 


Between host sex 


2-44** 


5-48 


2-04** 


4-58 


1-02** 


2-81 



Between parasite sex 



NS 



NS 



NS 



the adults (David et al 1980). So, the qualitative variation in the nutrient conter 
due to interspecific variation and quantitative variation as influenced by the size 
the host affected the reproductive capacity of the parasite. 

Even within a host, the quantitative variation in nutrition, as influenced by the si 
of the host had an effect on the parasite weight and fecundity. The data presented < 
the weight of shoot borer pupa, parasite puparia and adult in table 8 reveal 
that the puparia and adult parasite developed from female pupae of the ho 
C. infuscatellus were bigger irrespective of the sex, than that developed from mf 
pupae. Again weight of the adult parasite had a positive association with fecund 
(r = 0-3824*). 



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Biology of pests and their natural enemies of sugarcane 251 

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indicus K. on an artificial diet; Indian Sucj. 28 263-266 
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(Hymenoptera: Trichogrammatidae), an egg parasite of sugarcane and maize borers in India. I. Effect 

of superparasitism; Proc. Indian Acad. Sci. B45 122-128 
Narayanan E S and Mookerjee P B 'J955 Effect of nutrition on the longevity and rate of reproduction in 

Trichogramma evanescens minutum Riley; Indian J. Entomol. 17 376-382 
Navarajan Paul A V 1980 Nutritional and ecological factors of importance in breeding Trichogramma 

species; in Biological Control of Sugarcane Pests in India (eds) S Sithanantham and A R Solayappan 

(Madras: Tamil Nadu Co-op. Sug. Fed.) pp 21-28 
Navarajan Paul A V, Mohanasundaram M and Subramaniam T R 1975 Studies on the effects of different 

larval diets of rice meal moth on its egg parasites, Trichogramma australicum Gir. and T. japonicum 

Ashm.; Madras Agric. J. 62 190-198 
Qureshi Z A, Anwar M, Ashraf M, Chata N V and Arif M D 1975 Rearing, biology and sterilization of the 

pink rice borer, Sesamia infer ens\ Proc. Int. Symp. Trop. Res. Cent. Tokyo 1971 5 75-79 
Ramachandra Rao M 1955 Longevity and rate of reproduction of Trichogramma evanescens minutum Riley, 

an egg parasite of stem and root borers of sugarcane with various sugars and polyhedric alcohol, 

Associateship Thesis, Post-Grad. School, Indian Agric. Res. Inst., New Delhi, p 85 
Seshagiri Rao C 1954 Notes on rice moth, Corcyra cephalonica St.; Indian J. Entomol. 16 95-114 
Seshu Reddy K V and Davies J C 1978 A new medium for mass rearing of the sorghum stem borer, Chilo 

partellus Swinhoe (Lepidoptera: Pyralidae) and its use in resistance screening. Indian J. Plant Prot. 6 

48-55 
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with a view to improving its efficiency as a biological control agent, Ph.D. thesis, Post-Grad. School, 

Indian Agric. Res. Inst., New Delhi, p 145 J 

Thorpe Trevor A 1981 Plant tissue culture Methods and Application in Agriculture (New York: Academic 

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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 253-273. 
Printed in India. 



Nutrition and reproduction in haematophagous arthropods 

RSPRASAD 

Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695 581, India 

Abstract. Hosts of haematophagous arthropods range from amphibians to mammals. 
Blood meal is essential for egg production. Quantity and quality of ingested blood are 
important in realization of optimum reproductive potentials. In terms of egg production, 
the lowest nutritive value is for human blood. This inequality is probably based on 
differences in proteins and their constituent amino acids. Carbohydrates in the diet have no 
direct contribution to reproduction. Very little is known about sterol, lipid, vitamin and salt 
requirements for reproduction. In the mosquito Aedes aegypti some substance from the 
blood meal is thought to initiate vitellogenesis and is sustained by a complex hormonal 
mechanism in which median neurosecretory cells, corpus allatum, fat body and ovary 
participate. Mechanical stimuli from gut distension also has a role in hormone induction. 

Autogenous egg production seen in many dipterans is controlled both by the nutrition of 
the immature stages and genetic mechanisms. 

Quantity and the rate of utilization of the ingested blood decide the frequency of host 
visitation and feeding. Vertebrate blood sera contain substances which stimulate (secreto- 
gogue) as well as inhibit digestion. Nutrition plays some role in male maturation in insects, 
though not in spermatogenesis. In many ticks even spermatogenesis is influenced by adult 
blood meal. 

Keywords. Haematophagous arthropods; insects; nutrition; reproduction; blood meal; 
autogeny; nutrient metabolism; host visitation. 



1. Introduction 

Among haematophagous endopterygotes only the adults are parasitic whereas 
haematophagous exopterygotes, often spend their entire life cycle on the host. 
Examples of the former are Diptera (Nematocera, Brachycera and Cyclorrhapha) 
and Siphonaptera. The latter has Anoplura and Hemiptera. Except in cases where 
autogenous egg production is seen (e.g. certain spp. of mosquitoes, Culicoides, 
Phlebotomus, simuliids etc) blood meal is essential for ovarian maturation in all 
haematophagous endo and exopterygotes. Even in those which are autogenous, 
blood meal is necessary to mature the second and subsequent batches of eggs. 
Typical examples of haematophagous nematocerans are the Culicidae (mosquitoes); 
Ceratopogonidae (biting midges); Simuliidae (black flies) and Psychodidae (sand 
flies). Among mosquitoes, except in autogenous forms, blood meal is essential for egg 
production and they feed on a variety of hosts ranging from amphibians to 
mammals. Culicoides (Ceratopogonidae) and simuliids feed on birds and mammals, 
while psychodids have a wide range of hosts from amphibians to mammals. Nectar 
feeding is a common feature in both the sexes (Downes 1958; Lewis and Domoney 
1966). The only large group of Brachycera which habitually suck blood is those 
belonging to the family Tabanidae feeding mostly on mammals. Like Nematocera 
feeding dichotomy is seen, both sexes feeding on honey dew or nectar. Only females 
are haematophagous and blood meal appears necessary for ovarian maturation and 
egg production (Wilson 1967). According to Downes (1958) nectar feeding in 

253 



254 R S Prasad 

tabanids is a secondary development these being originally obligate blood suckers. 
Stomoxynae, Glossinidae and Pupipara (the louse flies Hippoboscidae, batflies 
Nycteribidae and Streblidae) are important groups of blood feeding Cyclorrhapha. 
Vertebrate blood is the only food taken by both sexes. Glossina feeds mainly on 
mammals whereas Pupipara have birds and mammals as hosts. Stomoxys and 
Haematobia are anautogenous blood suckers feeding mainly on ungulates and 
requiring several blood meals to complete ovarian development. Both sexes of the 
order Siphonaptera are obligate blood feeders parasitizing birds and mammals. 
Though Pthiraptera as a rule are obligate ectoparasites of birds and mammals, 
habitual haematophagy is found only among Anoplura (sucking lice) and in these 
cases blood is the only food. Hemiptera is a large assemblage exhibiting both 
phytophagy and zoophagy. Obligate haematophagy is exhibited by certain members 
of Reduviidae and Cimicidae. In these cases both sexes feed on blood and vertebrate 
blood is the only food. Cimicids feed on both birds and mammals. Polyctenids are 
exclusive parasites of bats. Triatominae of Reduviidae feed exclusively on vertebrate 
blood using mammals as hosts. Ticks are all obligate blood suckers at all stages of 
their life cycle, having a wide variety of hosts ranging from reptiles to mammals. 
Autogeny is reported in some ticks, but barring these, blood meal is essential for 
reproduction and a critical minimum amount of blood is needed for oviposition 
(Oliver 1974). Members belonging to families Psoroptidae, Dermanyssidae and 
Macronyssidae are probably the only haematophagous mites (DeLo^ch and 
DeVaney 1981; DeLoach and Wright 1981; Wright and DeLoach 1980, 1981). 

One gonotrophic cycle may be said, to consist of 3 phases: (i) locating a host and 
feeding on it; (ii) digestion of the blood meal and egg production and (iii) search 
for suitable oviposition site and egg laying (Detinova 1962). This review is focussed 
mainly on the second phase. 



2. Blood meal and reproduction 

Both quantity and quality of the blood meal influence the reproductive potentials of 
haematophagous insects. The size of blood meal influences not only the fecundity, 
but acquisition and transmission of pathogens (Hovanitz 1947; Jeffrey 1956), 
midgut protease activity (Shambaugh 1954) and refeeding behaviour (Edman et al 
1975; Klowden and Lea 1978) as well The number of blood meals required (related 
to the quantitative requirement?) to lay full complement of mature eggs varies from 
one to several. Though gonotrophic concordance [a condition where there is a strict 
alternation of blood feeding and oviposition (Kettle 1984)] may be seen in several 
blood sucking Diptera including certain mosquitoes, simuliids, tabanids and phle- 
botomines, certain others are shown to require more than one blood meal to complete 
one gonotrophic cycle (Beach et al 1983; Corbet and Smith 1974; Detinova 1962; 
Gillies 1954, 1955; Killick-Kendric et al 1977; Rao 1947; Thomas and Gooding 1976). 
The number of pre-ovipositional blood meals required by several floodwater 
mosquitoes like Aedes vexans, Psorophora confmnis and Psorophora cyanescens vary 
from 1-14 (Breeland and Pickard 1963, 1964; Chapman and Woodard 1965). Larval/ 
nymphal nutrition could have a role in deciding the number of pre-ovipositional 
blood meal required by the adult. MacDonald (1956) showed that female Aedes from 
well fed larvae needed only one pre-ovipositional blood meal to lay one raft of eggs 



Nutrition and reproduction in haematophagous arthropods 255 

whereas those from poorly fed larvae had to feed twice before laying eggs. There is a 
close correlation between quantity of blood ingested and the number of eggs laid on 
the one hand and degree of host association on the other. Those which take small 
meals (usually less than their own body weight) at frequent intervals to lay small 
batches of eggs are more closely associated with the host, example Haematobia 
irritans (Diptera) and Xenopsylla cheopis and X. astia (Siphonaptera). Oviposition in 
these cases, occurs daily and they would always have some follicles in the process of 
maturation. Gravid females ready for laying would have 2nd set of follicles already 
well advanced in vitellogenesis. In other words once initiated, vitellogenesis in these 
cases is a continuum until the life-time egg production potential is reached (Krafsur 
and Ernst 1983; R S Prasad, unpublished observations). Mosquitoes which require 
only one blood meal to lay a full complement of eggs of one batch (a full batch of 
eggs is one egg/ovariole and the number is limited by the number of ovarioles) ingest 
blood 1 to 1-2 times their own body weight and lay eggs numbering 100-150. In these 
cases ingestion of suboptimal quantity of blood does not result in oviposition. For 
example no ovarian development was seen in Anopheles maculipennis which took 
incomplete blood meal (Detinova 1962). 

Ticks which ingest large meal (about 120 times their own body weight) lay 
thousands of eggs. The quantity of the reserve yolk packed into individual egg (size 
of the egg) and the amount of nutrient available to the system are important aspects 
to be reckoned with this type of correlative studies. For example, Rhodnius which 
takes a very heavy blood meal compared to lice or fleas lays on an average 200-300 
eggs in its life time of 4-5 months, whereas probably a similar number or more is laid 
by a female of Pediculus humanus or X. cheopis during its life time of a little over a 
month. But then the egg of Rhodnius is 3-5 times larger than Pediculus or Xeno- 
psylla. The same or larger number of eggs laid during a shorter period suggest that 
the interphase between two layings in Rhodnius must be more protracted than the 
other two. The possibility that this is an adaptation for slower digestion of a larger 
meal and more efficient utilization of the nutrient content would be a good guess. 
What exactly is the correlation (if there is any) between the weight of the blood meal 
and that of the egg is not known. That there is considerable interspecific variation is 
evidenced by the findings of Goodchild (1955) who showed that the minimum 
amount of blood which allowed some eggs to mature was about 51 mg in Rhodnius, 
149 mg in Triatoma and according to Colles and Chellappa (1960) and Spielman and 
Wong (1974) 0-4-0-5 mg in Aedes aegypti. Several insects when given a chance to ingest 
more quantity of blood would lay more eggs upto a limit permitted by the number of 
ovarioles (Kamala Bai and Prasad 1981; Khalifa 1952). 

Reports show that blood from different vertebrates influence fecundity rates of 
haematophagous insects. Human blood has a poor nutritive value compared to 
blood from several other vertebrates, for Culex pipiens (Tate and Vincent 1936; 
Woke 1937b), Cx. p. berbericus and Ae. aegypti (Woke 1937a), Cimex lectularius 
(Johnson 1942) and the fox strain of the flea Pulex simulans (Hudson and Prince 
1958). Judson (1986) found the critical period (the interval following blood meal 
when the head of a female mosquito must be present for initiation of follicular 
development leading to maturation) of Ae. aegypti females fed on guinea-pig blood 
was considerably shorter than those fed on human blood. Despite its higher protein 
content human blood has the lowest efficiency in protein utilization and incorpora- 
* tion of nitrogen into egg. Total nitrogen excretion after ingesting human blood was 



256 R S Prasad 

78% whereas with guinea-pig blood it was 53% (Briegel 1985). On the other hand 
Simulium underhilli produced fewer eggs from rat blood offered, as enema than they 
did from equal volume of human blood (Klowden and Lea 1979c). Ikeshoji (1965) 
showed that Cx. pipiens laid more eggs when fed on avian host. The fecundity rate of 
Culiseta melanura was more on blood meals from chicken than from rabbit (Nasci 
and Edman 1981) when 3 }A blood meals were administered by enema. This suggests 
a physiological adaptation to avian hosts. Several similar studies have indicated that 
mammalian blood has a low nutrient value for mosquitoes compared to blood from 
amphibians, reptiles and birds. Fecundity and longevity of Cimex hemipterus was 
greatly reduced in those fed on blood of Calotes versicolor, compared to blood from 
man, rat and fowl (Sreelatha 1984). However, Nayar and Sauerman (1977) showed 
that though egg yield of Anopheles quadrimaculatus, Ae. aegypti, Culex nigripalpus 
and Psorophora columbiae fed on chicken blood was more than those fed on human 
blood (18-38% more oocytes were developed on chicken blood) in general on a per 
mg basis avian blood did not promote increased egg production in these mosquitoes. 
The reason for this kind of nutritional inequalities of blood from different vertebrate 
hosts could be many. Several artificial feeding experiments have pointers to blood 
proteins as the first suspect. As early as 1938, Yoeli and Mer, based on their 
experiments on Anopheles elutus suggested protein to be the factor which trigger egg 
development. Spielman and Wong (1974) found that enemas of 2-0 mg or more 
serum resulted in oocyte maturation and that the defibrinated human blood, washed 
cell components, serum alone and bovine albumin and globulin were adequate to 
stimulate oogenesis in Ae. aegyptL Unlike the mosquito Ae. aegypti (Woke 1937b) rat 
fleas X. cheopis and X. astia could not mature their ovaries on rat blood plasma diet, 
but the blood cell fraction was nutritionally adequate to trigger oocyte maturation. 
For these rat fleas washed blood cells resuspended in normal saline was an adequate 
diet to trigger vitellogenesis whereas blood plasma alone was found inadequate. 
Addition of blood-cell-bound-substances like ATP, serotonin and glutathione to 
plasma did not make it adequate diet for initiation of ovarian maturation, but 
reinforcing plasma with rabbit albumin thereby increasing its protein concentration 
made it an adequate diet for triggering vitellogenesis. If a gonoactive female of rat 
flea is put on a plasma diet, extensive oocyte resorption could be seen. These findings 
indicate that dietary protein concentration is a decisive factor for vitellogenesis 
(Kamala Bai and Prasad 1976). Similarly over 2 dilutions of rat blood with 
physiological saline inhibited vitellogenesis (Bella 1978). Glossina palpalis requires 
serum albumin for norml digestion of RBC and normal fecundity (Takken 1980). 

Insects show specificities to protein species for egg development. For example, in 
their study Lea et al (1956) found that only certain protein or their enzyme hydro- 
lyzates stimulate egg production in Ae. aegyptL Lactalbumin was found to be superior 
for the mosquito Aedes taeniorhynchus (Nayar. 1966) whereas purified egg albumin was 
found to be a better protein source for several other mosquitoes (Lang et al 1972). 
Rat fleas X. cheopis and X. astia were not able to utilize bovine albumin in place of 
bovine haemoglobin in an artificial diet for yolk synthesis (Kamala Bai and Prasad 
1979a). Greenberg (1951) found that for Ae. aegypti bovine plasma albumin, casein, 
gelatin or globulin had a superior nutritive value than erythrocytes (haemoglobin) 
with regard to egg production. 

The biological (nutritional) value of any protein is a function of its amino acid 
composition. Digestibility and proportion of complement of amino acid it provides 



Nutrition and reproduction in haematophagous arthropods 257 

govern the adequacy of a protein. Greenberg (1951) found that addition of isoleucine 
to a variety of dietary proteins improved egg laying in Ae. aegypti. Additions of amino 
acids to a suboptimal mixture of essential amino acids promoted greater egg produ- 
ction in Ae. aegypti (Lea et al 1956). Ae. aegypti requires 8 essential amino acids 
(arginine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan and valine) 
plus histidine, methionine and cystine are required in the diet for optimum egg 
production. Deletion of the 8 essential amino acids resulted in no egg production 
while the deletion of the other 3 caused lesser number of eggs to be produced. For 
optimum egg production, it is not only the presence of these 1 1 amino acids, but the 
proper balance between the amino acids in the mixture was also important (Dimond 
et al 1956). Attempts to replace protein with amino acids in an artificial diet for fleas 
X. cheopis and X. astia failed (Bella 1978). Amino acids present in the diet should be 
the utilizable isomer. For example D-isomers of isoleucine, threonine and valine are 
not metabolised and so inhibit egg production. The principle of nutrient propor- 
tionality suggests that metabolically suitable proportions of nutrients are needed for 
normal nutrition. Disproportionately abnormal amounts of even a single amino acid 
can reduce the efficiency in converting food stuff and can inhibit feeding and growth. 
Balance of nutrients can be one of the factors that determine choice of a food 
material (House 1974). If a single essential amino acid is absent from the diet or 
present in suboptimal levels, the amino acid in question rapidly disappears from the 
haemolymph, others increase in amount, protein synthesis comes to a halt and uric 
acid excretion rises rapidly (Dadd 1985). 

The utility of carbohydrates in the diet has been fairly well established in the case 
of mosquitoes and certain other dipterans. Sugar meal enhances longevity and 
supplies energy needed for flight activity in many blood sucking dipterans (Corbet 
1964; Davies 1953; De Meillon et al 1967; Lee and Davies 1979; Lewis 1953). 
Clements (1955) showed that subsequent to sugar ingestion there is accumulation of 
fat and glycogen. Correlation between glucose absorption and synthesis of glycogen 
and triglycerides was shown by Van Handel (1965). Glycogen is used as a source of 
energy mainly for flight while triglycerides for general maintenance functions such as 
respiration, excretion, digestion etc (Nayar and Van Handel 1971). In the case of 
males only small quantities of triglycerides are synthesized from sugar (Van Handel 
and Lum 1961). According to Nayar and Sauerman (1975a) glucose absorption, tri- 
glyceride accumulation and depletion are faster in Ae. aegypti and An. quadrimaculatus 
and the duration of survival shorter. In Aedes sollidtans and Ae. taeniorhynchus, on the 
other hand, these processes are slower and survival (longevity) longer. Sugar does 
not appear to have any direct role in egg production in mosquitoes (Lea et al 1956), 
but it does influence egg production. Magnarelli and Anderson (1981) showed fruc- 
tose and sucrose as essential dietary nutrients for Chrysops sp., Hybomitra sp. and 
Tabanus sp. which feed extensively on sugars. They also suggested that some sugars 
may be converted and deposited as yolk during early oocyte development, but 
compared to vertebrate blood, these supplementary carbohydrates probably have 
little importance in the vitellogenic process of anautogenous tabanids. Hecht (1933, 
cited by House 1958) found carbohydrates beneficial for reproduction of An. maculi- 
pennis at suboptimal temperature but had no effect at optimum temperature. It is 
possible that variation in metabolic rates of insects would have corresponding differences 
in dietary needs of nutrients. There are only 3 known cases (Culex quinquefasciatus, 
Aedes atropalpus and Ae. vexans) of carbohydrate diet inhibiting oviposition (De 



258 R S Prasad 

Meillon et al 1967; Hudson 1970; Shroyer and Sanders 1977). De Meillon et al (1967) 
found that by the 5th day after the blood meal only 45% of the sugar fed Cx. p. quin- 
quefasciatus laid eggs compared to the 98% in the sugar free group. Carbohydrate 
starved Ae. vexans laid 49 eggs/oviposition whereas females allowed to ingest sucrose 
deposited approximately 22 eggs/oviposition (Shroyer and Sanders 1977). As has 
been pointed out by these authors carbohydrate-dependent delay in oviposition and 
the attendant increased longevity could have adaptive values when oviposition sites 
are difficult to find during certain periods or for spp. which utilize temporary 
breeding sites. However in yet others sugar feed prior to blood meal resulted in 
laying more eggs and substantially longer survival than starved female mosquitoes. 
The latter was also shown to require more wet weight of blood to lay 100 eggs/female 
(ranged from 9-3-15-7 mg in different spp) compared to the former (sugar fed) group 
(ranged from 3-4-5-2 mg in different spp) (Nayar and Sauerman 1975b). Lee and 
Davies (1979) suggested that in stablefly Stomoxys calcitrans sugar meal before 
having the first blood meal appears important in the general well being of the flies. 
Digestion of blood meal was delayed when a 10% sugar solution was also fed com- 
pared to those which fed on blood alone. Haematophagous insects appear to com- 
pensate for the low dextrose level of blood by resorting to feeding on plant nectars 
(Dubose 1975, cited by Fritz 1983). Very little information is available on those 
which feed exclusively on blood. For streblids which feed exclusively on blood, 
frequent feedings appear to provide the required sugars for adults. If these insects are 
removed from their hosts for long periods of time they may be literally starved to 
death. Streblids may be used in nitrogen for both longevity and reproduction (Fritz 
1983). Deletion of sugars from the basal holidic diet of fat fleas X. cheopis and 
X. astia resulted in failure of yolk deposition in the former species, but about 13% of 
the latter showed yolk deposition. This interspecific difference may be linked up with 
larval nutrition as small percentage of X. astia could mature their ovaries in the 
absence of vitamins, salts, sugars and cholesterol (Kamala Bai and Prasad 1979a). All 
the carbohydrates are not equally utilized. Survival of adult Ae. aegypti was good on 
glucose, fructose, sucrose, maltose, raffinose etc., but poor on arabinose, cellobiose 
etc (Galun and Fraenkel 1957). 

Rather poorly known are the requirements of sterols, lipids, vitamins and salts of 
haematophagous insects for reproduction. An interesting example of sterol require- 
ment for reproduction is that of the rabbit fleas Spilopsyllus cuniculi and the hare flea 
Cediopsylla simplex. Rothschild and her colleagues did extensive studies on the 
reproduction of S. cuniculi and discovered that they could breed only when the blood 
level of corticosteroids of their host rabbit increases. They also found that the natural 
condition can be stimulated by external application (spraying) of hydrocortisone on 
the fleas. Progesterone induced ovarian regression in the rabbit fleas (Rothschild and 
Ford 1964a,b; 1966). However, reproduction of rat fleas X. cheopis and X. astia are 
not under the control of host's hormones (Prasad 1969, 1973, 1976). Deletion of 
cholesterol from an artificial diet for these rat fleas resulted in no yolk deposition 
(Kamala Bai and Prasad 1979a). Dimond et al (1958) found sterol having no 
influence on egg production of Ae. aegypti. Whether this sterol independence is the 
result of microbial action or not is not known. Lipid associated with serum proteins 
are important in nutrition and reproduction of tsetse fly Glossina morsitans. Flies fed 
on delipidated serum or delipidated albumin failed to reproduce. There was an 
apparent correlation between lipid content of the different albumins and the size of 



Nutrition and reproduction in haematophagous arthropods 259 

the offsprings produced by the flies fed on them (Kabayo 1982). Ae. aegypti can 
produce eggs in lipid-free diets (Dimond et al 1956). According to House (1958) it is 
unlikely that the adults are dependent on dietary lipids because lipids can be synthe- 
sized from carbohydrates and proteins. Recently it was found that arachidonic acid 
or certain structurally related long-chain polyunsaturated fatty acid appears to be a 
general mosquito requirement and must be present in the larval diet for production 
of viable adult Cx. pipiens (Dadd 1981; Dadd and Kleinjan 1978, 1979; Sneller and 
Dadd 1981). Arachidonic acid, a precursor of prostaglandin E2 (PGE2) are known to 
influence reproduction also (Dadd 1985). Dimond et al (1958) found no stimulation 
of egg production upon addition of vitamins and nucleic acids. Nayar and Sauerman 

(1977) concluded that nucleated RBC of amphibians, reptiles and birds did not 
confer any additional nutritive quality. Feeding the bed bug C. lectularius on folic 
acid deficient host resulted in reduced egg production. De Meillon and Golberg 
(1946, 1947a,b) found considerable reduction in egg production of the tick Ornitho- 
doros moubata fed on thiamine deficient rats. However, Wanchinga and Sonenshine 

(1978) found no effect on the tick Amblyomma maculatus fed on thiamin deficient 
rats. The major difficulty in interpreting this type of experiment is to draw a direct 
relation between deficiency and egg production. Vitamin deficiency could have 
several other secondary effects on the systems of the host such as for example accu- 
mulation of pyruvate in the blood due to thiamine deficiency and which could be 
toxic to the blood sucking insect (Nelson 1984). Filial and Madhukar (1969) found 
that adult Ae. aegypti emerging from biotin fed larvae showed follicular degeneration 
during postovipositional period and suggested that biotin interferes with certain 
metabolic processes of egg maturation. They also showed effect of biotin to be 
specific to ovary and not to testis. Vertebrate blood is deficient in B-vitamins. For 
those insects which subsist only on vertebrate blood, symbiotic microorganisms 
mostly concentrated in mycetomes in different parts of the abdomen including gut 
(Hippoboscid, Nycteribiid, Glossina, Cimicid, Reduviid and Anoplura) supply the 
necessary B-vitamins (Brookes 1964). Dimond et al (1958) found the presence of 
sodium and potassium in the diet increased egg production of Ae. aegypti. Cx. pipiens 
could lay eggs when fed on milk or on plant proteins, if Fe ions are present (Huff 
1929; De Boissezon 1933). Wanchinga and Sonenshine (1978) found calcium defici- 
ency in rats caused the ticks A. maculatus to lay fewer eggs and in these egg hatcha- 
bility was reduced. High concentration of calcium in the diet of rabbits was toxic for 
C. lectularius (De Meillon et al 1948). This mineral appeared to interfere with water 
excretion (Nelson 1984). De Meillon and Golberg (1947b) and De Meillon et al 
( 1 947) found no effect on egg production when C. lectularius and the tick 0. moubata 
were fed on ribofiavin deficient rats. 



3. Physiological aspects of nutrient influence on reproduction 

Engelmann (1970) suggested several pathways by which nutrition or nutritional 
factors may affect egg maturation. According to his scheme (p. 128), on the one hand 
food can directly influence egg maturation by supplying either the raw materials or 
food factors essential for the synthesis of yolk. On the other, food can have an 
indirect influence -on yolk synthesis via the central nervous/endocrine systems. With 
regard to haematophagous insects blood proteins are factors without which sus- 



260 R S Prasad 

tained oocyte development becomes impossible. Regression of developed oocytes 
when gonoactive female rat fleas were put on rat blood plasma diet which is sub- 
optimal for protein requirement is one of the examples (Kamala Bai and Prasad 
1976). 

Gut distension as a result of feeding (mechanical stimuli) appears to play a role in 
the egg maturation of certain haematophagous arthropods like the 5 mosquitoes 
An. maculipennis (Detinova 1953), Cx. pipiens (Larsen and Bodenstein 1959) and the tick 
0. tholozani (Gabby and Warburg 1976). In the two species of mosquitoes ingestion 
of a non-proteinaceous meal after anal occlusion resulted in initiation of egg 
maturation. Activation of corpora allata via the nervous system is suggested. 
However, in rat fleas X. cheopis and X. astia the role of gut distension is question- 
able. These fleas require more than one blood meal for the induction of yolk 
deposition. Milk (whole cow milk/milk diluted with normal saline) failed to induce 
yolk deposition. These fleas also feed on blood plasma if ATP is added, but the low 
protein concentration of this diet failed to induce yolk deposition (Kamala Bai and 
Prasad 1976). It is probably the haemolymph protein concentration or certain 
particular blood fractions (some peptides/amino acids?) which trigger yolk deposi- 
tion. Total haemolymph proteins increased by almost 2 times after feeding rat fleas 
X. cheopis and X. astia on a live rat. Whereas in fleas membrane-fed on 'plasma or 
sucrose, total haemolymph protein remained at unfed teneral level and showed no 
increase whereas sucrose fed fleas showed a very slight increase in the total 
haemolymph proteins. Fleas membrane-fed on whole blood of white rat and those 
fed on live rats showed almost comparable levels (slightly less in the former) of total 
haemolymph proteins (Narayana Prllai and Prasad 1986). 

As the endocrine influence is not the subject theme of this essay the reader is 
referred to excellent accounts on the influence of hormones on reproduction by 
Clements (1963), Engelmann (1970) and Hagedprn (1985). Much of the existing 
information on endocrine control of reproduction of haematophagous insects come 
from the studies on mosquitoes. As early as 1936, Wigglesworth demonstrated 
through decapitation and parabiosis experiments that corpora allata are necessary 
for egg maturation in Rhodnius prolixus. He also demonstrated a critical time after 
which removal of corpora allata, did not inhibit egg maturation. Later studies have 
shown this finding to be true for mosquitoes also. Though working with Ae. taenio- 
rhynchus, Ae. aegypti, Ae. sollicitans and Aedes triseriatus, Lea (1963) showed that allatec- 
tomy had varied effects on egg development within a single genus Aedes -and even 
within one species, in general both autogenous and anautogenous mosquitoes need 
hormones from the brain and corpora allata for egg maturation (Lea 1967). Lea 
'1970) showed that in autogenous forms the hormones were in circulation even 
without a blood meal while in anautogenous forms only the corpus allatum hormone 
was in circulation before a blood meal. Median neurosecretory cell (MNC) hormone 
iccumulates in the corpora cardiaca after emergence and it is released into haemo- 
.ymph only after a blood meal. The existing information on hormonal control of 
Dogenesis in mosquitoes may be summed up as follows. Two stages in oogenesis can 
De recognised. The 1st stage involves the previtellogenic development leading to 
resting stage after emergence in which they remain until after a blood meal. This 
stage is mediated by juvenile hormone from corpora allata (Gwadz and Spielman 
1973). In the second stage, blood meal triggers the release of a hormone from the 
MNC [egg development neuro-hormone (EDNH) described by Lea 1972] which in 



Nutrition and reproduction in haematophagous arthropods 261 

turn stimulate the ovaries to synthesize and release ecdysone. The ovarian ecdysone 
released into the haemolymph gets converted into 20-hydroxyecdysone (20-HE) in 
the fat body and triggers vitellogenin synthesis by the fat body cells (Hagedorn 1974, 
1985) and separation of the secondary follicles. Beckemeyer and Lea (1980) in 
Ae. aegypti and Redfern (1982) in Anopheles stephensi have shown that the presence of 
20-HE is necessary to stimulate the separation of a new follicle from the germarium. The 
general scheme on the action of hormones on gonotrophic cycles of anautogenous 
mosquitoes is given by Hagedorn (1985). In this scheme the process of egg matura- 
tion in mosquito is shown to require the sequential appearance of juvenile hormone 
(JH), EDNH and ecdysone for each gonotrophic cycle. JH has a direct stimulatory 
effect on vitellogenin synthesis even when the JH titer is declining, but this effect is 
seen only in the presence of 20-HE (Hagedorn 1986). Blood meal triggers the 
synthesis of both messenger and ribosomal RNA necessary for vitellogenin synthesis 
in Ae. aegypti (Hagedorn et al 1973). 

Van Handel and Lea (1984) showed that neither corpora allata nor the cerebral 
neurosecretory system, including the corpus cardiacum nor any ovarian factor is 
required to induce synthesis of vitellogenin in Ae. aegypti. However, continuous 
synthesis of vitellogenin depends on factors from both the head and ovaries. It is 
concluded that blood meal itself and not stretching of the gut is the stimulus needed 
to initiate vitellogenin synthesis and that a digestion product of blood either stimu- 
lates the fat body directly or some of the endocrine-like cells in the midgut which 
serve as transducers between the blood meal and fat body. Nayar and Sauerman 
(1977) working on Ae. aegypti, Cx. nigripalpus and Ps. columbiae found that blood 
clotted in the gut in 30 min and absorption of the separated serum components 
resulted in immediate initiation of oogenesis, but promotion of initiated oocytes 
followed the digestion of the coagulated blood. In the stable flies S. calcitrans sex 
pheromone secretion is under dietary control. Comparing blood or glucose-fed 
5. calcitrans Meola et al (1977) showed that only blood diet induced sexual 
behaviour and that sex pheromone (cuticular hydrocarbons) are produced only in 
the blood fed group. 

In Ae. aegypti JH level is high before feeding but declines after feeding. Meola and 
Petralia (1980) showed that JH influences blood feeding activity of Cx. pipiens and 
Cx. quinquefasdatus. JH deprivation caused by surgical removal of corpora allata 
shortly after adult emergence blocked the initiation of biting behaviour in these 
mosquitoes. 



4. Autogeny and nutrition 

Autogeny is defined as capacity to develop and lay eggs without a proteinaceous 
meal. Several haematophagous Diptera among others exhibit autogeny (Spielman 
1971). Since in such cases the adults lay eggs without a blood meal, the major source 
of yolk must be derived from the reserve nutrients carried over from the larval stages 
(Briegel 1969; Clements 1956; Spielman 1957). Twohy and Rozeboom (1957) found 
higher rates of lipids and glycogen in the larvae and pupae of the autogenous 
Culex molestus. Autogenous mosquitoes contain large stores of nutrient than 
anautogenous mosquitoes (Clements 1956; Lang 1963). Deficiencies in the larval diet 
can also prevent expression of autogeny in mosquitoes (O'Meara and Evans 1976). 



262 R S Prasad 

Chen (1967) during his electrophoretic studies on larval haemolymph of autogenous 
and anautogenous populations of Cx. pipiens discovered a protein fraction present in 
the former but not in the latter and suggested this to be involved in the endocrine 
control of autogeny. However, Spielman (1971) does not contribute to this idea. Lea 
(1964) found that the expression of autogeny in the highly autogenous strain of 
Ae. taeniorhynchus may be reduced by altering the quality and quantity of larval diet 
and withholding sugar from newly emerged females. 

Allatectomy within 1 h after emergence prevented autogenous yolk deposition in 
Ae. taeniorhynchus (Lea 1963). Larsen and Bodenstein's (1959) conclusion that in 
anautogenous forms corpora allata were inactive until activated by a blood meal 
whereas corpora allata of the autogenous females were active soon after emergence 
without the necessity for blood feeding is not supported by the findings of Lea (1963, 
1969) who showed that corpora allata of anautogenous female were active prior to 
the blood meal. Van Handel (1976) showed that the mafti source of yolk protein in the 
autogenous Aedes atropalpus is not the flight muscle but fat body itself. The contri- 
bution to yolk protein synthesis is negligible. Flight muscle may form a potential 
source of nutrient for autogenous production of yolk as has been suggested by 
Hocking (1954) in the case of non-haematophagous mosquito Aedes communis. This, 
however, is not the case in Ae. atropalpus. According to O'Meara and Evans (1977) 
the salt marsh mosquito Ae. taeniorhynchus has two types of autogenous females. One 
that requires a stimulus from mating and the other that does not. A substance from 
the male accessory glands (matrone?) appear to stimulate autogenous egg produc- 
tion. Autogeny, however, is also under poly genie control (Spielman 1957, 1971). 
Autogenous egg production can be viewed as an insurance against unfavourable 
situations. 



5. Nutrient metabolism and host visitation/feeding 

One gonotrophic cycle, bounded by two host seeking activities and feeding, is 
designated in the present context as an event of feeding and nutrient utilization 
culminating in oviposition. Frequency of feeding in the case of haematophagous 
insects, irrespective of whether they are permanent residents on the body of the host 
or spend short time on the host is controlled by the quantity of blood ingested and 
the pace of utilization of the ingested nutrient. In other words rate of utilization of 
the ingested nutrient or that which is already stored decides the frequency of host 
visitation and feeding. 

Factors which can regulate the pace of host visitation, feeding and utilization of 
ingested blood are outlined in figure 1. Obviously the first step in utilization would 
be the rate of digestion as most of the dietary components are to be first broken 
down before absorption and utilization for house keeping metabolic functions and 
reproduction. Permeability of the insect intestine to large peptides is restricted 
(Turunen 1985). Time required for oogenesis and blood meal digestion are inter- 
related in most gonoactive haematophagous insects and duration of oogenesis esta- 
blishes the minimum time between blood meals (Roberts et al 1983). 

Diet and dietary components appear to have very important role to play in rates 
of enzyme secretion and digestion. A secretogogue (specific dietary components elicit 
a direct response in the intestinal cells involved in enzyme synthesis) control of 



Nutrition and reproduction in haematophagous arthropods 



263 




Rote of utilization 



Rate of digestion 
Rate of vitellogenin synthesis ? 
Rate of oocyte development 
Spacing of successive 
gonotrophic cycles 
Environmental influence 



Frequent 



Frequencies of host 
visitation and feeding 



Required quantity of blood 
ingested in one feed 



Required quantity of 
blood not ingested in 
one feed 



Physical or other 
restraints 



Defensive behaviour 
of the host 

Limitation in extensibility 
of abdomen 



Figure 1. Role of nutrient intake on host visitation and feeding frequencies. 



protease production is indicated in the work of Akov (1972), Briegel an4 Lea (1975), 
Fisk and Shambaugh (1952), Gooding (1973, 1974), Prasad (1979), Shambaugh (1954) 
and others. The active principle which induce protease activity appears to be in the 
serum (Gooding 1974; Langley 1966). G. palpalis requires serum albumin for 
normal digestion of ingested RBC and normal rate of fecundity (Takken 1980). 
Chapman (1985) has compiled results of various workers on factors of host's blood 
which induce protease Activity in Rhodnius, Aedes and Glossina. Results have also 
suggested a control by MNC possibly through ecdysone (Briegel and Lea 1979). 
Secretogogue mechanism is now thought to be an immediate response to food con- 
sumption and control by hormones a later development (Applebaum 1985). Protein 
quality influences production of protease (Akov 1972; Gooding 1974). However, 
Yang and Davies (1968) showed trypsin activity to be almost identical in female 
Simulium venustum ingesting human, cow or duck blood or blood cells suspended in 
sucrose. There are reports for and against the possibility of source of blood meal 
influencing rate of digestion. Downe et al (1963) found that the source of blood had 
little effect on digestion rates of several spp. of Aedes, but did influence the digestion 
rate of Mansonia perturbans. Tempelis and Lofy (1963) found Culex tar sails digesting 
blood from 3 different spp. of birds at different rates. No marked difference was 
noticed by Downe (1957) in the rate of digestion of horse and guinea-pig blood by 
several spp. of blackflies. Louse, P. humanus a highly host specific species showed 
difficulty in digesting guinea-pig blood. Weitz and Buxton (1953) found C. lectularius 
digesting human blood slowly. There is some objection that precipitin test used for 
the study of rate of digestion actually demonstrates the differential holding of blood 
fractions in the gut rather than rate of digestion. 

It was already mentioned earlier that serum of vertebrate blood contain some 
substance which stimulate protease synthesis. Paradoxically enough, experiments 
have revealed that certain vertebrate blood sera also contain enzyme inhibitors. 



264 RSPrasad 

Trypsin inhibitor is present in the sera of 17 vertebrates. Two of these have been 
purified and were shown to be associated with a-globulin fractions of the serum 
(Huang 1971a,b). An inhibitor associated with a-globulin of horse and human sera 
was shown to inhibit chymotrypsin from Ae. aegypti (Yang and Davies 1971). How 
the stimulatory and inhibitory factors present in the serum interact in vivo conditions 
and how these influence the rate of digestion of blood from different hosts needs 
clarification. For example chick blood serum contains more Ae. aegypti trypsin 
inhibitor than any other blood sample, yet chicks are good hosts for laboratory 
rearing of these mosquitoes. According to Gooding (1972) as digestion of only a 
small fraction of the ingested blood proceeds at a time, the chances of inhibitor being, 
titrated out or destroyed would be more than if the enzyme were to mix with the 
whole blood meal at the same time. Whatever may this be, what biological function, 
if any at all, do these inhibitors play in the nutritional physiology of haematopha- 
gous insects is not clear. Judson (1986) mentions that inclusion of trypsin inhibitor in 
blood reduces or blocks the initiation of vitellogenesis. 

Feeding and ovarian development inhibit host seeking behaviour (HSB) and 
subsequent feeding until after oviposition. Inhibition of HSB is related to the 
quantity of blood ingested. For effective inhibition the meal size should be more than 
2-5 fil (Klowden and Lea 1979b). According to them this postprandial inhibition 
consists of two phases probably controlled by two different mechanisms. An imme- 
diate phase (distension inhibition) in Ae. aegypti is due to the distension of the ante- 
rior portion (1st 3 segments) of the abdomen. The second phase (oocyte inhibition) 
does not appear to be related to distension but due to humoral factor(s), as trans- 
fusion of haemolymph from gravid Ae. aegypti females 48 h post-blood-meal (PBM) 
into sugar-fed females inhibited host seeking behaviour (Klowden and Lea 1979a). 
These authors also state that it is unlikely that the increase in haemolymph amino 
acids/protein as a result of feeding (Armstrong 1968) is contributing to inhibition of 
host seeking behaviour, because saline enemas had the same effect as blood enemas. 
However, in rat fleas X. cheopis and X. astia, Geetha Devi and Prasad (1980) showed 
that injection of 50% bovine albumin into haemocoel reduced the quantity of blood 
ingested by unfed teneral females, while injection of a 10% albumin was sufficient to 
cause a quantitative reduction in blood intake by 7-day-old female fleas. This 
difference between unfed teneral and older females is attributed to the increase in 
total haemolymph protein in gonoactive females (Narayana Pillai and Prasad 1986). 
Surgical manipulations of ovaries have shown that these are not the source of the 
inhibitor in Ae. aegypti (Klowden and Lea 1979a). However, transplantation of fat 
body from blood-fed Ae. aegypti females into sugar-fed, inhibited the HSB indicating 
this as a source of the factor(s) (Davis and Bowen 1986). Beach (1979) showed that 
high titer of ecdysone inhibit feeding. If vitellogenins play any role in inhibition of 
HSB, it is not known. Inhibition of HSB starts about 30 h after blood meal, reaches a 
peak by 48 h and terminates after oviposition (Davis and Bowen 1986). Haemo- 
lymph titer of vitellogenin reaches a peak in Ae. aegypti around 27 h after the blood 
meal and remains in detectable level up to about 45 h (Ma 1986). Speaking of 
Ae. taeniorhynchus O'Meara and Evans (1977) state that as long as the ovaries remain 
undeveloped the females continue to be active blood feeders. Briegel and Lea (1979) 
showed that nonoogenic female Ae. aegypti secrete a quantum of nitrogen equivalent 
to total nitrogen normally observed in maturing ovaries. This shows that in non- 
oogenic females there is no protein build up necessary for vitellogenesis. If this has 



Nutrition and reproduction in haematophagous arthropods 265 

any effect on feeding inhibition, does it have any role to play in rat fleas where female 
specific lipoglycoprotein fractions are detectable even in newly emerged unfed female 
fleas (Narayana Pillai and Prasad 1986)?. There appears to be a similarity between the 
condition of cecropia (Pan 1971) and rat fleas (Narayana Pillai and Prasad 1986) 
where vitellogenins appear in the haemolymph before vitellogenesis starts. Rapid 
diuresis soon after feeding concentrates the protein and a meal quantitatively 
adequate to initiate egg maturation is retained in the gut until shortly before 
oviposition. This retention may be due to cohesive nature of the meal, peritrophic 
membrane, physical' barrier between mid and hind gut and/or hormonal feed back 
from the ovary. This retention at any rate ensures continued supply of nutrient for 
vitellogenin synthesis (Freyvogel and Staubli 1965; Nijhout and Carrow 1978; 
Rosenberg 1980). 

Frequencies of host visitation and feeding would increase if adequate quantity of 
blood is not ingested. This may be due to the defensive behaviour of the host or 
physical restraints like limitations in the extensibility of the abdomen. There is 
considerable interspecific variation in defensive behaviour of hosts and the studies of 
Edman and his coworkers have shown these variations influence the success of 
feeding and quantity of blood ingested by mosquitoes (Edman and Kale 1971; 
Edman et al 1974; Webber and Edman 1972). Under such conditions if the mosquito 
has to lay eggs, repeated feedings would be necessary. Rat fleas X. cheopis and 
X. astia are typical examples where extensibility of the abdomen constraints inges- 
tion of large quantity of blood required for the complete development of eggs. The 
small quantity of blood ingested along with low protein storage capacity of haemo- 
lymph 'make it necessary for these fleas to have frequent blood meals. In other words 
they have to live in close proximity of the host if they are to reproduce normally 
(Prasad 1986). 



6. Influence of nutrition on male maturation 

In general, spermatogenesis is completed by the time adult insect emerges. So it is 
unlikely that in most cases adult nutrition has any influence on spermatogenesis. 
However, in the testes of certain fleas, the exit of sperms into epididymis is blocked 
by a cellular plug which is dissolved only after feeding. Rothschild et al (1970) repor- 
ted this to be the case in X. cheopis, Nosopsyllusfasciatus and S. cuniculi. They showed 
that male maturation (dissolution of the plug) is controlled by hormones of the host 
in S. cuniculi. Whereas in X. cheopis such a hormonal relationship is not seen. Blood 
feeding resulted in dissolution of the plug in 100% of X. astia and 75% of X. cheopis 24 h 
after the meal. When on a saline diet only 55% of X. astia and 40% of X. cheopis 
matured. These studies indicated a dependency of proteinaceous diet for male 
maturation. If those matured on a saline diet probably needed only a feeding 
stimulus and if this condition has any relation to larval diet is not known (Kamala 
Bai and Prasad 1979b). In the case of most ticks, feeding is intimately associated with 
male maturation. In most Metastriata, meiosis and spermatid production occurs 
only after an adult blood meal. Adult blood meal accelerates germinal development 
in males of Argas and Ornithodoros but not in Otobius and Antricola. As males of 
many Ixodes spp. do not feed, adult blood meal has no role to play in spermato- 
genesis (Oliver 1974). Many cyclorrhaphous Diptera (e.g. S. calcitrans, H. irritans) do 



266 R S Prasad 

not become sexually active or become capable of inseminating females until after a 
proteinaceous meal. What exactly are the nutritional implications in these observa- 
tions are not known (Anderson 1974). 



7. Conclusions 

Vertebrate blood as a diet has many fascinating facets. Arthropods have successfully 
adopted this as a source of nourishment probably even since Permian. Among insect 
orders Diptera, Siphonaptera, Anoplura and Hemiptera exhibit haematophagy. 
Feeding dichotomy both phytophagy and zoophagy is characteristic of nematoceran 
and brachyceran Diptera. But in these cases proteins derived from blood meal are 
essential for egg production, with exceptions of autogenous forms. These insects use 
a wide variety of hosts ranging from amphibians to mammals. Ticks are obligate 
blood feeders and blood is essential for egg production. Among mites members 
belonging to families Psoroptidae, Dermanyssidae and Macronyssidae exhibit 
haematophagy. 

Both quantity and quality of the blood meal influence reproduction. Size of blood 
meal affect refeeding behaviour, midgut protease activity and of course fecundity. 
With certain exceptions, strict gonotrophic concordance is seen in many haemato- 
phagous insects where there is an alternation of blood feeding and oviposition. There 
is a positive correlation between quantity of blood ingested and number of eggs laid. 
Nutritionally (measured as rate of egg production) human blood appears to hold the 
least value compared to blood from other hosts for many blood sucking insects, but 
for C. hemipterus, blood from the reptile Calotes versicolor gave the least value. 
Nutritional inequalities can arise as a result of differences in the constituent amino 
acids. The nutritional value of any protein is a function of its amino acid composi- 
tion. 

Carbohydrates derived from nectar/plant juices enhance longevity and supplies 
energy for flight and does not have a direct role in egg production. But sugar diet in 
some cases inhibit or increase egg production. Rather poorly known are the roles of 
sterols, lipids, vitamins and salts in reproduction. An interesting example of sterol 
requirement is the case of rabbit flea S. cuniculi where the hormones of the host 
influence reproduction of the fleas. While sterols did not influence egg production of 
Ae. aegypti, deletion of cholesterol from the diet of rat fleas inhibited yolk deposition. 
Since lipids can be synthesized from carbohydrates and proteins, their absence from 
the diet does not adversely affect reproduction. However, arachidonic acid, a pre- 
cursor of prostaglandin, is essential in the larval diet of Cx. pipiens for production of 
viable adults. Vertebrate blood being poor in vitamin B complex, the deficiency is 
made good by the symbiotic microorganisms. However, folic acid, thiamine and 
biotin are shown to influence egg production in certain species. But experiments on 
measuring the influence of vitamin B deficiency by feeding insects on deficient hosts 
should be viewed with caution as vitamin deficiency in the host can produce side 
effects which themselves could be injurious to the insects. Certain metallic ions like 
sodium, potassium^ iron and calcium appear to influence reproduction of certain 
species. 

Feeding in addition to supplying raw materials for egg production also induce the 
release of hormones from the endocrine glands. At least in some cases, the mecha- 



Nutrition and reproduction in haematophagous arthropods 267 

nical stimuli from gut distension could trigger release of gonadotrophic hormones. It 
is possible that this could be achieved by chemical stimuli (peptides, amino acids?) 
from the diet. 

Autogenous egg production is under the control of larval diet and genetic consti- 
tution. 

Quantity of blood ingested and its rate of utilization decide the frequency of host 
visitation and feeding. Rate of digestion is one of the important aspects in utilization 
of the ingested food. Source of blood meal, rate of enzyme production, presence of 
enzyme inhibitors in the blood of hosts, environmental factors etc could affect the 
rate of digestion and so the rate of utilization. There is a definite postprandial inhibi- 
tion of HSB at least in mosquitoes. The immediate cause may be abdominal dis- 
tension and later inhibition is thought to be mediated by humoral factor(s). 
Ecdysone is known to inhibit feeding. If vitellogenin in the haemolymph has any 
influence, it is not proved experimentally. 

Adult nutrition has no direct influence on spermatogenesis in insects, male 
maturation is influenced by hormones of the host in rabbit fleas and feeding on a 
proteinaceous diet (nutritional?) in rat fleas. Cyclorrhaphous Diptera become 
sexually competent only after a blood meal. Adult blood meal has a decisive role to 
play in spermatogenesis and maturation of many species of ticks. 

A scan through the literature cited in the present review would show that a large 
quantum of work on nutrition and reproduction has turned out centering around 
mosquitoes. Much more work is needed to understand the mechanisms involved in 
other groups like fleas, bugs and ticks. 



Acknowledgements 

I am grateful to my students Sri R Sreekumar and Sri R Rajendran for the help 
rendered in preparing the manuscript. 

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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 275-280. 
Printed in India. 



Feeding and reproduction in vector mosquitoes 

R REUBEN 

Centre for Research in Medical Entomology, Madurai 625 003, India 

Abstract. Mosquitoes depend on high fecundity to compensate for heavy immature 
mortality. Important factors affecting fecundity are (1) Body size, dependant on larval 
nutrition (2) Amount of blood ingested (3) type of blood ingested. With the development of 
highly sensitive techniques for determination of bloodmeals of wild mosquitoes many 
puzzling variations in host selection behaviour are now capable of explanation. 

Keywords. Mosquitoes; fecundity; bloodmeal identification. 



1. Introduction 

The mosquitoes are r-strategists, falling within Odum's definition of species which 
display a' high ratio of reproductive to maintenance effort (Odum 1983). How 
successful this strategy is for species survival is borne out by the fact that strenuous 
human efforts have totally failed to eradicate even a single species of mosquito from 
any part of the world, except in the case of Anopheles gambiae from Brazil and Egypt, 
where the species was a recent entrant and had not established itself. Mosquitoes 
depend on a high fecundity to compensate for heavy immature mortality. Mean 
mortalities of 99-1 and 97-6% respectively were recorded for Culex vishnui and 
A. peditaeniatus larvae in Philippine paddy fields, of which about 90% could be shown 
to be due to predators, including Anisoptera (Mogi et al 1984). Predation was not 
important in C. quinquefasciatus breeding in wells in Delhi villages, where mortality 
was strongly density dependant (Rajagopalan et al 1976). In both cases the key to 
survival was high fecundity, which made it possible for the species to tide over 
adverse periods and to build up high population levels in favourable seasons, in spite 
of the operation of strong mortality factors. As in other insects, fecundity in 
mosquitoes is closely associated with nutrition both at the adult as well as at the 
larval stage. These relationships have been extensively studied, particularly since 
their blood-feeding habit is linked with the transmission of pathogens of medical 
importance. 



2. Fecundity 

The average number of eggs laid during one gonotrophic cycle is a species 
characteristic, and varies considerably between species. In general the larger species 
lay more eggs than the smaller species. Even within the- same species, individual egg 
batches show considerable variation. Factors affecting fecundity can be grouped 
under 3 heads; (i) body size, (ii) amount of blood ingested and (iii) type of blood 
ingested. 

275 



276 R Reuben 

2.1 Body size 

The number of eggs laid shows a positive correlation with the size of the female in 
several genera of mosquitoes. In Aedes aegypti the total number of ovarioles 
available for development in both the ovaries showed a positive correlation with the size 
of the female, varying from 50-150 (Colless and Chellapah 1960). In addition, large 
females of this species ingested more than twice as much blood as small females (Bar- 
Zeev 1957). Undersized females of A. aegypti generally need 2 blood meals before 
they can develop eggs (Macdonald 1956). Body size itself is determined by nutritional 
status, large females being produced from well fed larvae, and undersized adults .. 
under conditions of starvation and overcrowding. This provides a neat feed-back 
mechanism for reducing egg production when food resources are a limiting factor. 

Larval nutrition plays an important role in the phenomenon of autogeny, which is 
recorded in several genera, but has been best studied in C. pipiens molestus. 
Autogenous mosquitoes are able to lay a single egg raft without feeding, but require 
a blood meal for maturing subsequent egg batches. Autogenous females use reserves 
carried over from the larval stage to develop the first batch of eggs. Some workers 
have found that poor larval nutrition reduces the number of eggs laid by autogenous 
females, but that even larvae near the starvation limit produced females which were 
all autogenous. Other workers found that poor larval nutrition lowered both the 
percentage of laying females as well as the number of eggs produced by an 
autogenous population (Clements, 1963). 

2.2 Amount of blood ingested 

In autogenous mosquitoes engorging by the female stimulates the secretion of a 
gonadotrophic hormone, resulting in the development of the ovaries. The hormone 
thus links ovary development with the supply of nutrients. It has been shown 
experimentally that distention of the abdomen for a sufficient period, even with 
liquids other than blood, stimulates afferent nervous impulses leading to the release 
of gonadotrophic hormones by the neurosecretory cells of. the brain and the corpora 
allata (Larsen and Bodenstein 1959). The subsequent development of individual 
oocytes depends on the availability of nutrients. After a sufficiently large blood-meal 
the majority of the follicles in the resting stage begin to develop, but a number of 
them degenerate during the early- stages of oocyte development. The number of 
follicles degenerating is greater after a nutritionally inadequate meal (Hosoi 1954) 
and it is also high in older females which have passed through several gonotrophic 
cycles (Detinova 1949). 

Competition between follicles for available nutrients has been demonstrated by 
transplanting ovaries at the resting stage into the abdomens of females of the 
autogenous females of C. pipiens molestus. When the host was less than 16 h old 
the transplanted ovaries developed to maturity and suppressed development of the 
host ovaries. When the host was however, 50-65 h old, its own ovaries continued to 
develop, but the transplanted ovaries did not (Larsen and Bodenstein 1959). The 
exact nature of the inhibition was not clear. 

2.3 Type of blood ingested 

Early work in which blood fractions were fed to mosquitoes suggested that protein 



Feeding and reproduction in vector mosquitoes 211 

was the only essential requirement for egg production, and this was confirmed when 
synthetic diets of amino acids were fed to female A. aegypti (Dimond et al 1956). 
Eight amino acids were essential for complete development. These were arginine, 
isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan and valine. In 
addition, when either histidine or methionine was omitted from the diet, a few eggs 
were laid a few days after the start of the experiment, but not later. This was 
interpreted to mean that these two amino acids were carried over from the pupa in 
small quantities, but when these stores were depleted no further egg production 
occurred. Ten amino acids .were therefore essential. Addition of cystine and glutamic 
acid to the diet enhanced egg output. 

It is generally believed that mosquitoes will develop more eggs when fed on the 
preferred natural host. However, little work has been done on this aspect. C. pipiens 
laid on an average 40 eggs/mg of human blood and 82 eggs/mg of bird blood 
(Woke 1937). It is possible that the nucleated erythrocytes of bird blood are more 
nutritive than the anucleated erythrocytes of mammals. However, addition of nucleic 
acids to the diet of A. aegypti did not raise egg production. In the same species, egg 
production was lower when females were fed on whole blood of man, sheep or ox, in 
which the isoleucine content of the haemoglobin is low, as compared with the high 
egg production when fed on whole blood of pig or rabbit, which have a higher 
isoleucirfe content. Addition of isoleucine to human, sheep or ox-blood raised the 
level of egg production to that obtained with pig or rabbit blood (Lea et al 1956). 

3. Feeding preferences of wild mosquitoes 

Host selection by adult mosquitoes in nature will, therefore, play a role in population 
dynamics by influencing fecundity. It has also a direct bearing on vectorial capacity, 
species showing a higher degree of anthropophily being more efficient in transmitting 
pathogens to man. Much attention, has therefore been devoted to the determination 
of the source of blood meal of wild mosquitoes. Basically the test consists of 
antigen-antibody reactions between serum antigens of mosquito blood-meals of 
unknown origin and specific antisera, resulting in visible precipitates. Specific 
antisera of representing animals of the area .can be raised in cockerels and rabbits. 
The test can be conducted either in solution in small glass tubes (precipitin test) or 
in semi-solid agarose medium (agarose gel diffusion technique and radial immuno- 
assay). The speed and sensitivity of the agarose gel assay can be enhanced by 
electrophoretic methods. In the precipitin test the antigen-antibody reaction is seen 
as a ring; in agar gel diffusion and electrophoretic methods as bands/arcs; and in 
radial immunoassay as circular zones. Of the 4, the electrophoretic method is the 
most sensitive, followed by gel diffusion, radial immunoassay and precipitin test 
(Bheema Rao 1984). 

For most purposes it is sufficient to have relatively broadly reacting antisera, and 
to distinguish between bloods of humans, birds, bovines and so forth. However, it is 
possible to raise antisera which are order specific and even species specific (Tempelis 
and Reeves 1962a, b), and this has been used in the study of vectors of viral 
encephalitides, where particular orders and families of birds are known to be 
amplifying hosts of viruses. Thus Reeves et al (1963) studied the feeding habits of C. 
tarsalis, the vector of Western equine and St. Louis encephalitis, in California. 3,310 
engorged females were tested. In summer 84-4% of the females had fed on birds, 
which included chickens, doves and passerines. Cattle, dogs, horses, rabbits and cats 



278 R Reuben 

accounted for 13-9% of the feeds. During winter, 98% of the feeds were on birds, 
mostly passerines, while only 14% were on mammals. This reflects the flocking 
together of passerines during winter, and the absence of doves and chickens. It is also 
technically feasible to distinguish different haemoglobin types of mosquito blood- 
meals of human origin. Thus, by testing blood-fed A. qambiae and C. quinque- 
fasciatus collected inside bed-nets under which mother and baby pairs with different 
haptoglobin types slept, it was possible to show that the mothers were fed on much 
more by mosquitoes than the babies (Boreham et al 1978). 

The choice of host in nature depends on (i) host availability as well as (ii) innate 
species preference. The influence of host availability is clearly demonstrated in the 
results of precipitin tests carried out on blood meals of C. quinquefasciatus collected 
in villages near Delhi. Among specimens collected in the centre of the village 93-3% 
was fed on man and 1-7% on birds. At the periphery 64-2 and 25-2% were fed on man 
and bird respectively, while among those mosquitoes collected in pump houses 
outside the village only 17-7% were fed on man while 53-0% were fed on bird. During 
the hot season, when these collections were made, heavy mosquito breeding was 
found in agricultural wells, which were not in use at this season. Mynas and pigeons 
were found breeding in the Persian wheel containers and mud embankments, and 
presumably the mosquitoes were able to maintain a cycle independent of the blood 
sources within the village (unpublished data of the ICMR-WHO Research Project 
on Genetic Control in Mosquitoes, New Delhi). Seasonal shifts in choice of hosts, as 
in the case of C. tarsalis in California cited above, are often due to seasonal shifts in 
host availability; in this case due to migration of some species of birds out of the area 
and tethering of domestic stock indoors during the winter. A. culicifacies, a normally 
zoophilic species with a strong preference for bovine blood, shows seasonal changes 
in feeding behaviour in Pakistan, where there is a higher proportion of manfeeding in 
the summer months when villagers sleep out of doors, as compared with the winter 
months when they sleep indoors under quilts (Reisen and Boreham 1979) a 
different kind of host non-availability. This opportunistic host selection behaviour of 
mosquitoes, particularly those species which are basically zoophilic, can be exploited 
to divert them from human hosts (zooprophylaxis). It was shown that keeping cattle 
under the same roof as man (mixed dwelling) increased contact between A. aconitus 
and man, while if the cattle were kept in open cattle sheds, without roof or -walls, 
away from human dwellings, man-vector contact fell to extremely low levels, as 
shown by the human blood index as well as direct collections from human bait 
(Kirnowordoyo and Supalin 1986). 

While host availability can alter mosquito feeding behaviour according to 
circumstances, there are nevertheless strong innate preferences for particular hosts, 
which are species specific. Mosquitoes may be broadly classified as anthropophilic or 
zoophilic according to their preference for human blood or animal blood 
respectively. Some of the most dangerous disease vectors are anthropophilic, for 
example A. gambiae, A. minimus and A. fluviatilis, vectors of malaria, A. aegypti, the 
vector of dengue haemorrhagic fever and C. quinquefasciatus, the vector of 
bancroftian filariasis. There are also efficient zoophilic vectors of malaria, for 
example, A. culicifades, and zoonotic diseases of man are primarily transmitted by 
zoophilic mosquitoes, for example C. tritaeniorhynchus and allied species which transmit 
Japanese encephalitis virus. The feeding preferences of these species were extensively 
investigated in North Arcot District and clear differences in the feeding behaviour of 



Feeding and reproduction in vector mosquitoes 279 

various common species of mosquitoes emerged. In the same villages, with the 
identical hosts available to them, species of the C. vishnui subgroup, all important 
vectors of JEV, were strongly attracted to cattle, and there were few man feedings 
reported. On the other hand C. bitaeniorhynchus showed a higher percentage of 
feedings on birds than on cattle, and 4-6% were fed on man (Christopher and Reuben 
1971). 

Blood-meal testing has been carried out most extensively on species of Anopheles, 
particularly in the early years of the National Malaria Control and Eradication 
Programmes. A number of puzzling anomalies were revealed of the same species 
exhibiting anthropophilic behaviour in one geographical area and zoophilic 
behaviour in another. These differences could not always be related to host 
availability. It has now been shown that many taxa, previously considered to be 
species, are in fact complexes of sibling species which are morphologically identical, 
but can be differentiated by cytological or electrophoretic analysis. A. culicifacies is 
in reality a complex of 4 sibling species provisionally named A, B, C and D. In the 
Thenpennai riverine tract, 434 specimens of species A were tested, of which 21-4% 
had fed on man, while on 1-8% of 398 tests on species B were positive for human 
blood. Since species A predominates in the dry season, and species B in the wet 
season, the population of A. culicifacies shows an apparent seasonal change in feeding 
behaviour which is due to the change in proportion of the different component 
sibling species (Tewari et al \ 984). Research in this area is rapidly advancing and 
many anomalies in host selection patterns will soon be capable of explanation. 



References 

Bar-Zeev M 1957 The effect of density on the larvae of a mosquito and its influence on fecundity; Bull 

Res. Counc. Isr. 6 220-228 
Bheema Rao U S 1984 A rapid method for identification of mosquito blood meal; Indian J. Med. Res. 79 

836 
Boreham P F L, Chandler J A and Jolly J 1978 The incidence of mosquitoes feeding on mothers and 

babies at Kisumu, Kenya; J. Trop. Med. Hyg. 81 63 
Christopher S and Reuben R 1971 Studies on the mosquitoes of North Arcot District, Madras State, India: 

Host preferences as shown by precipitin tests; J. Med. Entomol. 8 314 
Clements AN 1963 The Physiology of Mosquitoes; (London: Pergamon Press) pp 174-175 
Colless D H and Chellapah W T 1960 Effects of body weight and size of blood-meal upon egg production 

in Aedes aegypti (Linnaeus) (Diptera, Culicidae); Ann. Trop. Med. Parasitol 54 475-482 
Detinova T S 1949 Physiological changes in the ovaries of female Anopheles maculipennis; Med. Parasit. 

Moscow 18 410-420 
Dimond J B, Lea A O, Hahnert W F and DeLong D M 1956 The amino acids required for egg production 

in Aedes aegypti; Can. Entomol. 88 57-62 
Hosoi T 1954 Egg production in Culex pipiens pallens Coquillett. III. Growth and degeneration of ovarian 

follicles; Jpn. J. Med. Sci. Biol 7 111-127 
Kirnowordoyo S and Supalin 1986 Zooprophylaxis as a useful tool for control of A aconitus transmitted 

malaria in Central Java, Indonesia; J. Commun. Dis. 18 90 
Larsen J R and Bodenstein D 1959 The humoral control of egg maturation in the mosquito; J. Exp. Zool. 

140343-381 
Lea A O, Dimond J B and DeLong D M 1956 Some nutritional factors in egg production by Aedes 

aegypti; Proc. Tenth Int: Congr. Entomol 3 793-796 
MacDonald W W 1956 Aedes aegypti in Malaya II. Larval and adult biology; Ann. Trop. Med. Parasitol 

50 399-414 
Mogi M, Miyagi I and Cabrera B D 1984 Development and survival of immature mosquitoes 

(Diptera: Culicidae) in Philippine rice fields; J. Med. Entomol. 21 283-291 



Reuben 

ium E P 1983 Basic Ecology, 3rd edition (Tokyo: CBS College Publishing) 

jagopalan P K, Yasuno M and Menon P K B 1976 Density effect on survival of immature stages of 

Culex pipiens fatigans in breeding sites in Delhi villages; Indian J. Med. Res. 64 688 
eves W C, Tempelis C H, Bellamy R E and Lofy M F 1963 Observations on the feeding habits of Culex 

tarsalis in Kern Country, California, using precipitating antisera produced in birds; Am. J. Trop. Med. 

Hyg. 12 929 
isen W K and Boreham P F L 1979 Host selection patterns of some Pakistan mosquitoes; Am. J. Trop. 

Med. Hyg, 28 408 
mpelis C H and Reeves W C 1962a The production of a specific antiserum to bird serum; Am. J. Trop. 

Med. Hyg. 11 294 
mpelis C H and Reeves W C 1962b The production of immunological unresponsiveness in the chicken 

to produce a species specific antiserum to bird serum; Am. J. Trop. Med. Hyg. 11 298 
wari S C, Mani T R, Suguna S G and Reuben R 1984 Host selection patterns in anophelines in riverine 

villages of Tamil Nadu; Indian J. Med. Res. 80 18 
oke P A 1937 Comparative effects of the blood of man and of canary on egg production of Culex pipiens 

Linn.; /. Parasitol. 23 311-313 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 281-291. 
Printed in India. 



Host preferences in some pentatomids and related impact on the 
fecundity of their parasitoids 

R VELAYUDHAN 

Entomology Research Institute, Loyola College, Madras 600 034, India 

Abstract. Two species of polyphagous parasitoids, Gryon sp and Telenomus lucellus, 
parasitising the pentatomids Acrosternum gratninea and Agonoscelis nubila were subjected to 
host preference studies and consequent impact on the rates of parasitism of these host eggs. 
Chemical composition of these eggs were correlated with the age specific fecundity of the 
parasitoids. Polyphagous pentatomids like Acrosternum graminae had varying fecundity on 
different host plants like Cleome viscosa, Gynandropsis pentaphylla, Leucas aspera, Croton 
sparciflorus, Acalypha indica etc and this variation was found to depend on the chemical 
parameters of these host plants. The overall egg output of the pentatomids was also found 
to influence the percentage of parasitism. 

Keywords. Parasitoids; pentatomids; tritrophic interactions; life table studies; intrinsic rate 
of natural increase; fecundity index; biochemical analysis. 

1. Introduction 

The conceptual and factual basis for understanding the multifaceted nature of 
trophic interactions between plants, insects and parasitoids/predators has been 
established very clearly in the long progression of experiments that has led to our 
modern understanding of Batesian-Mullerian mimicry complexes (Rothschild 1972; 
Gilbert 1983; Duffey et al 1986). A more dramatic, but seemingly unnatural example 
of the movement of chemicals through multiple trophic levels was demonstrated by 
showing the ability of insecticides to bioaccumulate at given foci in the food chain 
(Kilgore and Li 1976). The nutritional suitability of a host was proposed by Flanders 
(1937) and Salt (1938) to be important for the survival of a parasitoid. House (1961) 
concluded that the nutritional needs of parasitoids are probably similar to most 
other organisms, and the problem of nutritional suitability may not be due to lack of 
specific nutrients or accessory growth factors in the host, but the quality and ability 
to obtain certain nutrients present in the host tissues at the proper time and to 
compete with the host tissues for the available nutrients. Yet another important 
factor is the amount of nutrients available to the parasitoid since a host (egg) is a 
finite food resource which because of its size, age and nutritional history may affect 
parasitoid development and reproduction (Vinson and Iwantsch 1980). Smaller host 
eggs yield smaller parasitoids with reduced emergence, fecundity and longevity or 
more males (Vinson and Iwantsch 1980). Female parasitoids may have the ability to 
assess host size and deposit fertilised or unfertilised (male) eggs accordingly (Nazato 
1969), or they may deposit eggs of both sexes with preferential survival of one sex in 
larger hosts (Holdaway and Smith 1932). Within solitary endoparasitoids, Miles and 
King (1975) reported decreases in the parasitoids developmental time with increasing 
host age at the time of parasitoidism, which may be due to nutrients in older hosts 
being more abundant (Vinson 1984). The present paper attempts to highlight the 
tritrophic level of interactions between the host plant, the pentatomid host and its 

281 



282 R Velayudhan 

natural enemies especially with reference to the flow of lipids through the system in 
greater detail. 

2. Materials and method 

2.1 Biochemical analysis 

The chemical composition of the various host plants and the eggs of pentatomids 
were analysed using the following standard procedures. Total carbohydrates (Dubois 
et al 1956); total phenols (Bray and Thrope 1954); total proteins (Lowry et al 1951) 
and total lipids (Folch et al 1957). 

In order to analyse the qualitative fatty acid profile of the host plants and the eggs 
of the host, the total lipids were extracted using the following procedure to the 
material was added 10 ml of ethanol, 3 ml of 28% ammonium hydroxide, 25 ml of 
petroleum ether and 25 ml of diethyl ether in a separating funnel, this mixture was 
shaken for 5 min and allowed to stand for separation for 20 min. The bottom layer 
was drained off and the ether fraction was dried to which 3 ml of 0-5 N NaOH in 
methanol was added and heated in a steam bath for 15 min. To this 5 ml of distilled 
water was added and 2 N HC1 were added slowly until the pH reached approxi- 
mately 2. The fatty acid methyl esters were then extracted into 5 ml of petroleum 
ether and 5 ml of diethyl ether from the acidified methylated lipid extract. Fatty acids 
were analysed by the Hewlett Packard HPLC system at 230 nm using an Hypersil 
ODS 5 ,um column with water and acetonitrile as solvents at a flow rate of 0-45 ml 
according to the gradient programme as per Schuster (1985). The retention time and 
area percentage of fatty acid methyl esters were recorded. 

2.2 Life table 

Five adult female parasitoids of both species (Gryon sp and Telenomus lucellus) soon 
after emergence were allowed to mate with their male counterpart ad lib and these 
pairs were isolated and kept in separate plastic vials along with 25 host eggs. They 
were also provided with a cotton bud soaked in dilute honey. Every day these 
females were provided with 25 fresh host eggs and the previous egg masses were 
labelled and kept for observation of the emergence of the parasitoids. The use of low 
parasitoid/host ratio resulted in high rates of parasitism. Thus the number of eggs 
laid by the parasitoids were estimated by summing up the following values the 
number of emerging parasitoids and also their sex ratio. These observations were 
made till the death of the female parasitoid. In the case of the death of the n^ale 
partner it was substituted by another male. 

The life tables were constructed with the help of fecundity and later the intrinsic 
rates of increase of population of parasitoids were calculated using Birch's (1948) 
formula as elaborated by Watson (1964). 

e~ r m x l x m x = 1, 

where e is the base of natural logarithms, X is the age of the individuals in days, l x is 
the number of individuals alive at age X in proportion of one and m x is the number 
of female offspring produced per female in the interval x while R is the sum of the 



Host plants pentatomids parasitoids interaction 283 

products of l x m x the rate of multiplication of population for each generation 
measured as the female offspring produced. The approximate value of cohort gene- 
ration time T c was calculated as follows 



The arbitrary value of innate capacity for increase r c was calculated from the formula 

log/?,, 
r <~ T c ' 

This was an arbitrary value for r m and value of r m upto two decimal places was sub- 
stituted in the formula until the two values of the equation were found immediately 
above or below 1096-6. The two values of I*e~ r m x l x m x = 1 were plotted on the 
horizontal axis against their respective arbitrary r m value on the vertical axis. The 
two points were joined to give a line which intersected a vertical line drawn from the 
desired value of 



The point of intersection gave the value of r m accurate to 3 decimal places. The 
precise generation time T was then calculated from the formula: 



r m 
The finite rate of increase (A) was calculated as e" r m. 

2.3 Fecundity studies 

To study the fecundity of the pentatomids, they were reared on their respective host 
plants i.e. Acrosternum graminea (Fabricius) on Cleome viscosa L, Gynandropsis 
pentaphylla DC, Acalypha indie a L, Croton sparciflorus Mor. and Leucas aspera Spr; 
while Agnoscelis nubila (Fabricius) being monophagous was reared on Leucas aspera^ 
Fecundity of the parasitoids were analysed by providing 25 eggs per day for 3 days 
after which the parasitoids failed to lay any eggs. 

3. Observations 

The results showed that the longevity of ovipositing Gryon females ranged from 5-18 
days (average 12-5). The number of progeny produced ranged from 46-78 (average 
58). The male : female ratio of progeny ranged from 1 : 6 (average 4-6). The first adult 
mortality within the cohort occurred on the fifth day after qviposition and mortality 
increased thereafter (figure 1). The duration of the immature stages lasted for 8 days. 
The adults attained maximum mean progeny production per day (m x ) of 6:250 female 
per female on the third day and production ceased by the twelfth day after ovi- 
position (figure 1). The innate capacity for increase was 0-2810 (figure 2) per female 



284 



R Velayudhan 



1-0 



o 0-8 



0-6 



0-4 




-JO 



12 16 

Time (days) 



20 



Figure 1. Daily age specific survival and age specific fecundity of Gryon sp. 



0-3100 - 



0-2800 - 



- 0-2500 - 




1500 



Figure 2. Determination of the intrinsic rate of increase (r w ) of Gryon sp. 



per day and the population of this parasitoid would tend to multiply by 31-5581 in a 
generation time of 12*2841 days in the present conditions. The finite rate of increase 
was 1-324 during the reproductive period of 12 days. 

In contrast to the above analysis the life table analysis of T. lucellus on the same 
eggs of Acrosternum graminea showed that the longevity of the ovipositing female 
ranged from 3-15 days and the number of progeny produced ranged from 30-64 
(average 36-8). The sex ratio was 1 male : 9 females (average 6-9) and the first adult 
mortality within the cohort occurred on the third day after the commencement of 
oviposition and the length of time spent in immature stages was 7 days. The adults 
attained maximum mean progeny production per day (m x ) of 9-66 females per female 



Host plants pentatomids parasitoids interaction 



285 



on the fourth day (figure 3) and production ceased by the tenth day and the overall 
innate capacity for natural increase (r m ) was found to be 0-3640 per female per day 
(figure 4). This study was mainly done to calculate the intrinsic rate for natural 
increase because it has been used as a bioclimatic index for rating the efficiency of 
these parasitoids. It was also observed that the population of T. lucellus (Nixon) 




0-4 



11 14 

Time (days) 



17 



Figure 3. Daily age specific survival and age specific fecundity of T. lucellus (Nixon). 



$ 0-3800 



o 0-3600 - 



5i 0-3400 - 




800 



1000 



1200 



1400 



1600 



Figure 4. Determination of the intrinsic rate of increase (r m ) of T. lucellus. 



286 



R Velayudhan 



would tend to multiply 53-231 in a generation time of 10-919 days in the present 
conditions. The finite rate of increase was 1440. 

Further observations revealed that though the r m value was more for T. lucellus, 
Gryon sp was more efficient in the rates of parasitisation because of a radical diffe- 
rence in the general behaviour. It was found that Gryon could tolerate other indi- 
viduals of the same species, parasitising simultaneously on the same egg mass, while 
a female T. lucellus would not tolerate another individual within the vicinity of the 
egg mass. Scanning electron microscopic studies also highlighted the presence of 
some receptors on the ovipositors of these parasitoids which in turn would aid in an 
analysis of the egg content, prior to feeding. It was also observed that these parasi- 
toids spent the initial time on the act of host feeding which was presumed to suppli- 
ment its natural diet (honey dew). 

The chemical composition of the host plants (table 1) revealed that an optimum 
proportion of all the dietary constituents is a basic requisite for the fecundity of the 
pentatomids. The data presented in the table clearly shows that high carbohydrates, 
proteins, phenols and lipids as found in C. viscosa results in the maximum egg pro- 
duction of A. graminea (126 8-75) (table 2). In A. indica there was a higher 
percentage of carbohydrates and protein than in C. viscosa and almost equal quan- 
tity of phenols, but the fecundity on this host was comparatively less (102 4-9) (table 
2) and this can be attributed to the low dosage of lipids present within A. indica. Egg 
laying was the least in C. sparciflorus (46 5-9) (table 2) though it had a high level of 
carbohydrates, but low amount of other components. Lower fecundity in L. aspera 
could be due to the low amount of proteins and lipids, while in G. pentaphylla, it 
could be due to the least amount of carbohydrates, low proteins and phenols though 
it had a higher amount of lipids. On the other hand the fecundity of A. nubila was 
140 10-8 on its host L. aspera and this pentatomid was found to be monophagous. 
The fecundity index and the average number of eggs per oviposition (table 2) of 
A. graminea also shows a higher value on C. viscosa over all the other host plants. 

Table 1. Chemical composition of the host plants (mg/g). 



Host plants 


Family 


Carbohydrates 


Proteins 


Lipids 


Phenols 


C. viscosa 


Capparidaceae 


125 


62 


22 


28 


G. pentaphylla 


Capparidaceae 


55 


50 


21 


12 


A. indica 


Euphorbiaceae 


190 


65 


10 


27 


C. sparciflora 


Euphorbiaceae 


150 


39 


11 


19 


L. aspera 


Labiatae 


160 


51 


15 


30 



Table 2. Fecundity of pentatomids A. graminae. 



Host plants 


Fecundity Fecundity index 


Oviposition 


Average number of 
eggs per oviposition 


C. viscosa 


126 8-75 


21 


4-7 


25-2 


A. indica 


102 4-99 


17 


4-8 


19-5 


G. pentaphylla 


81 11-60 


13-6 


6-12 


9-09 


C. sparciflorus 


46 5-9 


7-8 


6-9 


5-11 


L. aspera 


67 10-8 


11-16 


5-8 


8-0 


A. nubila 










L, aspera 


140 10-8 


11-16 


3-5 


8-0 



Host plants pentatomids parasitoids interaction 



287 



Experiments on the fecundity of the parasitoids in order to analyse the importance 
of supplementary feeding clearly shows that both the egg parasitoids i.e. Gryon sp 
and T. lucellus preferred the eggs of A. graminea over A. nubila, though in texture the 
chorion of the former was thicker than the latter. Hence this enhanced fecundity of 
both the parasitoids (table 3) within the eggs of A. graminea and this could be due to 
some dietary constituent derived from the egg at the time of host feeding. 

Figures 5 and 6 show the results of the HPLC analysis of fatty acid methyl esters 
and it clearly depicts the presence of stearic acid in all the host plants as well as the 
chemical content of the egg. Based on the retention time it was also observed that the 
unsaturated fatty acids which got separated within the column before 5 min also 
formed a high percentage within the plants (except C. sparciflorus) and eggs. The low 

Table 3. Fecundity of the parasitoids. 



Eggs 



A. graminae 



A. nubila 



Gryon sp 


203-16 


16l-5 




123-16 


72 




6 2-34 


21-41 


T. lucellus 


182-14 


14 2-34 




8 148 


6l-62 




41-21 


2l-48 



Fresh eggs given at 25 eggs per day for 3 days. 



Fatly acid methyl esters 



Eggs of A. nubila 



VJ 



RT 


Area (%l 




2-30 
4-87 
7-28 
16-49 

X_ . 


80-201 
10-890 
0-697 
8-212 

j 



2S 




Retention time (min) 



25 



Figure 5. HPLC analysis of fatty acid methyl esters of eggs of A. graminea and A. nubila. 



288 R Velayudhan 



C. viscosQ 



C.pentaphyllq 



A. indica 



RT 
2-41 
2-51 
2-61 
2-87 
3.28 
4-58 
9-99 
15-25 
16-00 
22-15 





25 



Area 14 
0.274 

0-134 
0-409 
10- 127 
59.OI1 
2-541 
O.OM 
14-785 < 
12.707 : 
O-OO2 



Area % 
15.440 
57. 162 
0-091 
0-002 
O-036 
0-026 
0-009 
0-OO7 
0.018 
0-004 
O.I69 P 
1.361 P 
0-0950 
6-OIO S 
19-338 
O-233 



8-017 
-556 
0-894 

3-128 



C. sparciflorus 



RT 
0.67 
1.34 
1.63 
1-95 
2-62 
7.46 
8-08 
10-79 
12.31 
16-34 
2O-00 









3-29 
5.52 
6.70 
7.12 
15-93 


1 ' 


La 


_J 


L^ 


1 

:> 25 



Area % 

0-005 

0.333 

0-617 

0-711 

0.268 

7-423 L 
11.585 L 

1-965 

O-59I 
75.092 S 

1-410 A 



Area * 

1.245 

1-895 
86-435 
0-026 
0. 134 
0- 174 L 
10-090 S 



Figure 6. HPLC analysis of fatty acid methyl esters of host plants. 



concentration of these fatty acids within C. sparciflorus could be the cause of low 
fecundity of A. graminea on this host plant. 



4. Discussion 

The development of general theories on plant defensive strategies against insect 
herbivores has received considerable attention because the plant/herbivorous insect 
relationship has provided a moderately tractable experimental system with which to 
investigate the role of biotic factors as a potentially main determinant in regulating 
animal abundance. Of particular importance is the role of plant quality (i.e. nutri- 
tional value, texture and architecture and defensive characteristics) in determining 
the population dynamics of herbivorous insects and their coevolution with plants 
(Atsatt and O'Dowd 1976; Denno and McClure 1983; Kareiva 1983; Price 1983; 
Rhoades 1983; White 1978). Price (1983), Whitham (1983) and McClure (1983) indicated 
that an assessment of plant fitness as well as insect fitness must be integrated into the 
nature of tritrophic interactions. The macroscopic architecture of individual plants or 
communities of plants are known to influence the ability of parasitoids to locate 
hosts (Hassell and Waage 1984; Kareiva 1983; Price 1983; Price et al 1980; Vinson 
1976). At a more microscopic level, physical factors such as leaf-shape, leaf colour, 
the presence of trichomes etc also modify the ability of parasitoids to locate and 
utilise hosts (Price 1983; Price et al 1980; Vinson 1976). Integrated with the above 



Host plants pentatomids parasitoids interaction 289 

modifiers is the ability of plant odours (damaged and undamaged states), host-insect 
produced kairomones and/or host-insect behaviour/physiology conditioned by 
established parasitoids or host-plant geno/phenotype to modify the ability of 
parasitoids to effectively locate and utilise hosts (Duffey et al 1986; Beckage and 
Riddiford 1983; Fritz 1982; Greany et al 1977; Hagstrum and Smittle 1977; Holmes 
and Bethel 1972; Levin et al 1982; Lewis et al 1977; Loke et al 1983; Nordlund et al 
1977; Prokopy and Webster 1978; Roth et al 1982; Pair et al 1983). Finally at the 
microscopic chemical level is the ability of plant natural products, after ingestion by 
the host insect, to modify the efficacy of parasitoids. 

This paper has thus highlighted the passage of fatty acids through 3 tropic levels 
and also shows that the host feeding behaviour of the parasitoids could enhance its 
fecundity. Insects are well known to accumulate fatty acids in high concentrations, 
especially at physiological stages of development preceding periods of non feeding or 
in maturing females for deposition in eggs. Certainly the quality of these host's food 
has a great bearing on the growth, development and survival of parasitoids because 
it is thought that the food modifies the quality of the host for a given parasitoid 
(Cheng 1970; Shapiro 1956; Smith 1957; Zohdy 1976). At present, the paucity of work 
in these directions has resulted in the lack of knowledge regarding these complicated 
mutual interactions which could give an idea of a comprehensive physiological 
mechanistic theory of tritrophic interactions. Studies on the role of host nutrition as 
in A. graminea has indicated that the addition/subtraction of specific nutrient could 
alter the reproductive capacity of the host insect. Further studies on other dietary 
components could throw more light on these interactions. 



Acknowledgements 

The author is extremely grateful to Prof. T N Ananthakrishnan for his continuous 
guidance and critical suggestions during,the course of the work. He is also grateful to 
Dr G Suresh and Mr R Senrayan for the help rendered during chemical analysis. 
Thanks are due to COSIST facilities provided by the UGC. 



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Can. Entomol. 93 1041-1044 
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Kilgore W W and Li M Y 1976 Environmental Toxicology; in Insecticide biochemistry and physiology (ed) 

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management of entomophagous insects. V. Moth scales as a stimulus for predation of Heliothis zea 

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chogramma praetiosum to two suitable hosts; J. Chem. Ecol. 3 513-518 
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with sesame interplanting in cotton, 1971-74: Implications of host-habitat interaction: Environ. 

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in natural and managed systems (eds) R F Denno and M S McClure (New York: Academic Press) 
i Chapter 6, pp 155-220 
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sequence of the tachinid Lixophaga diatraeae\ Environ. Entomol. 11 273-277 

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pp 59-83 



Host plants pentatomids parasitoids interaction 291 

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Application (Hewlett Packard) Pub. No. 12-5954-0826 
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 293-304. 
O Printed in India. 



Impact of predation and food utilization on reproduction of Diplonychus 
indicus and Ranatra filiformis 

P VENKATESAN and S MUTHUKRISHNAN 

Department of Zoology, Loyola College, Madras 600 034, India 

Abstract. Impact of predation and food utilization on reproduction of the water bugs 
Diplonychus indicus and Ranatra filiformis was studied. In Diplonychus indicus, the food 
quality affects the rate of predation as well as food utilization. Allometric growth of various 
body parts and the longevity of each nymphal instar of the bug showed distinct variation, 
when exposed to individual prey item viz Culex, Anopheles and Aedes larvae and fish fing- 
erlings. In Ranatra filiformis, a distinct pattern of oviposition in relation to the increased 
rate of predation on Culex larvae was observed. The significance of such variations in 
reproductive activities of these water bugs was discussed. 

Keywords. Allometric growth; longevity; energetics; oviposition. 



1. Introduction 

Nutritional ecology is central to proper interpretations of manner of feeding, repro- 
duction, defense, habitat selection and such other life history phenomena in arthro- 
pods (Scriber and Slansky 1981). With regard to factors that promote oviposition, 
embryonic development, growth and sexual maturity, starvation or nutritional 
deficiency is felt to be a major cause. Davis (1964) has reported that resorption of 
oocytes in hemipterous insects is governed and promoted by the nutritional status 
even in mated females. When food quality is changed, the subsequent rate of 
development, body composition and growth of arthropods differed significantly 
(Waldbauer 1968; M.ueller et al 1973; Blumberg and Swirski 1974). To predators, the 
mobility is vital among properties of the prey item. In aquatic hemipterous bugs, the 
predation is categorised as chase and capture the prey (Peckarsky 1982), sit and wait 
in ambush (Hoffmann 1927) and resting-questing attitude (Menke 1979). 

If nitrogen proved to be a limiting nutrient for the growth as suggested in Pieris 
rapae (Slansky and Feeny 1977), then the first objective of the present study was to 
verify if growth during postembryonic development is related more closely to the 
nutritional status and to the energetics of an active predator Diplonychus indicus. 
The second objective was to study the interaction between the sex of the bug and the 
efficiency of utilization of the food provided. 

The third objective was to examine the impact of specific diet on life fecundity of a 
matured female and ovipositional strategy in Ranatra filiformis. 

2. Materials and methods 

2.1 Longevity and allometric growth 

To record the impact of nutrition on longevity and allometric growth on D. indicus, 
the encumbered males were maintained in the laboratory. First nymphal instar that 

293 



294 P Venkatesan and S Muthukrishnan 

emerged and its successive stages of development till adulthood were fed with Culex, 
Anopheles or Aedes larvae with fish fingerlings as control. The life span of each 
nymphal instar with specific diet was recorded. Various body measurements of all 
nymphal instars fed with specific diets were measured. Data obtained were fitted to 
Huxley's (1924) exponential formula Y=rjX k , where Y is the allometrically growing 
organ, b the growth index, X the body length taken as reference measurement and k 
the equilibrium constant by which Y grows in relation to X throughout the onto- 
genetic stages and the significance of growth was derived statistically. 

2.2 Energetics of nymphal instars 

All nymphal instars of D. indicus were starved for 24 h prior to experiments. Their 
predatory efficiencies were tested against 4th age group of Culex and Aedes larvae at 
different densities. To assess the food utilization, the scheme of energy balance of 
Petrusewicz and Macfadyen (1970) namely C = P + jR-bF+l7 was followed, wherein 
C, food consumed; P, growth attained; R, respiration; F, faeces and U, nitrogenous 
wastes. From the data obtained, the assimilation and the conversion efficiencies, 
rates of consumption, assimilation and conversion were calculated for all develop- 
mental stages. Tissue samples of these experimental ones were dried at 105C. The 
calorific value of each sample was determined by incinerating them in a semi micro 
oxygen bomb calorimeter following the procedure of Pandian and Madhavan (1974). 

2.3 Food utilization in adults 

Freshly moulted males and females of D. indicus were maintained in the laboratory 
with 4th age group of Culex larvae. After feeding them for 20 days, the experiment 
was suspended and the bugs were treated to estimate the assimilation and conversion 
efficiencies, rate of feeding, assimilation and conversion as cited earlier. 

2.4 Statistical analysis 

Data obtained were subjected to statistical analysis of variance to derive the 
significance of differences in the above experiments. 

2.5 Life fecundity and oviposition 

Fifth nymphal instar of R. filiformis were collected and maintained in the laboratory 
with 4th age group of Culex larvae as the specific diet at 28 1C with a daily regime 
of 12 h light and 12 h darkness. 

Males and females obtained soonafter moulting from 5th nymphal instar were 
kept in pairs and were fed normally with Culex larvae. Stems of Hydrilla were 
provided as ovipositional cites in order to record the percentage of ovipositive 
females, rate of reproduction, total number of eggs produced during its life time, 
distribution of eggs on the floating vegetation, adult longevity and the percentage of 
hatching success. Number of eggs per batch of a female served as the index of repro- 
ductive potential. First nymphal instar that emerged and the successive stages it 
moulted with Culex larvae as diet were also recorded. 



Nutrition and reproduction in water bugs 295 

3. Results 

3.1 Nutrition and life span 

Table 1 shows that the duration of post embryonic stages of D. indicus in the labo- 
ratory reared population simulating natural conditions and providing food ad 
libitum varies from 80-90 days. The longevity of I nymphal instar and II were 8-7 
days, III for 10-6 days, IV for 24-5 days and V for 37-5 days. However, when they 
were fed with a specific test food, the longevity of each nymphal instar varied signi- 
ficantly. On comparison, the duration of Culex fed ones was higher throughout the 
post embryonic development than those fed with other test food. Duration of deve- 
lopment of all nymphal instars was found to be very low when fed with fish finger- 
lings. 

3.2 Allometric growth in D. indicus 

Figure 1 shows the measurement of the total body length of all nymphal instars 
plotted against time in days of the predator stages. Second and third instars of 
D. indicus showed maximum body length when fed with Culicine larvae. The fourth 
nymphal instar showed" maximum body length when fed with anopheline larvae. The 
fifth nymphal instar showed maximum body length when fed with culicine larvae. 
The second and third instars showed minimum body length when fed with anophe- 
line larvae and fish fingerlings. The fourth nymphal instar showed minimal body 
length when fed with aedes larvae and the fifth nymphal instar with anopheline 
larvae. Measurement of individual organ was subjected to statistical analysis of 
variance. The rostrum length, head width, 1st leg, 2nd leg and 3rd leg showed stati- 
stically significant differences among the members of third nymphal instars that were 
fed on different test food. At other nymphal stages such differences were not 
observed. 

3.3 Food utilization in the post embryonic development 

Data on energy budget of nymphal instars of D. indicus when fed with different test 

Table 1. Duration (in days) of the nymphal instars of D. indicus fed with different food 
types. 

Quality of food provided 



Nymphal instar 


Mixed 
diet 


Culicine 
larvae 


Anopheline 
larvae 


Aedes 
larvae 


Fish 
fingerlings 


I 
II 
III 
IV 

V 


8-7 049 
8-7 1-29 
10-6 2- 11 
2442 1-0 
37-67 6-06 


4-9 049 
5-9 0-7 
8-6 2-07 
2242 1-0 
35-33 6-62 


6-0 
5-0 0-33 
12-1 3-84 
11-0 2-56 
18-36l-8 


6-1 0-35 
5-55 0-74 
11-73 1-94 
23-33 5-75 
11-27 3-0 


540-53 
4-5H2 
647 1-51 
7-55 0-62 
13-0 1-61 



90-09 77-15 5246 57-98 36-92 



296 



P Venkatesan and S Muthukrishnan 



.12 



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en 
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o 

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75 



95 



Figure 1. Mean body length against mean duration of nymphal stages of D. indicus fed 
with specific and mixed diets. 



1OO 



6O 



Assimilation efficiency 




A Culex 

o Anopheles 

Aedes 

A Fish fingerling 

ill! 



I II III IV V 

Figure 2. Effect of specific diet on the assimilation efficiency of D. indicus. 



foods were recorded. In general, assimilation efficiency was almost 90% in the first 
nymphal instars of the predators and remain constant with others. In particular, 
assimilation efficiency of fifth nymphal instar of the predator was 82% with aedes 
larvae, 90% with anopheline larvae and 93% with culicine larvae (figure 2); whereas 



Nutrition and reproduction in water bugs 



297 



the conversion efficiency at all stages of development was almost constant except the 
fifth nymphal instar but it is inversely related to the assimilation efficiency when fed 
with fish fingerlings (figure 3). The feeding rate was minimum when fed with fish 
fingerlings and maximum with culcine larvae. Among all nymphal instars, the second 
one showed the highest rate of feeding with culex and anopheles larvae. However, the 
feeding rate declined in other developmental stages (figure 4). The assimilation rate 
was almost equal in those fed on culex larvae, anopheles larvae or fish fingerlings; 
but those fed with aedes larvae showed 2 peak values with the first and third 
nymphal instars (figure 5). Similarly, the conversion rate was also noted to vary in 
relation to the quality of food provided, being higher at the second nymphal instar 



Conversion efficiency 




I II III IV V 

Figure 3. Effect of specific diet on the conversion efficiency of D. indicus. 



500O 



o 

0) 
<fi 

30OO 

0) 



1OOO 



'A Feeding rate 




I I I 



I II III IV V 

Figure 4. Effect of specific diet on the feeding rate of D. indicus. 



298 



P Venkatesan and S Muthukrishnan 



1800 



o 

CD 
crt 
C 



Q> 



IOOO - 



Assimilation rate 




2OO - 

I I L 

I II III IV V 

Figure 5. Effect of specific diet on the assimilation rate of D. indicus. 



250 ~ 



o 
-o 



o 

<D 
to 

15O 

0) 



o 

E 50 



Conversion rate 




I 



I 



I 



I 



I II III IV V 

Figure 6. Effect of specific diet on- the conversion rate of IX indicus. 



when fed with larvae of Culex, Anopheles or fish fmgerlings. It was considerably high 
when fed with fish fmgerlings at all stages (figure 6). 

Data on the energetics of the predator stages throughout their feeding periods is 
summarised in table 2. It may be noted that the feeding rate was minimum 
(1060 mg/g live insect/day) in fingerling fed individuals and very high (4300 mg/g live 
insect/day) in those fed with Culex larvae but the assimilation rate was maximum in 
Aedes fed ones and minimum when fed with Anopheles or fish fingerlings. The 
conversion rate was highest (171 mg/g live insect/day) when fed with fish fingerlings 



Nutrition and reproduction in water bugs 299 

Table 2. Energy budget of the nymphal instars of D. indicus fed with specific diet. 



Quality of food 




Culicine 


Anopheline 


Aedes 


Fish 




larvae 


larvae 


larvae 


fingerling 


Duration (days) 


77-15 


5246 


57-98 


36-92 


Feeding rate (mg/g-live 










insect/day) 


4300 


2532 


1550 


1060 


Assimilation rate (mg/g 










live insect/day) 


955 


841 , 


1170 


845 


Conversion rate (mg/g 










live insect/day) 


128 


143 


124 


171 


Assimilation 










efficiency (%) 


95-2 


92-8 


89-6 


68-8 


Conversion 










efficiency (%) 


5-0 


7-0 


9-6 


29-4 


Calorific value of the 










converted food (g cal) 


234-85 


"185-81 


115-1 


154-9 



Table 3. Energy budget of adult (male and female) D. indicus fed with culicine larvae. 



Sex 


Conversion 
efficiency 


Assimilation 
efficiency 


Feeding 
rate 


Assimilation 
rate 


Conversion 
rate 


Male 
Female 


20-36 2-62 
18-75 4-25 


47-28 2-66 
38-92 5-03 


497-24 65-14 
558-11 140-08 


233-81 17-67 
213-09 34-74 


45-6 5-85 
41-98 9-54 



and lowest (124 mg/g live insect/day) in Aedes fed ones. The calorific value of the 
converted body tissue was 234-859, 185-81, 115-1 and 154-9 g/cal with Culex, 
Anopheles and Aedes larvae and fish fmgerlings respectively. 

3.4 Energy budget in adult 

Table 3 shows that the assimilation efficiency of the male was 47-28% which is higher 
than that of the female. Similarly, the conversion efficiency was 20-36 in males and 
18-75 in females but the feeding rate was low in males than in females. However, the 
assimilation and conversion rates were noted to be higher in males than in females. 



3.5 Egg viability in Ranatra 

The hatching success of eggs deposited by R.filiformis was recorded (figure 7). There 
was a great difference between the number of eggs laid among the batches. The 
number of eggs in 4 batches was 7-86, 9-93, 4-07 and 0-14 respectively. Also, the time 
interval between any 2 batches of the eggs varied significantly. The percentage of 
hatchability ranged from 64-82 in these batches. Data shows that the time interval 
between batches of eggs and percentages of hatchability were found to be inversely 
proportional with first and the last batch of eggs. Of the eggs laid in each batch, the 
hatching success was 74%, 62%, 78% and 84% respectively. 



300 



P Venkatesan and S Muthukrishnan 



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S s 











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Figure 7. To show the hatching process of eggs oviposited by a female R. filiformis. 



14 - 


<? 






l 






l 






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n 




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n 


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n IW 

11 




1 1 1 1 1 1 1 I 1 I I I I I 


5 


10 15 20 




Batch number 



-6 



4 ^ 



-2 



Figure 8. To show the rate of oviposition by a female R. filiformis. 



3.6 Rate of oviposition in Ranatra 

The number of batches of eggs laid by a female during its life time ranged from 17- 
23. The number of eggs laid was from a maximum of 12-33 4-4 in the first batch to a 
minimum of 2 in the 23rd batch (figure 8). However, the trend in the fluctuation of 
number of eggs deposited batchwise during the life span of Ranatra was not highly 
significant; whereas the time interval between successive batches of eggs laid was 
significantly high. Generally, the hatching period was noted to be more or less 
constant. The bug took 5-987 3-585 min to get acclimatised for oviposition. Time 
taken for actual oviposition of any egg in one batch ranged from 0-56-1-17 min. 
However, the time interval between successive eggs laid increased from the first to 
the last egg deposited (figure 9). When the percentage of success of successive stages 
of development and the percentage of success of development of egg to adult "were 



Nutrition and reproduction in water bugs 



301 




n Time interval between 

L - ' successive eggs laid 

Isjsj Actual oviposition period 

1-11 Egg number 



n 1 1 1 1 i r 

1 234567 



Figure 9. Time interval between successive eggs laid, actual oviposition period and 
number of eggs laid by a female R. filiformis. 



Table 4. Success (%) of successive stages of development and of egg to adult in R.flliformis. 

Percentage success 



Egg to I 



I to II 



II to III III to IV IV to V V to Adult 



Stages of 

development 89-05-95 91-962-65 81-363-60 78-424-80 74-784-70 19-673-53 
Egg to adult 89-0 5-95 8 1 -80 54 66-60 5-8 52-40 6-92 39-20 6-08 7-80 2-08 



calculated, it was noted to be more or less constant, till the fifth nymphal instar but 
there was a sudden decline from fifth nymphal instar which was recorded as 19-6% 
irrespective of the sex moulted (table 4). 



4. Discussion 

Aquatic insects exhibit the ability to use a wide variety of food sources, exploit and 
develop them into a variety of macro- and microfeeders that operate from deep 
within the substrate to the top of the surface film (Resh and Solem 1978). Results of 
the present study reveal the ability of belostomatid bugs feeding on variety of prey 
items. However, a preferential selection was noted which has a direct influence on 
their longevity. Probably, prey mobility and morphology may suppress the preda- 
tory efficiency of the bugs concerned. Difference in mobility between Ephemerella 
altana and Bactis tricaudata was pivotal in producing the higher consumption of the 
stoneflies (Molles and Pietruszka 1983). 

Rate of predation and food quality were observed to alter the duration of post- 
embryonic development in D. indicus. Clark (1963) has shown that some strains of 
Habrobracon females when fed on white cloves honey, lived much longer than 
Ephestia fed females. But in other strains, the adult life span was identical on either 
diet. Report of Alpatov (1930) on Drosophila and Strong and Kruitwagen (1969) on 



302 P Venkatesan and S Muthukrishnan 

Lygus support the present observations of the impact of nutrition on the life span 
and ageing of insects. 

The results of energetics of developmental stages of D. indicus in relation to food 
quality show many interesting observations. When feeding is greater with Culex than 
with Aedes larvae, the conversion and assimilation efficiencies do not show much 
difference but with fish fingerlings, the conversion- and assimilation efficiencies during 
development differed significantly. Such a feature is not uncommon in herbivorous in- 
sects as is known from the report of Bailey and Mukerji (1976) on Melanoplus sangrinipes 
and Smyth (1962) on Analis mail. However, there appears to be no relationship between 
rate of feeding with that of rate of assimilation and conversion. But the finding that 
the high conversion rate of second nymphal instar that fed on culcine larvae is ref- 
lected in the increased body size of the penultimate instar is of considerable interest. 
Also, relationship between conversion rate and allometric growth including total 
body length was noted. This may be due to the difference in the nutritive require- 
ments responsible for the growth of various organs (Kasting and Mcginnis 1959; 
Rockstein 1964). 

Results on egg viability of R.filiformis show that the number of eggs laid in earlier 
batches is more than that in the later ones. The prolonged period of mating in the 
early period might have resulted in such increased oviposition and on experience, the 
females might have avoided repeated copulation which is well reflected in the 
minimum number of eggs laid in the later batches. Siew (1966) in Galeruca tenaceti 
has suggested that ovipositing females possess neurosecretory cells that produce 
more neurohormones resulting in an increased production of batches of eggs than do 
those of maturing females which further supports the present view that oviposition is 
frequent at a later stage irrespective of the number of eggs laid in each batch. 

Results further indicate that the time interval between oviposition of successive 
batches of eggs is less in the later batches. This may reflect on the ability of females to 
store sperms (Parker 1970) and ageing (Spence and Scudder 1980). Data on oviposi- 
tional period show that the bug takes a minimum time to lay the eggs in the begin- 
ning and a maximum period at the end in a single batch. Demand for floating vege- 
tation, energy lost during oviposition and the mobilisation of the matured eggs from 
the terminal part of the ovariole may be attributed as the causal factors for such 
variations in oviposition period during oviposition of eggs in a single batch. 
However, such an interpretation needs to be confirmed based on histological and 
histochemical studies. 

Among the successive instars, a sudden fall is noted in the percentage of emergence 
of adults from fifth nymphal instar. This may be owing to an increased rate of mor- 
tality during the moulting of adults from fifth nymphal instar. This in turn may be 
either owing to the influence of increase in temperature that can eliminate a species 
by disrupting emergence patterns as is suggested by Nebeker (1971) in Pteronarcys 
dorsata or to the susceptibility of low oxygen concentration in the medium in other 
aquatic insects (Nebeker 1972). 

The quantitative nutritional approach consists of measuring the amount of food 
consumed, digested and assimilated, excreted, metabolised and converted into 
biomass (Woodring 1979). Analysis of these measurements in D. indicus and the 
impact of nutrition on oviposition strategy of R. filiformis reveal how organisms 
respond to different foods and which food component exerts the greatest effects on 
growth. Further investigations on mobilisation of biochemical components from 



Nutrition and reproduction in water bugs 303 

haemolymph to various body parts may highlight the significance of food quality on 
reproductive biology of water bugs. 



Acknowledgements 

The authors are highly thankful to Rev. Fr. Leo Correya, Principal for the facilities 
provided, to Prof. T K Raghunatha Rao for the constant encouragement, to Prof. 
T N Ananthakrishnan for accepting our paper and to the University Grants Com- 
mission, New Delhi for financial support. 

References 

Alpatov W W 1930 Experimental studies on the duration of life. The influence of different feeding during 

the larval and imaginal stages on the duration of life of the image of Drosophila melanog aster; Am. Nat. 

64 37-55 
Bailey C G and Mukerji M K 1976 Consumption and utilisation of various host plants by Melanoplus 

bivittatus Say and Melanoplus femurrabnm Degeer (Orthoptera: Acrididae); Can. J. Zool. 54 1044-1050 
Blumberg D and Swirski E 1974 Prey consumption and preying ability of three species of Cybocephalus 

(Coleoptera: Cybocephalidae); Phytoparasitica 2 3-1 1 

Clark A M 1963 Impact of nutrition on Habrobracon; Ann. Entomol. Soc. Am. 56 616 
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maturation; J. Insect Physiol. 10 947-964 

Hoffmann W E 1927 Biological notes on Laccotrephes; Lingnan Agric. Rev. 4 77-94 
Huxley J 1924 Constant differential growth ratio and their significance; Nature (London) 114 895 
Kasting R and McGinnis A G 1959 Nutrition of the pale western cutworm, Agortis orthogonia Morr 

(Lepidoptera: Noctuidae). 1. Effect of underfeeding and artificial diets on growth and development and 

a comparison of wheat sprouts of Thatcher, Triticum aestivum L. and Golden Balls T. durum Desf. as 

food; Can. J. Zool. 37 259-266 
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Calif. Insect Sur. 22 1-166 
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prey morphology, behaviour and predator hunger; Oecologia (Berlin) 57 25-31 
Muller A J, Sharma R K, Reynolds H T and Toscano N C 1973 Effect of crop rotations on emergence of 

overwintered pink Bollworm populations in the Imperial valley, California; J. Eco. Entomol. 67 

227-228 
Nebeker A V 1971 Effect of water temperature on nymphal feeding rate, emergence and adult longevity of 

the stonefly Pteronarcys dorsata; J. Kansas Entomol. Soc. 44 21-26 
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Am. Fish Soc. 4 675-679 
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trivialis (Rambur) and some other aquatic insects (Anisoptera: Libelluidae); Odonatologica 3 241-248 
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525-567 

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Blackwell) p 190 
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An Introduction to the aquatic insects of North America (ed) R W Merritt and K W Cummins (Iowa: 

Kendall/Hunt Pub Comp) p 441 

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ScriberJMand Slansky F 1981 The nutritional ecology of immature insects; Annu. Rev. Entomol. 26 

183-211 
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(Coleoptera: Chrysomelidae); Trans. R. Entomol. Soc. London 118 359-374 



304 P Venkatesan and S Muthukrishnan 

Slansky F and Feeny P 1977 Stabilization of the rate of nitrogen accumulation by larvae of the cabbage 

butterfly on wild and cultivated food plants; Eco. Monogr. 47 209-228 
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(Heteroptera: Gerridae) on the Fraser Plateau of British Columbia; Can. Entomol. 1 1 2 779-792 
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Physiol. 25 903-912 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 305-309. 
Printed in India. 



Observations on feeding propensities, growth rate and fecundity in 
mayflies (Insecta: Ephemeroptera) 

K G SIVARAMAKRISHNAN and K VENKATARAMAN 

Department of Zoology, Madura College, Madurai 625 Oil, India 

Abstract. The relative importance of organic detritus and algae in promoting the growth 
and fecundity of some species of Baetidae is assessed on the basis of the nutritional 
differences among them. Taking into consideration the ephemeral aspect of adult life, an 
understanding of the relative importance of such substances as carbohydrate, protein and 
lipid in relation to their growth and fecundity is also examined. 

Keywords. Feeding propensities; growth rate; fecundity; alga; detritus; mayflies. 

1. Introduction 

A basic facet of the structure and function of a freshwater ecosystem is the material 
cycling and energy flow. In turn, a significant portion of such cycling and flow 
involves the processing of various forms of organic matter by freshwater invertebra- 
tes, especially insects. This constitutes a basis for interest in trophic relations of 
aquatic insects (Cummins 1973). The nymphs of Ephemeroptera do not play the 
same part in the trophic structure of the communities in which they occur, and in 
view of this detailed knowledge of their feeding propensities is highly desirable. 

The majority of mayfly nymphs are herbivorous, feeding on detritus and 
periphyton. The herbivorous mayflies fall into two main categories: collectors and 
scrapers (Edmunds 1978). Among the collectors, several genera are filter-feeders, with 
setae on the mouthparts or forelegs acting as filters. Within the Oligoneuridae, 
Leptophlebiidae, Siphlonuridae and the Heptageniidae, there are several genera that 
are probably filter-feeders (Wallace and Merritt 1 980). By using their gills to produce 
a current of water through their burrows, several of the Ephemeridae and Polymitar- 
cyidae may, at least for part of their food supply, be regarded as filter-feeders 
(Brittain 1982). Most mayflies, however, are fine-particle detritivores. These include 
many Siphlonurinae, Baetidae, Leptophlebiidae, Metretopodidae, Ephemerellidae, 
Caenidae and Baetiscidae, as well as some Heptageniidae (Edmunds 1978). The other 
major feeding group within the mayflies, the scrappers, utilize the periphyton present 
on mineral and organic surfaces. These include representatives of several mayfly 
families, notably the Baetidae, Heptageniidae, Leptophlebiidae and Caenidae 
(Edmunds et al 1976). Shredders are probably also represented among mayflies. 

Earlier observations reveal that even within the detritivore/herbivore category, 
diet may change with season, habitat and stage of development. Seasonal differences 
are often a reflection of food availability (Brown 1961; McClure and Stewart 1976; 
Moore 1977), thus emphasizing the opportunistic nature of mayfly nutrition. How- 
ever, within the range of food available, there is often evidence of selection (Brown 
1961; Cianciara 1980). Food selection may clearly be advantageous, as growth may 
be influenced by different kinds of food (Anderson and Cummins 1979; Cianciara 
1980; McCullough et al 1979). 

305 



306 K G Sivaramakrishnan and K Venkataraman 

Reproduction in insects is very closely related to nutritional factors, the qualitative 
and quantitative aspects of which have an impact not only on fecundity, but also on 
the rates of growth and development. Notable publications in this direction include 
those of Johansson (1958), Slansky (1980a,b), Ananthakrishnan et al (1982) and 
Raman and Sanjayan (1983). Whereas there are some investigations on feeding pro- 
pensities (Brown 1960, 1961; Gupta and Michael 1981; Venkataraman 1984), food 
habits (Baekken 1981), trophic relations (Winterbourn 1974), food preference and 
dependence of growth on the type of food (Cianciara 1980) and nutritional dynamics 
(Zimmerman and Wissing 1980), work on the impact of nutrition on reproduction of 
mayflies is conspicuous by its absence. The present investigation aims at understand- 
ing and comparing the feeding propensities, growth rate and fecundity of two species 
of Baetis and one species of Cloeon available in and around Madurai, based on 
laboratory observations. 

2. Material and methods 

Early instar nymphs of Baetis sp. A and B were collected from Vaigai river near 
Thiruvedagam, 10 km west of Madurai. Early instar nymphs of Cloeon sp. were also 
collected from the temple tank of Thirumohur, 10 km north of Madurai. These 
nymphs were acclimated to the laboratory conditions (temperature 261C) for 
24 h. Nymphs of constant length (approximately 2-0 mm), which are in the same 
physiological age were assorted. Twenty nymphs of each species were reared indi- 
vidually in separate petridishes of 9 cm diameter. Mortality was around 50% and 
dead nymphs were replaced by the nymphs of the same physiological age. A batch of 
10 nymphs of each species was fed on crushed alga (Spirogyra sp.) and the remaining 
batch of 10 of each species on detritus collected from respective habitats. The two 
types of food (organic detritus and &}ga-Spirogyra sp.) used in the cultures were 
selected on account of abundant occurrence in the natural environment and of being 
well known from the literature (Brown 1960; Minshall 1967; Cianciara 1980). Food was 
supplied in excess. Filtered water brought from their natural habitats was used for 
rearing. A fresh supply of food was added every day when the water was changed. 
The head width and the body length of each nymph excluding antennae and cerci 
were measured after every moult. Since adult mayflies usually live for only a day or 
two and all the eggs are produced prior to the subimago stage (Clifford and Boerger 
1974) total potential fecundity was determined accurately by counting the eggs in 
subimagos soon after emergence. The alga (Spirogyra sp.) and detritus were 
quantitatively analysed for proteins, carbohydrates and lipids by following the 
methods of Lowry et al (1951), Dubois et al (1956) and Kok (1971) respectively. 

3. Results and discussion 

It is evident from table 1 that Baetis sp. A and B grow faster in detritus than in alga 
(Spirogyra sp.) whereas it is vice versa in Cloeon sp. Apparently present observations 
indicate preference of detritus by the two species of Baetis under investigation and 
preference of alga by Cloeon sp. These are in conformity with the observations of 
some previous workers. Studies of Badcock (1949), Brown (1961) and Baekken (1981) 
on Baetis rhodani and of Gupta and Michael (1981) on Baetis sp. in Shillong, 



Feeding propensities, growth rate and fecundity in mayflies 307 

Table 1. Growth rate of different species of mayflies fed on detritus and alga. 



Detritus 


Alga 




Body length 
(mm) 


Head width 
(mm) 


Body length 
(mm) 


Head width 
(mm) 


Baetis sp. A. 
Baetis sp. B. 
Cloeon sp. 


0-36 0-08 
0-50 0-05 
0-20 0-03 


0-1 5 0-01 
0-14 0-01 
0-09 0-01 


0-27 0-08 
0-33 0-03 
0-66 0-1 5 


0-100-01 
0-09 0-004 
<M40-Ol 



Table 2. Quantitative profile of carbohydrate, protein and lipid as well as calorific values 
of different types of food. 





Carbohydrate 
(mg/lOOmg) 


Protein 
(mg/lOOmg) 


Lipid 
(mg/lOOmg) 


Calorific value 
(cal/mg) 


Alga 
Detritus 


5-1 0-42 
1-09 0-16 


43-5 6-3 
17-80-35 


ll-20-8 
8-2 0-6 


4,100 
3,950 



Meghalaya revealed them to be mainly detritivorous whereas Brown (1960) has 
shown that several species of algae were thoroughly and rapidly digested by Cloeon 
dipterum in the laboratory. 

It is a well known fact that the type of food may have a substantial effect on the 
growth and fecundity of an organism. This is especially so in the nymphal span of 
mayflies whose adults are ephemeral and do not feed. Quantitative analysis of alga 
and detritus given as food for nymphs reveals that alga (Spirogyra sp.) is richer in 
carbohydrates, proteins and lipids when compared to detritus (table 2), though the 
calorific values of both types of food are approximate [Spirogyra 4,100 cal/mg, 
Ivanova (1958); detritus 3,950 cal/mg, Coffman et al (1971)]. When the nymphs of 
mayflies are feeding upon filamentous algae, the mandibles play a more important 
part in the collection of food than when this is fine detritus. Apparently the mandible 
tips of Cloeon sp. are more suitable for algal diet than those of Baetis spp. However, 
previous studies have shown little or no cellulose activity in mayflies (Monk 1976). 
Nevertheless, organic compounds leaked or secreted by the algae may be of nutri- 
tional importance (Cummins 1973). 

The retarded growth of Baetis sp. A and B reared on algal diet in spite of its 
richness of nutrients may be correlated with their inability to consume large 
quantities of algae. The tip of the mandible of Baetis sp. A and B is more tapered 
than that of Cloeon sp. and the straight canines project towards the substratum 
rather than into the preoral cavity. Obviously the molar surfaces of Baetis spp. and 
of Cloeon sp. show constant differences in the details of their structure correlated 
with the nature of their respective diet though the feeding mechanisms of Baetis spp. 
and Cloeon sp. are adapted to allow the ingestion of a heterogenous diet in a variety 
of habitats as pointed out by Brown (1964). 

It is also obvious from table 3 that the difference in fecundity of Cloeon sp. fed on 
alga and detritus is much more conspicuous than those observed in Baetis sp. A and 
B. Moreover, the quantity of food ingested also is of value in improving fecundity. 
Apparently it is the larger quantity of algae ingested by Cloeon sp. and the 
considerable quantity of detritus ingested by Baetis sp. A and B that might have 



308 K G Sivaramakrishmm and K Venkataraman 

Table 3. Mean number of eggs/individual in 
different species of mayflies in relation to the type of 
food. 





Detritus 


Alga 


Baetis sp. A. 
Baetis sp. B. 
Cloeon sp. 


303 1-41 
200 2-82 
101-52-12 


245-5 7*77 
151 3-0 
539-633-2 



resulted in increased fecundity of alga-fed Cloeon sp. and of detritus-fed Baetis sp. A 
and B in the present investigation. 

Acknowledgement 

The authors are indebted to Prof. T N Ananthakrishnan, Entomology Research 
Institute, Loyola College, Madras for having initiated this study and for many 
valuable suggestions. They are also thankful to Prof. K Srinivasan for his encourage- 
ment. This work was supported by a grant from the University Grants Commission 
(F. 3-95/86 SR-II), New Delhi the receipt of which is gratefully acknowledged. 



References 

Ananthakrishnan T N, Raman K and Sanjayan K P 1982 Comparative growth rate, fecundity and 

behavioural diversity of the dusky cotton bug, Oxycarenus hyalinipennis Costa (Hemiptera: Lygaeidae) 

on certain malvaceous plants; Proc. Indian Natl Sci. Acad. B48 577-584 
Anderson N H and Cummins K W 1979 Influences of diet on the life histories of aquatic insects; J. Fish. 

Res. Board Can. 36 335-342 

Badcock R M 1949 Studies on stream life in tributaries of the Welsh Dee; J. Anim. EcoL 18 193-200 
Baekken T 1981 Growth patterns and food habits of Baetis rhodani, Capnia pygmaea and Diura nanseni in 

a West Norwegian river; Holarctic EcoL 4 139-144 
Brittain J E 1982 Biology of mayflies; Annu. Rev. Entomol 27 119-147 
Brown D S 1960 The ingestion and digestion of algae by Chloeon dipterum L. (Ephemeroptera); 

Hydrobiologla 16 81-96 
Brown D S 1961 The food of larvae of Chloeon dipterum L. and Baetis rhodani Pictet (Insecta, 

Ephemeroptera); J. Anim. EcoL 30 55-75 
Brown D S 1964 The morphology and functioning of the mouthparts of Chloeon dipterum L. and Baetis 

rhodani Pictet (Insecta, Ephemeroptera); Proc. Zool. Soc. London 136 147-176 
Cianciara S 1980 Food preference of Cloeon dipterum (L) larvae and dependence of their development and 

growth on the type of food; Pol. Arch. Hydrobiol. 27 143-160 
Clifford H F and Boerger H 1974 Fecundity of mayflies (Ephemeroptera), with special reference to 

mayflies of a brown-water stream of Alberta; Can. Entomol. 106 1111-1119 
Coffman W P, Cummins K W and Wuycheck J C 1971 Energy flow in a woodland stream ecosystem; 

Arch. Hydrobiol. 68 232-276 

Cummins K W 1973 Trophic relations of aquatic insects; Annu. Rev. Entomol. 18 183-206 
Dubois M, Gilles K A, Hamilton J K, Rebers P A and Smith F 1956 Colorimetric determination of sugars 

and related substances; Anal. Chem. 28 351-356 
Edmunds G F Jr 1978 Ephemeroptera; in An Introduction to the Aquatic Insects of North America (eds) 

R W Merritt and K W Cummins (Dubuque, Iowa: Kendall/Hunt) pp 57-80 
Edmunds G F Jr, Jensen S L and Berner L 1976 The Mayflies of North and Central America (Minneapolis: 

Univ. Minn. Press) pp 330 
Gupta A and Michael R G 1981 Population ecology and feeding propensities of two co-existing species of 

Baetidae (Insecta: Ephemeroptera); Proc. Symp. EcoL Anim. Popul. Zool. Surv. India Pt. 2, pp 95-104 



Feeding propensities, growth rate and fecundity in mayflies 309 

Ivanova S S 1958 Feeding of larvae of some mayflies; TV. Mosk. Tekhnol. Inst. Ryhn. Promsti. Khoz. (in 

Russian) 9 102-120 
Johansson A S 1958 Relation of nutrition to endocrine reproductive functions in the milkweed bug, 

Oncopeltus fasciat us (Dallas) (Heteroptera: Lygaeidae); Nytt. Mag. Zool. 1 3-132 
Kok L T 1971 Fungal symbionts of Xyleborous spp. certain chemical components and their nutritional 

significance to the ambrosia beetles, Ph.D. dissertation, Univ. of Wisconsin, Madison 
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with phenol reagent; 

J. BioL Chem. 193 265-273 
McClure R G and Stewart K W 1976 Life cycle and production of the mayfly Choroterpes 

(Neochoroterpes) mexicanus Allen (Ephemeroptera: Leptophlebiidae); Ann. EntomoL Soc. Am. 69 

134-144 
McCullough D A, Minshall G W and Gushing C E 1979 Bio-energetics of a stream collector organism, 

Trychorythodes minutus (Insecta: Ephemeroptera); Limnoi Oceanogr. 24 45-58 
Minshall J N 1967 Life history and ecology of Epeorus pleuralis (Banks) (Ephemeroptera: Heptageniidae); 

Am. Midi Nat. 78 369-388 
Monk D C 1976 The distribution of cellulase in freshwater invertebrates of different feeding habits; 

Freshwater BioL 6 471-475 
Moore J W 1977 Some factors affecting algal consumption in subarctic Ephemeroptera, Plecoptera and 

Simuliidae; Oecologia 21 261-273 
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dusky cotton bug, Oxycarenus hyalinipennis (Costa) (Hemiptera: Lygaeidae); Proc. Indian Nati Sci. 

Acad. B49 23 1-236 
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Oncopeltus fasciatus; Physiol. EntomoL 5 73-86 
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milkweed bugs, Oncopeltus fasciatus\ J. Insect Physiol. 26 79-84 
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Heptageniidae), Ph.D. dissertation, Madurai Kamaraj University, Madurai 
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103-132 
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(Plecoptera) and Deleatidium sp. (Ephemeroptera) in a New Zealand river; Freshwater BioL 4 507-524 
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 311-316. 
Printed in India. 



Impact of chemicals on feeding and reproduction in insects* 

KUMUDA SUKUMAR 

Regional Research Laboratory, Hyderabad 500 007, India 

Abstract. Pest control is today an important segment of entomology supported by 
objective programs of research, education and business management. In the continuous 
search for newer strategies for pest suppression, certain unique ideas have emerged and 
concretized to definite principles and practice. The two themes which merit relevance and 
recognition here are the use of chemicals directed to interfere with feeding and reproduction 
in insects. The impact of chemicals derived from natural and synthetic sources has been well 
utilized. by entomologists to suppress pests effectively either by inhibiting the gustatory 
stimulus or the reproductive potential. 

Keywords. Feeding behaviour; antifeedants; alkylating agents; reproductive .potential. 

1. Introduction 

Insect pest suppression is a basic need and several strategies have been employed. 
Chemicals which control the complex behaviour of insects in choosing and rejecting 
plants for food can be intricately designed and used to achieve a considerable degree 
of pest suppression. Manipulation by chemicals results in interference with the 
gustatory stimulus by modifying the sensory input of the insect eventually causing 
starvation death. Another approach is to control the future pest generations by 
chemically disrupting the normal insect reproductive process. The impact of certain 
chemicals on insect feeding and reproduction will be discussed in this paper. 

2. Impact of chemicals on feeding 

The intricate insect chemoreceptors surrounding the mouth region are hypersensitive 
to different chemicals present in the potential host plants and thus the insects have 
the capacity to discern the presence of phagostimulants or phagodeterrents (anti- 
feedants). The chemical composition of plants is the fundamental basis for acceptance 
or rejection of host plants as food in the phytophagous insect. The insect feeding 
behaviour is governed first by the host plant recognition and orientation, second by 
initiation of feeding and continuation of that activity and third by cessation of 
feeding. An antifeedant will inhibit the insect feeding by blocking the gustatory 
stimulus and thus disrupting the activity of initiation and maintenance of feeding. 
Some of the secondary plant products function as chemical defences against poly- 
phagous insects, and in the presence of these natural antifeedants insects may 
perhaps take to feeding on less distasteful weeds or eventually starve themselves to 
death. 

Antifeedants have been known for many years. As early as 1928, Eulan New was 
used for moth proofing woollens. Later triphenyl phosphorium salts, Mitin FF were 
also successfully used for protection of woollen against feeding of moth larvae. A 



*RRL(H) Communication No. 2054. 

31: 



312 Kumuda Sukumar 

series of compounds were later screened and the compound 24,055, i.e. 4-(dimethyl- 
triazeno) acetanilide was found to inhibit feeding in army worms. Many chemicals 
came under investigation, including inorganic salts of sodium, potassium, calcium, 
ammonium, arsenic, antimony and tin, aromatic hydrocarbons, quinones and 
naphthoquinones, alcohols, aldehydes, ketones and amines, amino acids, carbohy- 
drates, acids and bases, sesquiterpene lactones, oils, limnoids, quassinoids, saponins, 
flavonoids, isoflavonoids, phenols, tannins, coumarins and furocoumarins, alkaloids, 
steroids, terpenes, terpenoids and organometallic fungicides and most of these were 
found useful in inhibiting feeding in various leaf eating and sucking pests. Certain 
insecticides also proved to possess antifeedant activity to some extent. 

Our investigations showed that those chemicals which exhibited strong 
antifeedant activity induced similar patterns of behaviour in the test insect Achoea 
Janata larvae. After the first few bites of the treated leaf discs the test insects were 
deterred from feeding. At no stage did the insect orient away from the treated leaf 
discs and this condition is a prerequisite for development of a good antifeedant. 
Catharanthus root and leaf alkaloid exhibited absolute antifeedant activity against 
Achoea larvae. So was the case with methanol extracts of Nerium leaves. Meisner 
et al (1981) also found that the leaf extracts of C. roseus were effective antifeedants 
against Spodoptera. Ascher et al (1980, 1981) showed that certain ergostane-type of 
steroids, with anolides isolated from solanaceous plants had a considerable anti- 
feedant effect to Spodoptera. Coumarins are already known as good antifeedants 
(Chapman 1974; Koul 1983). A compound structurally similar to coumarin known as 
6,7, dimethoxy isochroman-3-one showed relative antifeedant activity (Reena et al 
1983). Triphenyl tin acetate a well-known antifeedant proved very effective against 
Achoea larvae and also against nymphs of H eiroglyphus banian. Joshi et al (1971) and 
Raghupathy (1973) have shown triphenyl tin acetate as a good antifeedant against 
Prodenia litura and Pericallia ricini. 

The mechanism of perception is dependent on the relevant receptors which diffe- 
rentiate stimulatory and inhibitory materials. Caterpillars have been shown to 
respond to inhibitory chemicals at several points in the feeding behaviour sequence 
and various ablation experiments have shown that sensilla on the maxilla are 
particularly important in perception of inhibitors not excluding other receptors. 
Dethier (1937) found that the response of caterpillars to odours was eliminated after 
removal of the antennae and maxillae and maxillary palps and the larvae of Sphinx 
liquistri after ablation fed on oleander and cabbage which were otherwise normally 
rejected. Removal of the palps from the larvae of Leptinotarsa also resulted in 
increased intake of normally unacceptable plants (De Wilde 1958). Fentin acetate an 
established antifeedant ceased to deter feeding in Spodoptera larvae by acting on the 
sensory organs situated on or near the mouth; this was proved by maxillectomy and 
extirpation of the labrums and antennae (Ascher and Ishaaya 1973). Kennedy (1967) 
and Ma (1972) interpret the results involving maxillectomy as indicating that the 
normal effect of the maxillary palpi of caterpillars is perhaps to inhibit feeding 
probably spontaneously and their removal disposes off this source of inhibition, 
while sensilla on other areas of mouthparts are responsible for the perception of 
inhibitory chemicals. The occurrence of other receptors of inhibitory materials 
elsewhere on the mouthparts was recognized by Ito et al (1959), Waldbauer (1962) 
and Ma (1969) since the intake of distasteful materials was not sustained and the 
insects were still able to differentiate degrees of distastefulness following 



Impact of chemicals on feeding and reproduction in insects 313 

maxillectomy. All experiments thus far conducted indicated that the maxillae and 
especially the palps are particularly important in the recognition of inhibitory 
chemicals although the receptors elsewhere on the mouthparts are also involved. 

The fact that insects may be prevented from feeding and damaging crops by 
interfering with their sensory inputs gains tremendous significance in the future pest 
control programme. 



3. Impact of chemicals on reproduction 

Insect reproduction is invariably influenced by many chemicals. By manipulating the 
dispersion of these chemicals during times detrimental to the insect, effective control 
can be established. 

3. 1 Chemosterilants 

Chemicals which directly affect the reproductive system bringing about sterility in 
insects by interfering and interrupting the steps in the reproductive cycle are usually 
known as chemosterilants. As early as 1942, it was reported that Mustard gas caused 
sterility in Drosophila. Then followed chemicals showing similar action, wherein 
ethylene glycol, nitrogen mustard, formaldehyde and tretamine also were reported as 
reproductive inhibitors. Attention was soon diverted to specific groups of chemicals 
and now presently the chemosterilants could be fairly categorised into 3 groups 
(i) alkylating agents, (ii) antimetabolites and (iii) miscellaneous chemicals. We 
confine our discussions mainly only to these chemosterilants and to certain chitin 
synthesis inhibitors although hormones and ecdysones also interfere with insect 
reproduction. 

3.2 Physiological effects on the female 

Although the female insect presented a more complex reproductive system, most of 
the research on the physiological effects by chemosterilants was carried out on 
females. Since the newly emerged female possessed a partially developed ovary, the 
interference of chemicals with the process of maturation and Oogenesis was more 
observable. The first investigations in this aspect could be attributed to Goldsmith 
et al (1948), when they found the inhibition in ovarian development in Drosophila 
due to chemical interferences, and later many chemicals like amethopterin, 
methotrexate and 5-flurourasil were found to share this property of ovarian inhibition. 
Soon after LaBrecque (1961) reported a group of chemicals belonging to the alky- 
lating group and commonly known as Apholate, Tepa and Metepa showing excep- 
tional promise in affecting both sexes. 

The most often observed physiological effect on female insects due to the chemical 
interferences was the retardation or complete cessation of ovarian development. The 
changes produced ranged from almost complete necrosis and disappearance of the 
organ to a slight reduction in size. The dose undoubtedly had a profound effect on 
the severity of damage. In general, the inhibition of ovarian development is the most 
outstanding characteristic feature of female chemosterilants particularly alkylating 



314 Kumuda Sukumar 

agents and antimetabolites. Too many chemosterilants cause varying degrees of 
ovarian inhibition and it is impossible to discuss all. We will only see certain 
examples now, since the number and diversity of compounds that inhibit oogenesis is 
truly astounding. Some chemosterilants although caused sterility, did not affect the 
reproductive physiology adversely. We could not induce any adverse effects on the 
Hemipteran or Lepidopteran ovary with Hempa or Hemel even at very high doses 
but there are reports wherein Dipteran ovary showed retardation of growth. Some 
miscellaneous compounds like colchicine also inhibited ovarian growth. Numerous 
triphenyl-tin derivatives also disturbed the ovarian physiology. 

3.3 Physiological effects on the male 

The testis unlike the ovaries seldom show drastic morphological changes. Although 
certain alkylating chemosterilants have been reported to cause testicular atrophy but 
more frequently the treated male continues to produce seemingly normal motile 
sperm throughout their life. But the sperms in spite of its outwardly normal chara- 
cteristics fail to produce adult progeny. The Zygotes present abnormal mitotic 
figures, chromosomal aberrations resulting in partial interrupted embryonic deve- 
lopment. Certain chemicals like the S-triazines (Hemel) and melamine derivatives 
showed a male sex specificity in causing sterility. In certain cases the male accessory 
glands showed some inhibition in development when chemosterilants were administe- 
red at younger stages. 

The chemosterilant effect is largely selective to the gonads. A direct interaction of 
the sterilant with the sperm and ovum has been demonstrated and this primary 
interaction seems to be the main mechanism of sterility (Borkovec 1969). Aziridines 
are known to inhibit various enzymes and also the synthesis of DNA of the gonads 
(Chamberlain and Barrett 1968; Madhukar et al 1971) as well as that of sterile eggs 
(Painter and Kilgore 1967). Ross (1962) showed that chemosterilants can cause 
cytostatic mutagenic and cytotoxic effects. Affected gonads exhibit various degrees of 
abnormalities. The spermatogenic activity may show disharmony and the direct 
interaction with the chemical can induce chromosomal breaks (Rai 1964). Such 
chromosomal anomalies find expression in dominant lethals in the embryo causing 
cell death (LaChance and Riemann 1964). 

3.4 Chitin synthesis inhibitors 

Chitin synthesis inhibitors mostly belonging to the group of benzoyl phenyl ureas 
were first developed as good insecticides around 1970 by Philips-Duphar Company. 
These compounds interfere with chitin biosynthesis during molting and hence are 
very effective as larvicides. Later, they were shown to possess a sterilizing activity on 
the adults. Diflurobenzuron commonly known as dimlin and its trifluroderivative, 
penfluron were the two chitin synthesis inhibitors which really gained immense 
popularity. In the case of these compounds also, the effect was more pronounced in 
females, than in males. Males perhaps transferred the compound to female during 
copulation which in turn acted as an ovicide. In certain Lepidopterans like nun moth 
and pink boll worm, adult treatment of benzoyl phenyl ureas presented no effects on 
spermatogenesis, mating or oviposition, but ovicidal effects were observed in codling 



Impact of chemicals on feeding and reproduction in insects 315 

moth, Egyptian cotton worm, soyabean looper etc. Boll weevil adults treated with 
benzoyl phenyl ureas primarily showed reduced egg hatch and mating behaviour was 
affected and the size of the testis also got reduced. Ovicidal effects without adverse 
effects on fecundity was observed in hornflies, stable flies, houseflies, faceflies and 
screw worm flies. Penfluron initiated adverse physiological changes in both sexes of 
Leptocoris and Dysdercus (Satyanarayana et al 1985; Satyanarayana and Sukumar 
1985). 



4. Action of certain miscellaneous agents 

4.1 Anti-juvenile hormones 

Following the discovery of juvenile hormones' (JH), hormonal research received an 
impetus and juvenile hormone-antagonists were envisaged. A JH antagonist will stop 
immature development resulting in a premature molt in an adult and an adult 
without JH cannot develop ovaries and would be sterile. Anti-JH are compounds 
which induced in insects precocious metamorphosis and Bowers named such com- 
pounds as Precocenes. Immature hemiptera undergo precocious metamorphosis by 
contact with precocenes. The resulting precocious adult females are sterile and the 
adult males are unable to inseminate normal females. Newly emerged females of 
cotton stainers and milk weed bugs treated with precocene show permanent sterility. 
But the application of exogenous JH to a sterilized female results in prompt ovarian 
development, showing that precocene acts to turn off the secretion of JH rather than 
interfering with JH action. 

Interfering with the presence or absence of JH during developmental stage will be 
disastrous for insect reproduction. Precocenes prevented ovarian development or 
regressed the already developed ovaries. Apart from the precocenes initially derived 
from Ageratum certain synthetic compounds like benzyl-3-benzodioxole derivatives 
also proved to be good anti-JH compounds. 

4.2 Antibiotics 

Antibiotics also captured the attention of agricultural entomologists and many 
compounds were used for insect control. 

Some of the ordinary solvents like benzene and acetone are reported to affect insect 
reproduction. Acetone at higher volumes 10 ^I/insect reduced fecundity in some 
Lepidopterans, but not at lower volumes. Coming to some of the commonly used 
insecticides, carbaryl and carbofuran stimulated females to lay more eggs. Methomyl 
also increased the fecundity of females of diamond back moth. 

It is already known that the Queen bee extract is found to reduce the fecundity of 
insects. Many amino acids tested against Corcyra showed promise as chemosteri- 
lants while certain other amino acids caused only partial sterility or at times 
increased fertility. Prostaglandins is also found to reduce eggs hatch and interfere 
with fecundity and fertility in cotton stainers. 

Insect reproduction is a versatile area wherein the intricate mechanism causing 
insect sterility could be made use of in pest control programs. 



316 Kumuda Sukumar 

References 

Ascher K R S and Ishaaya I 1973 Antifeeding and protease and amylase-inhibiting activity of fentin 

acetate in Spodoptera littoralis larvae; Pestic. Biochem. Physiol. 3 326-336 
Ascher K R S, Nemny N E, Eliyahu M, Kirson I, Abraham A and Glotter E 1980 Insect antifeedant 

properties of withanolides and related steroids from Solanaceae; Experientia 36 998-999 
Ascher K R S, Schmutterer H, Glotter E and Kirson I 1981 Withanolides and related ergostane-type 

steroids as antifeedants for larvae of Epilachna vari'vestis (Coleoptera: Chrysomelidae); Phytoparasitica 

9 197-205 
Borkovec 1969 Alkylating agents as insect chemosterilants in Biological effects of alkylating agents; Ann. 

New York Ac ad Scl. 163 866-868 
Chamberlain W F and Barrett C C 1968 Incorporation of tritiatea thymidine into the ovarian DNA of 

stable flies: Effects of treatment with apholate; Nature (London) 218 471-472 
Chapman R F 1974 The chemical inhibition of feeding by phytophagous insects: a review; Bull. Entomol. 

Res. 64 340-363 
Dethier V G 1937 Gustation and olfaction in lepidopterous larvae; Biol. Bull Mar. Bioi Lab. Woods Hole 

727 
DeWilde J 1958 Host plant selection in the Colorado beetle larvae; Leptinotarsa decemlineata; Say. 

Entomol Exp. AppL 1 14-22 
Goldsmith E D, Tobias E B and Harnly M H 1948 Folic acid antagonists and the development of 

Drosophila melanogaster; Anat. Rec. 101 93 
Ito T, Horie Y and Fraenkel G 1959 Feeding on cabbage and cherry leaves by maxillectomized silkworm 

larvae; J. Seric. Sci. Tokyo 28 107-113 
Joshi B G, Ramaprasad G and Subrahmanyam M 1971 Effect of fentin acetate as an antifeedant against 

tobacco, caterpillar Prodenia litura Fabricius; Indian J. Agric. Sci. 41 1110-1114 
Kennedy J S 1967 Mechanisms of host plant selection; Ann. AppL Biol. 56 317-322 
Koul O 1983 Feeding deterrence induced by plant limnoids in the larvae of Spodoptera litura F. (Lepido- 

ptera: Noctuidac); Z. Any. Entomol. 95 160 
LaBrecque G C 1961 Studies with three alkylating agents as housefly sterilants; J. Econ. Entomol. 54 

684-689 
LaChance L E and Riemann J G 1964 Cytogenetic investigations on radiation and chemically induced 

dominant lethal mutations in oocytes and sperm of the screworm fly; Mutat. Res. 1 318-333 
Ma W C 1969 Some properties of gustation in the larvae of Pieris brassicae; Entomol. Exp. AppL 12 

584-590 
Ma W C 1972 Dynamics of feeding responses in Pieris brassicae Linn, as a function of chemosensory 

input: a behavioural ultrastructural and electrophysiological study; Meded. Landbouwhogesch. Wage- 

ningen 72 1 62 
Madhukar B V R, Pillai M K K and Borkovec A B 1971 Chemosterilization of the yellow-fever mosquito. 

1 Laboratory evaluation of aziridinyl compounds by larval and pupal treatments; J. Econ. Entomol. 64 

1024-1027 
Meisner J, Weissenberg M, Palevitch D and Aharonson N 1981 Phagodeterrency induced by leaves of 

Catharanthus roseus in the larva of Spodoptera littoralis; J. Econ. Entomol. 74 131-135 
Painter R R and Kilgore W W 1967 The effect of apholate and thiotepa on nucleic acid synthesis and 

nucleotide ratios in housefly eggs; J. Insect Physiol. 13 1105-1118 
Rai K 1 964 Cytogenetic effects of chemosterilants in mosquitoes. I. Apholate-induced abberrations in the 

somatic chromosomes of Aedes aegypti L.; Cytologia 29 346-353 
Reena C, Afzal J, Das K G and Sukumar K 1983 6,7~Dimethoxy-isochroman-3-one: An antifeedant for 

castor semilooper Achoea Janata L.; Indian J. Farm. Chem. 1 63-65 , 
Raghupathy A 1973 Antifeeding properties of two fentin compounds in the control of Pericallia ricini F. 

(Arctidae) and Spodoptera litura Boisd (Noctuidae) on castor; Madras Agric. J. 60 32-37 
Ross W C J 1962 Biological alkylating agents (Butte worth's and Co. Ltd.: London) p 232 
Satyanarayana K and Kumuda Sukumar 1985 Sterility and retardation of oocyte growth by penfluron in 

soapnut bug Leptocoris coiiribatorensis (Gross); Z. Ange\v. Entomol. 100 367-372 

Satyanarayana K, Reena C and Kumuda Sukumar 1985 Penfluron induced sterility and ovarian inhibi- 
tion in adults of Dysdercus cingulatus F.; Int. Pest Control (London) 27 47-48 
Waldbauer G P 1962 The growth and reproduction of maxillectomized tobacco horn worm feeding on 

normally rejected non-solanaceous plant; Entomol. Exp. AppL 5 147-158 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. %. No. 3, May 19X7. pp. 317-321. 
Printed in India. 



Impact of differential feeding on the reproduction of tiger beetle 
Cicindela cancellata DeJean (Cicindelidae: Coleoptera) 

T SHIVASHANKAR and G K VEERESH 

Department of Entomology, College of Agriculture, University of Agricultural Sciences, 
GKVK, Bangalore 560 065, India 

Abstract. Tiger beetles belonging to the family Cicindelidae are exclusively predaceous 
and have great impact on the natural eco-balance. Recent studies have revealed the exi- 
stence of more than 300 species all over India under divergent habitats and none have been 
exploited for biological control of insect pests. Studies on the impact of differential feeding 
on the reproduction of Cicindella cancellata revealed a definite relationship between the 
quality and quantity of food consumed by adults on the fecundity, egg size, longevity of the 
adults, larval emergence pattern and the survivability of first instar larvae. Corcyra cephalo- 
nica larvae fed 3 times a day (55-74 mg) resulted in maximum egg production, longer adult 
life and longer survival of first instar larvae, followed by Corcyra, one larvae per day 
(37-16 mg) and mixed food of spiders, hoppers and Corcyra (alternately) (32-81 mg). Least 
fecundity was observed when fed with ants alone and no eggs with no food. 

Keywords. Tiger beetle; differential feeding; fecundity; survivability. 

1. Introduction 

Approximately around 2000 species of tiger beetles are known all over the world of 
which 300 species are reported from India. Almost all tiger beetles are predaceous 
both in their sedentary larval forms and mobile adult stage. Tiger beetles are genera- 
lists in their food habits and as such none have been exploited for biological control 
of insect pests. However, the major contribution of tiger beetle is as indicators of 
habitat degradation (Holeski and Graves 1978). The limiting factors in the distri- 
bution and abundance of tiger beetles in any ecosystem are oviposition site and food 
(Shelford 1908). The food intake rate of larvae is known to affect the size of later 
instars and adult size which in turn affect the individual fecundity (Hori 1982; 
Pearson and Knisley 1985). It is also stated that the years of abundant tiger beetles 
are normally the years of exceptionally high rainfall followed by the availability of 
rich prey. Therefore the study on the ecology and feeding behaviour assumes greater 
importance for the environmentalists. 

The present study was aimed to know the adult food preference, and the impact of 
selective feeding on the reproductive potential and longevity of a commonly occu- 
rring riverbed tiger beetle Cicindela cancellata DeJean (Ganeshaiah and Belavadi 
1986). 

2. Material and methods 

C. cancellata which occurs throughout the year on the river bed near sangam, 
100km from Bangalore, was collected periodically and the cultures maintained in 
the laboratory in small plastic containers measuring 7x12 cm. Newly emerged 
adults were confined in plastic containers, filled with 3/4 fresh sand. Optimum 



318 T Shivashankar and G K Veeresh 

moisture was maintained throughout the experiment at room temperature (23-65 
058C). A male and a female was enclosed in each of the following 7 treatments and 
replicated 5 times. The treatments consisted of 7\ = one corcyra larva (37-16 mg) per 
beetle/day, T 2 = 3 corcyra larva (55-7 mg) per beetle, fed 3 times a day, T 3 = 4-5 ants 
(Componotus compressus, 77-0 mg) per beetle/day, T 4 = mixture of spiders, leaf 
hoppers and grass hopper nymphs (23-97 mg) per beetle/day, T 5 = one corcyra larvae 
(37-50 mg) per beetle once in two days, T 6 = mixture of hoppers and ants alternated 
with one corcyra larvae (32-81 mg) per day and T 7 = no food. 

Actual quantity of corcyra larva fed was measured by weighing the beetles before 
and after feeding. The weight of beetles was recorded once before the commencement 
of the experiment and again on 12th and 18th day to know the difference if any. The 
rearing containers were changed on 9th and 20th day to avoid crowding of eggs/ 
larvae. Corcyra larva head was crushed before feeding to prevent it from spinning. 
The weight of the ants and hoppers were recorded by freezing them for a few min 
before feeding. 

Observations were taken for the number of eggs laid by counting the larval 
burrows everyday, even after the death of the beetles, till the last larval burrow was 
counted. 

Female beetles were dissected to know the effect of differential feeding on the re- 
productive organs. 

The data was subjected to statistical analysis following RCBD for longevity and 
fecundity, and correlation, regression for the rate of food consumed and survivabi- 
lity. 



3. Results and discussion 

It can be seen from table 1 that fresh weight of the female beetles at the beginning of 
the experiment ranged from 67-9 5-08 to 77-98 5-6 mg, and the males weighed 
51-2 6-08 to 58-56 4-55 mg. No significant differences in the weight of the beetles 
was observed on the 12th and 18th day of experiment, compared to the weight before 
commencement of experiment. Initially a female beetle consumed 11-15 mg of prey 
(7\, T 2 and T 6 ), whereas males consumed only 7-13 mg of prey, suggesting that the 
amount of food consumed depended on the size and body weight. 

The egg laying extended upto 4 weeks in almost all treatments except in mixed 
feeding with hoppers and spiders, where the egg laying stopped in the second week 
itself (table 2). 

The number of eggs laid was maximum in beetles supplied with 3 corcyra larvae 
(T 2 ) per day. The next best was one larvae per day (7\) and mixed feeding (T 6 ) but 
statistically these 3 were on par and significantly different from those that were fed 
with ants (r 3 ), mixed feeding (T 4 ) and feeding with corcyra larvae on alternate days 
(r 5 ). Treatment of ants alone, mixed feeding and corcyra on alternate days were on 
par. Also treatments 7\, T 4 , T 5 and T 6 were on par and superior to ants alone (T 3 ) 
(figure 1). 

The number of eggs laid were directly related to the quantity and quality of food 
consumed by the adults (r = 096) (table 3). According to Pearson and Knisely (1985) 
adult females of 3 species of tiger beetles at a low feeding levels produced significantly 
lower eggs and larvae than females at higher feeding levels, further they found that 



Differential feeding on the reproduction of C. cancellata 



319 



Table 1. Body weight of the beetles (n = 5) in relation to the quantity of food consumed 
during the study period. 







Initial 
weight (mg) 


Av. food 
consumed at 
2 days (mg) 


Weight at 
12 days (mg) 


Av. food 
consumed at 
12 days 


Weight at 
18 days 


Corcyra (7\) 


Female 


72-60 7-6 


14-50 3-55 


72-15 5-21 


9-30 1-77 


72-90 7- 15 




Male 


51-20 6-08 


6-87 2- 16 








Corcyra (T 2 ) 


Female 


74-74 4-20 


13-94 2-60 


74-1 2-01 


11-00 0-21 


72-40 5-00 




Male 


56-22 6-86 


13-08 2-99 








Ants (7* 3 ) 


Female 


67-90 5-08 





73-103-30 





66-73 2-56 




Male 


58-56 4-55 










Spiders, leaf and 














grass hoppers (T 4 ) 


Female 


69-44 11-31 





69-20 15-56 





61-367-52 




Male 


53-08 3-87 










Corcyra once in two 














days(r 5 ) 


Female 


70-02 5-12 





66-00 5-58 





61-409-15 




Male 


53-88 2-19 










Mixed and alternate 














with corcyra (T 6 ) 


Female 


77-98 5-60 


10-41 2-68 


69-50 6-85 


5-90 5-36 


61-66 5-41 




Male 


54-84 6-64 


10-524-21 








No food (r 7 ) 


Female 


71-354-80 





69-50 3-9 





62-50* 




Male 


56-28 4- 11 














*One observation 



Table 2. Larval emergence holes of C. cancellata from the 14th day after enclosure. 



Days after 
enclouser 


14 


16 


18 


21 


24 


27 


34 


45 


46 


48 


Corcyra (7^) 





1-75 


8-75 


17-50 


7-3 


22-50 


28-00 


30-25 


31-25* 


31-75 


Corcyra (T 2 ) 


2-4 


5-40 


18-20 


21-80 


26-4 


29-20 


35-00 


40-60* 


40-60 




Ants (T 3 ) 





0-33 


2-66 


3-33 


4-0 


5-00 


5-00 


6-00* 


6-00 




Spiders, leaf and 






















grass hoppers (T 4 ) 


1-75 


7-25 


13-00 


16-75 


17-5* 












Corcyra once in 






















two days (T s ) 


2-20 


7-20 


8-80 


14-80 


17-4 


21-40 


22-80 


23-40* 


23-40 




Mixed and alternate 


4-00 


6-33 


9-33 


13-00 


16-33 


20-33 


23-33 


28-33* 


28-33 




with corcyra (T 6 ) 






















No food (7 7 ) 

































*Last larval emergence noticed. 



small adult females produced fewer eggs and larvae at low feeding levels than larger 
conspecific females at the same feeding levels. 

The longevity of the adults fed with ants was on par with no-food and the 
fecundity was least among the beetles that were fed (table 3). It is evident from this 
finding that the major constituents of the prey of tiger beetles in the riverbed is not 
ants as it was commonly believed. Irrespective of the quality and quantity of the food 
consumed and with and without food, the longevity of the male did not vary much 
(figure 2) whereas the longevity of the female seems to depend on the quality and 
quantity of food, females live for only 17-66 1-80 days when starved compared to 
beetles fed with corcyra thrice a day (45-25 7-46 days). 

There was reduction in the size of the egg as well in the number of eggs in the 



320 



T Shivashankar and G K Veeresh 
60 r 



3 20 



O 



n 



n 



T l T 6 T 5 



Figure 1. Fecundity of C. cancellata in different treatments. 7\, one corcyra; T 2 , 3 corcyra; 
T 3 , ants; r 4 , mixed; T 5 , alternate day corcyra; 7 6 , mixed + corcyra. 



Table 3. Effect of differential feeding on the longevity of adults and survivability of larvae 
in C. cancellata. 



Quantity of Mean longevity (days) 


Survivability 
Mean No. of of first instar 


Moulting 
to second 




prey given 


lalvac pci aiici uu ua^a 


instar 


per day (mg) Male 


Female 


female 


(%) 


(O/\ 


Corcyra (7\) 


37-16 


11-75 9-54 


37-50 6-03 


31-33 9-06 


55-20 


1-60 


Corcyra (T 2 ) 


55-74 


8-00 3-74 


45-25 7-46 


40-60 6-97 


73-41 


7-38 


Ants (T 3 ) 


77-00 


16-00 4-40 


17-664-ll* 


6-00 1-41* 


27-77 


0-00 


Spiders, leaf and 














grass hoppers (T 4 ) 


23-97 


9-50 2-30 


25-75 4-96 


17-5 4-10 


57-14 


1-43 


Corcyra once in 














two days (t s ) 


18-50 


8-00 4-84 


39-80 12-74 


23-40 5-23 


37-61 


1-71 


Mixed and alternate 














with corcyra (T 6 ) 


32-81 


12-33 3-85 


42-33 20-00 


28-33 3-30 


55-29 


2-35 


No food (r 7 ) 





7-00 1-41 


17-66 1-80 











Correlation value 




0-81** 


0-814** 


0-96** 


0-39 


0-282 



* Correlation value excluding T 3 . 
** Significant. 



follicle depending upon the duration of starvation. The initial mean number of eggs 
found was 8-0 3-08 which reduced to 2-6 2-57 in 5 days starved beetles and no eggs 
were found in completely starved beetles at 20 days. 

Survival of first instar larvae was maximum (73-41%) in treatment T 2 (3 corcyra/ 
day) followed by mixed feeding T 4 (57-00%), ^ and T 6 treatment (55%). Evidently 
increased food to adult has increased the vigour and survivability of the larvae. It is 
also evident that the first instar larvae from the maximum fed adults (T 2 ) can pass to 
second instar without feeding as is seen in table 3. 

Therefore quality and quantity of adult food intake has great influence on the 
number of eggs laid by the predator, on the longevity of adults, larval survivability 
and vigour. 



Differential feeding on the reproduction of C. cancellata 

D Female 
Male 



321 



~ 80 j 










r"! 




2 40 


" 











>> 












1 20 


_ 










o> 
c 

3 o 




1 






I, 



I. 



Figure 2. Longevity of male and female C. cancellata in different treatments. Same nota- 
tions as in figure 1. 



Acknowledgements 

The authors wish to thank Dr David L Pearson of Penn State University, USA and 
Mr A R V Kumar and Mr M Vijaendra for their help and encouragement. 



References 

Ganeshaiah K N and Belavadi V V 1986 Habitat segregation in four species of adult tiger beetles 

(Coleoptera: Cicindelidae); EcoL Entomol 11 147-154 
Holeski P M and Graves R C 1978 An analysis of the shore beetle communities of some channelized 

streams in north west Ohio (Coleoptera); Great lakes Entomol. 11 28-36 
Hori M 1982 The biology and population dynamics of the tiger beetle Cicindela japonica (Thunberg); 

Physiol. EcoL Jpn. 19 71-212 
Pearson D L and Knisley C B 1985 Evidence for food as a limiting resource in the life cycle of tiger beetles 

(Coleoptera: Cicindelidae); Oikos 45 161-168 
Shelford V E 1908 Life-histories and larval habits of the tiger beetles (Cicindelidae); J. Linn. Soc. London 30 

157-184 



Prop. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 323-327. 
Printed in India. 



Some observations on the nutrition reproduction correlation in grouse 
locusts (Orthoptera: Tetrigidae) 

A M BHALERAO, N M NAIDU and S Y PARANJAPE 

Post Graduate Research Centre, Department of Zoology, Modern College, Shiv'ajinagar, 

Pune411 005, India 

Abstract. The correlation between nutrition and reproduction with reference to two 
grouse locusts, Euscelimena harpago (Tetrigidae: Scelimeninae) and Potua sabulosa (Tetri- 
gidae: Cladonotinae) is assessed. As far as Euscelimena harpago is concerned the impact of 
nutrition on reproduction is presented with reference to ovarian development in certain 
nymphal stages and in the adult. On the other hand, the impact is evaluated from an al- 
together different angle as far as Potua sabulosa is concerned. This pigmy locust prefers 
moss as food and tides over winter and hazardous summer as adult. During this period it 
almost does not feed. This peculiar feature is used as a parameter to assess the nature of 
impact in P. sabulosa. 

Keywords. Nutrition; reproductive biology; insect-plant interaction; preferred and non- 
preferred host; Tetrigids (grouse locusts). 



1. Introduction 

The Tetrigids essentially are phytophagous caeliferan orthoptera, considered to be 
primitive and phylogenetically related to the acrididoid grasshoppers and locusts on 
the one hand and the tridactilids on the other (Paranjape et al 1986). Contributions 
by Bhalerao and Paranjape (1986), Gangwere (1961), Hodgson (1963), Hancock (1898), 
Paranjape (1976, 1985), Paranjape and Bhalerao (1985), Paranjape et al (1986) and 
Poras (1979) are the most significant ones while some acridological work with 
reference to sensillar pattern reported by Ananthakrishnan et al (1985), to plant 
chemistry and feeding behaviour by Bernays and Chapman (1978), and to host pre- 
ferences as well as biochemical parameters by Sanjayan and Ananthakrishnan (1986) 
are of considerable importance in relation to the grouse locusts: As no information is 
available on the impact of nutrition on reproduction in these insects an attempt has 
been made to study these aspects with reference to Euscelimena harpago Serville and 
Potua sabulosa Hancock. 

2. Material and methods 

Samples of live E. harpago (Subfam.: Scelimeninae) and P. sabulosa (Subfam.: Clado- 
notinae) were periodically collected from localities in Pune and Mahabaleshwar. The 
grouse locusts were kept in cages containing their natural food. The female speci- 
mens were periodically dissected to study mainly, the development of ovaries with 
reference to the nature and the number of ovarioles, the oocyte-ovariole index and 
accumulation of fats in the body; after measuring the body length. The different mea- 
surements were taken using calibrated eye-piece gratings, of a zoom type trinocular 
dissecting microscope. Since P. sabulosa, the pigmy locust shows an altitude-limited 



324 A M Bhalerao, N M Naidu and S Y Paranjape 

distribution (Paranjape and Bhalerao 1985), to verify the behavioural change, if any, 
during the overwintering period, these specimens were maintained in the laboratory 
in Pune before the onset of winter. They were also collected periodically from their 
natural habitat in Mahabaleshwar in Dist. Satara, the hill station located at an 
altitude of about 1573 m and about 120km from Pune. 



3. Results and discussion 

3.1 Food of grouse locusts 

The tetrigids are known to be feeding mainly on algae, bryophytes (especially moss: 
Funaria sps.), fungi and lichens, on detritous material and also on humus. E. harpago, 
a predominantly semi-aquatic grouse locust, shows preference to algae and detritous 
material (Bhalerao and Paranjape 1986) as is also revealed by the study of foregut 
contents. The scelimenids as shown by E. harpago, are known to be occasionally 
feeding on sproutings of paddy and also showing necrophagy. On the other hand, 
the pigmy locust, P. sabulosa shows its preference for moss (Funaria sps.) that luxu- 
riantly grows in the said habitat with humid and rather cool climate, and also feeds 
on humus. 

Although the grouse locusts possess the biting and chewing type or mandibulate 
type of mouth parts that are characteristic of orthoptera in general, the molar 
regions of the mandibles have been shown to be lacking in molar dentes that are 
present in other acridoids (Paranjape 1985). Instead, the SEM studies of the molar 
region in these insects (Paranjape et al 1986) have revealed an alternating grooved 
and serrated ridged pattern that is suitable for eating the soft, pulpy food. Moreover, 
this very pattern seems to be acting as a limiting factor when tough textured 
monocot leaves of cereals and grasses are considered as alternate host of these phyto- 
phagous orthopterans. 

The habitat together with the climatic conditions in a year, availability of the food/ 
host-plants and host plant preference by the two grouse locusts under study and the 
impact of these on their reproductive biology as is revealed by the observations done 
so far, are briefly given in the account to follow (table 1). 

3.2 Observations in E. harpago 

These grouse locusts are found in semi-aquatic habitat that also ensures their 
preferred food almost throughout the year excepting the summer months that are 
hazardous. E. harpago therefore shows ability to lay eggs almost from June through 
February/March in natural conditions that are available during major parts of the 
year. This is supported by the observation that one can find different nymphal stages, 
adults and periodically even gravid females, practically throughout the year. During 
the summer months however the population declines and consists of aestivating 
5th/6th instar nymphs and a few adults. As far as the impact of food is concerned the 
findings are as under: 

The zero day female adults when fed on paddy sproutings as an intermediate 
preferred food, died after 5/6 days. Furthermore, the maize plant turned out to be a 



Nutrition-reproduction correlation in grouse-locusts 

Table 1. Impact of seasonal variations on the ovaries of two grouse locusts. 



325 



Body length (mm) Ovariole No. Oocyte Ovariole 

Stage Month Mean Nature of ovary Average index <Mn\ 



E. harpago 


Nymph-III 


Nov. 


6-35 


Panoistic, 


32 


0-041 










translucent 








Nymph-V 


Nov.-Dec. 


9-04 


Panoistic, 


38 


0-057 






(Winter) 




translucent 








Zero day 


Nov.-Dec. 


22-56 


Panoistic, 


46 


0-092 










light yellow 








Adult 


Nov. 


24-26 


Panoistic, 


50 


0-148 










deep yellow 








Adult 


Jan. 


25-08 


Panoistic, 


50 


0-232 




(Gravid) 






deep yellow 






P. sabulosa 


Adult 


Nov.-Dec. 


7-50 


Panoistic, 


24 


0-110 




(Lab.) 


(Winter) 




almost transparent 








Adult 


Nov.-Dec. 


8-00 


Panoistic, 


23 


0-142 




(Nature) 






almost transparent 








Adult 


June 


8-20 


Panoistic, 


22 


0-519 




(Gravid) 


(Monsoon) 




deep yellowish 














brown 







non-preferred host because the zero day female adults died in about 26 h. The modi- 
led mandibular molar region makes it difficult for these insects to feed on the said 
^osts, by acting as some kind of a limiting factor. 



3.3 Observations in P. sabulosa 

rhe pigmy locust on account of its habitat peculiarities and relative preference for 
noss-humus complex, presents an altogether different case for study. The gravid 
'emales (table 1) lay eggs in June-July. The hatchlings metamorphose and become 
idults by the following October. The adults are seen to be overwintering (November- 
December/mid January or so). During this period there is very little food intake, 
arge quantities of fat accumulate in the body, while ovarial filaments show hardly 
my growth, save the large basal oocyte. The entire ovary appears transluscent indi- 
cating that the process of vitellogenesis as such is not active. The pigmy locusts kept 
n the laboratory from October through January showed similar ovarian develop- 
ment when compared with the specimens collected periodically from their natural 
labitat (table 1). During February through May, the adults try to overcome un- 
'avourable summer conditions by burrowing in soil (Paranjape and Bhalerao 1985). 
During this period they practically do not feed. With the beginning of monsoon the 
Dvarian development is activated and the adults copulate and lay eggs as stated 
earlier. Further the above aspects will throw more light on the impact of various 
environmental factors on the reproductive biology of the males and females of the 
locust. 



\. Conclusions 

i) The studies carried out so far indicate that in the grouse locusts nutrition has an 
mpact on reproduction. 



326 A M Bhalerao, N M Naidu and S Y Paranjape 

(ii) As compared to acridoids the grouse locusts have relatively specific habitat and 

trophic requirements. 

(iii) The peculiar modification of the mandibular molar region acts as an adaptation 

to the type of food that these insects prefer and at the same time as a limiting factor 

when it comes to their feeding on tough textured leaves. 

(iv) Along with food the climatic factors also influence reproductive biology of 

these insects although the degree is different in the grouse locusts under study. 

(v) The primitive phylogenetic status of the grouse locusts, their habitat and their 

peculiar feeding preferences on lowly evolved food plants, provides an interesting case 

for study from the point of view of plant-animal coevolution, since the importance of 

this topic has already been suggested by Swain (1978). 



Acknowledgements 

The authors are extremely grateful to Prof. T N Ananthakrishnan, Entomology 
Research Institute, Loyola College, Madras for his advice to undertake these studies 
in the grouse locusts and to Dr S N Navalgundkar for the encouragement. Thanks 
are also due to the staff at the research centre and to our colleague Dr H V Ghate in 
particular for his help in discussions. One of the authors (SYP) specially wishes to 
thank Director, Zoological Survey of India, Calcutta for the award of Contractual- 
Collaborative Project (No. 223-34(4)/85, Tech.) on grouse locusts under which this 
work has been carried out. 



References 

Ananthakrishnan T N, Dhileepan K and Padmanaban B 1985 Behavioural responses in terms of feeding 

and reproduction in some grasshoppers (Orthoptera: Insecta); Proc. Indian Acad. ScL (Anim. Sci.) 94 

443-461 
Bernays E A and Chapm ( an R F 1978 Plant Chemistry and Acridoid Feeding BehaviourMwiw0/ Proc. 

Phytochemical Society, Reading No. 15, (London: Academic Press) Chapter 5, 99-137 
Bhalerao A M and Paranjape S Y 1986 Studies on the bioecology of a grouse locust Euscelimena harpago 

Serv. (Orthoptera: Tetrigidae); Geobios 13 145-150 

Gangwere S K 1961 A monograph on food selection in Orthoptera; Trans. Am. Entomol. Soc. 87 67-230 
Hancock J L 1898 The food habits of Tettigidae; Entomol. Rec. J. Variat. 10 6-7 
Hodgson C L 1963 Some observations on the habits and life history of Tetrix undulata Swrb. (Orthoptera: 

Tetrigidae); Proc. R. Entomol. Soc. London A38 200-205 
Paranjape S Y 1976 Studies on the semi-aquatic grasshopper, Scelimena ( = Euscelimena} harpago Serv. 

Life history: Observations on egg-laying, the eggs, their growth and hatching, Abstract, Proceeding 

(Part IV) 63rd Session, Indian Sci. Congr. pp 47-48 
Paranjape S Y 1985 Behavioural analysis of feeding and breeding in Orthopteran insects; Proc. Indian 

Acad. ScL (Anim. Sci.) 94 265-282 
Paranjape S Y 1985 
Paranjape S Y and Bhalerao AM 1985 Bioecological observations on a pigmy locust, Potua sabulosa 

Hancock (Tetrigidae: Orthoptera); Psyche 92 331-336 
Paranjape S Y, Bhalerao A M and Naidu N M 1986 On etho-ecological characteristics and phytogeny of 

Tetrigidae Memoire delta', Societa Entomol. Italiana (In Press) 
Paranjape S Y and Bhalerao A M 1986 A review of Biosystematic and Ethoecological studies on the 

family Tetrigidae (Orthoptera); Special Proc. of III Oriental Entomol. Symp. Vol. 1 19-23 
Poras M 1979 Le cycle biologique d'un tetrigid bisannuel (Tetrix undulata Sowerb) hivernat a 1'etal 

larvaire et imaginal (Orthoptera: Tetrigoidea); Acrida 8 151-162 



Nutrition-reproduction correlation in grouse-locusts 327 

ayan K P and Ananthakrishnan T N 1986 Host preferences of some Acridoids (Insecta: Orthoptera) 
n relation to some biochemical parameters; Proc. Indian Acad. Sci. (Anim. Sci.) 96 15-21 
in T 1978 Plant- Animal Coevolution, a Synoptic View of the Paleozoic and Mesozoic: Plant-Animal 
interactions; in Biochemical Aspects of Plant and Animal Coevolution (ed) J B Harborne, (London: 
\cademic Press) 



roc. Indian Acad. Sci. (Anim. Sci,), Vol. 96, No. 3, May 1987, pp. 329-332 
) Printed in India. 



nfluence of biochemical parameters of different hosts on the biology of 
brias vittella (Fab.) (Noctuidae: Lepidoptera) 

R SUNDARARAJ and B V DAVID 

Fredrick Institute of Plant Protection and Toxicology, Padappai 601 301, India 
Abstract. The present study aims to understand the influence of biochemical parameters of 
the host plants viz Ahelmoschus esculentus L, Gossypium hirsutum L. and Abutilon indicum 
G. Den. on the reproductive biology of Earias vittella. The results indicate remarkable 
variation in the biochemical parameters of host plants which affected significantly the 
growth index and reproductive potential of the insect species. Decrease in body weight, 
fecundity, prolongation of larval period, percentage of larva becoming adult and adult 
longevity of both male and female was observed when reared on Abutilon indicum which 
had less amount of reducing sugars, protein and free arnino acids but had high amount of 
non reducing sugars. Decreased larval period, increased fecundity and adult longevity were 
observed when reared on cotton and okra which had high amount of reducing sugars, free 
amino acids and protein. There was no significant difference in the amount of total soluble 
sugars among the 3 hosts tested. The growth index and fecundity of Earias vittella are 
apparently high on okra than on cotton. It is observed that high amount of reducing sugars, 
free amino acids and protein seems to be favourable for increase in fecundity of Earias 
vittella. 

Keywords. Earias r\ttella\ reproductive biology; okra; cotton; Abutilon indicum; 
biochemical parameters; incidence. 



Introduction 

he pest status of an insect species depends on its ability to breed on a variety of host 
[ants, comparative growth rate, fecundity, population dynamics and distribution 
^nanthakrishnan 1977). It was reported by Khan and Rao (1960) that preferred 
3Sts have a great influence on oviposition, fecundity, developmental period and 
mgevity of adults of Earias vittella (Fab.) and E. insulana (Boisd.). Mehta and 
axena (1970) were the first to study the role of ovipositional responses of E. vittella 
( its establishment on different plants. Subsequently in 1973, they reported that 
*owth of E. vittella varies with the degree of consumption, nutritive value and 
tilization of food from different plants. The insect grows best on diet whose nutritive 
due and degree of conversion into body matter are high. Krishna et al (1977) 
ported on oviposition in E. vittella in relation to adult nutrition, mating and some 
ivironmental factors. Vishwapremi and Krishna (1974) working on E. vittella 
rought to light the need for a carbohydrate diet, especially raffinose during its adult 
age for achieving maximum fecundity. Hiremath (1984) investigated the host 
r eference of E. vittella on 7 different hosts under field conditions and the effect of 
icsc host plants on development, longevity and fecundity. Mani et al (1986) studied 
le effects of larval food quality of okra and Hibiscus rosasinensis on egg output and 
ability in E. vittella. The present study aims to relate the biochemical nature of 3 host 
:ants viz Ahelmoschus esculentus L., Gossypium hirsutum L. and Abutilon indicum 
. Den. with the reproductive biology of E. vittella. 



330 



R Sundararaj and B V David 



2. Material and methods 

Three plant species viz A. esculentus (okra), and G. hirsutum (cotton) grown in fields 
and A. indicum found along the bunds in the campus of the Fredrick Institute of 
Plant Protection and Toxicology, Padappai, were taken up for investigations. From 
the laboratory culture of E. vittella adequate number of larvae were obtained for the 
studies. On each of the host plants viz A. esculentus (epicarp removed okra fruit), 
G. hirsutum (opened tender bolls of cotton) and A. indicum (opened young fruits), 
hundred freshly hatched larvae were released and the duration of larval and pupal 
stages, adult longevity and fecundity were also recorded. The studies were replicated 
thrice. 

Simultaneously, samples of the larval food material were analysed for reducing 
sugars, total sugars, free amino acids and protein. Ethanol extracts of the larval food 
material were obtained by using 80% ethanol. The reducing sugars were determined 
first and subsequently the nonreducing sugars in the extract were hydrolysed to 
reducing sugars and the total sugars were estimated by Nelson's (1944) method. By 
subtracting the reducing sugars from the total sugars the nonreducing sugars were 
estimated and expressed as glucose equivalents. Free amino acids were estimated by 
Moore and Stein's (1948) method and protein by Lowry's (1951) method (Mahadevan 
and Sridhar 1982). 



3. Results and discussion 

The data presented in table 1 shows the developmental period, longevity, survival, 
weight of fourth instar larva, pupa and adult, fecundity and growth index of 
E. vittella on 3 hosts viz okra, cotton and A. indicum. The biochemical constituents 
of the larval food viz the seeds of the 3 host plants with regard to reducing sugars, 
nonreducing sugars, free amino acids and protein are furnished in table 2. 

The duration of larval and pupal period was considerably longer on A. indicum 
being 18-33 3-70 and 13-03 3-11 days respectively and the toal developmental 
period worked out was 32- 10 2-98 days. On the other hand, the total developmental 
period was the shortest on okra (26-90 3-20 days) while slightly more on cotton 
being 27-90 3- 12 days. The percentage survival has been found to be the lowest on 

table 1. Biology of E, vittella on three different hosts. 



Duration (days) 


Total deve- 
lopmental 
period (days) 


Survival 
(%) 


Growth 
index* 


Host 


Incubation 


Larva 


Pupa 


Okra 
Cotton 
A. indicum 


4-57 0-32 
4-55 0-41 
4-57 0-38 


12-73 2-41 
13-76 3-10 
18-33 3-70 


!H62-79 
12-36 2-54 
13-03 3-11 


26-90 3-20 
27-903-12 
32- 10 2-98 


68-00 5-72 
67-00 9-09 
26-33 3-29 


2-52 0-21 
2-40 9-09 
1-02 0-13 


Longevity (days) 


Weight (g) 




Hoist 


Male 


Female 


Fecundity 


IV instar 


Pupa 


Adult 


Okra 
Cotton 
A. indicum 


10-76 2-20 
9-33 2-84 
6-23 2-45 


14-603-12 
14-13 3-09 
9-90 2-98 


350-67 28-77 
301-66 14-52 
135-00 20-05 


0-084 0-011 
0-077 0-014 
0-050 0-01 3 


0-069 0-005 
0-068 0-009 
0-045 0-005 


0-024 0-004 
0-023 0-06 
0-0 14 0-003 



Survival (%) 



Biochemical parameters of different hosts on E. vittella 
Table 2. Biochemical constituents* in seeds of three hosts of E. vittella. 



331 





Reducing 


Non reducing 


Total soluble 


Free amino 




lost 


sugars 


sugars 


sugars 


acids 


Proteins 


)kra 


5-66 


1245 


17-58 


2-73 


15-10 


"otton 


5-88 


13-34 


18-62 


2-65 


14-64 


I. indicum 


1-74 


16-38 


18-12 


1-85 


12-02 



Values in mg/g (mean of 3 replications). 



1. indicum being 26-33 3-29 as against very high levels (67-00 9-09 to 68-00 
1-72% respectively) on cotton and okra. The growth index was also found to be poor 
a A. indicum and was fairly high in okra and cotton. The fecundity, longevity of 
.dults and the weight of fourth instar larva, pupa and adult on okra was compara- 
ively higher than that of cotton. In these aspects there has been considerable 
eduction in case of A. indicum. The duration of larval and pupal periods, adult 
ongevity and fecundity observed in the present study on okra, cotton and A. indicum 
.re in conformity with the observations of Hiremath (1984). Vedamoorthy and Reed 
1977) reported that the spotted bollworms E. vittella and E. insulana exhibits 
narked preference for various host plants. Vishwapremi and Krishna (1974) 
>bserved maximum fecundity in E. vittella when the larvae were reared on 
leveloping seeds of okra. 

It is obvious from the present study that okra and cotton are the most favoured 
tosts. This finds correlation with the high level of reducing sugars, free amino acids 
.nd proteins and low level of nonreducing sugars. In the unfavourable host namely 
1. indicum, reducing sugars and free amino acids are very low. The nonreducing 
ugars are considerably high and protein content is also comparatively very less, 
lowever, there is no much variation in the level of total sugars. Though Mehta and 
laxena (1973) studied the nutritive value of host plants, there is no quantitative 
ndication of the various biochemical parameters. Only qualitative assessments have 
>een presented. It is evident from the present study that a high level of reducing 
ugars, free amino acids and proteins and low level of nonreducing sugars in the food 
dants of okra and cotton favour high fecundity, survival and quicker development of 
I. vittella and these nutrients are found in less amount in the unfavoured A. indicum. 
n this connection it may be of interest to note that high level of reducing sugars in 
Irassica campestris has been correlated with high level of infestation of the 
.gromyzid pests viz Liriomyza brassicae Rilay and Chromatomyia horticola Gour 
[pe 1985) and in rice increased amount of free amino acids was correlated with its 
usceptibility to yellow stem borer (Vidyachandra et al 1981). Though A. indicum was 
Dund in large numbers in the campus and throughout the state, periodical surveys 
evealed no natural infestation and the nonpreferred nature of the host might 
irobably be responsible for this. In Karnataka, on the other hand infestation of 
I. vittella on A. indicum was observed and the mean number of fruiting bodies 
iamaged was 0-10 as against 5-74 on cotton and 60-68 on okra. 



icknowledgement 

'he authors are thankful to Miss V Jayanthi, for suggestions and to Mr S James 
r redrick for facilities provided. 



332 R Sundararaj and B V David 

References 

Ananthakrishnan T N 1977 Insects and host specificity (ed) (Madras: Loyola College) pp 1-4 

Hirernath I G 1984 Host preference of spotted bollworm Earias sp. (Lepidoptera: Noctuidae); Entomon. 9 

185-188 
Ipe M Ipe 1985 The Acjromyzidae: Infestation, host plant selection, Host chemistry and interactions (ed) 

S Singh (Agra: St. Johns College) pp 58-63 
Khan Q and Rao B P 1960 Insect and mite pests, cotton in India (Bombay: Central Cotton Committee) 

pp 2 17-301 
Krishna S S, Vishwapremi K K C and Shahi K P 1977 Studies on the reproduction in Earias fabia Stoll 

(Lepidoptera: Noctuidae); Oviposition in relation to adult nutrition, mating and some environmental 

factors; Entomon. 2 11-16 
Mahadevan A and Sridhar S 1982 Methods in Physiological plant pathology 2nd edition, (Madras: 

Sivakami Publications) pp 69-113 
Mani H C, Pathak P H and Krishna S S 1986 Effects of larval food quality on egg number and viability in 

Earias fabia Stoll (Lepidoptea: Noctuidae); Z. Amjew. Zool. 73 115-120 
Mehta R C and Saxena K N 1970 Ovipositional responses of the cotton spotted bollworm Earias fabia in 

relation to its establishment of various plants; Ento-mologyra Exp. Appl. 13 10-20 
Mehta R C and Saxena K N 1973 Growth of the cotton spotted bollworm Earias fabia (Lepidoptera: 

Noctuidae) in relation to consumption, nutritive value and utilisation of food from various plants; 

Entomol. Exp. Appl. 16 20-30 
Vedamoorthy G and Reed W 1977 A review of host specificity of the pests of cotton in India; in Insects 

and host specificity (ed) T N Ananthakrishnan (The Macmillan Company of India Ltd.) 
Vidyachandra B, Roy J K and Bhaskar Das 1981 Chemical differences in rice varieties susceptible or 

resistant to gall midge and stem borer IRRN 6 7-8 
Vishwapremi K K C and Krishna S S 1974 Variation in the reproductive potential of Earias fabia (Stoll) 

(Lepidoptera: Noctuidae) reared on whole fruit of okra or on its components; Proc. Indian Nati Sci. 

Acad. 13 440-445 



Proc. Indian Acad. Sci. (Anim. ScL), Vol. 96, No. 3, May 1987, pp. 333-340. 
Printed in India. 



Effects of feeding regime on energy allocation to reproduction in the 
silkworm Bombyx mori 

S MATHAVAN, G SANTHI and B NAGARAJA SETHURAMAN 

School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India 

Abstract. Pattern of energy allocation for reproduction was studied as a function of 
feeding regimes in the silkworm Bombyx mori. The larvae were restricted to feed for 6, 12, 18 
or 24 h (ad libitum}. Restriction of feeding duration resulted in slow growth and extended 
larval duration. Consumption, assimilation, conversion and metabolism and their rates 
showed steady increase with the increase in the feeding duration. On pupation, energy 
allocation for egg production was studied by dissecting the pupa and estimating the 
reproductive and body tissues separately during the entire pupal period. Length and 
biomass of ovary steadily increased with increasing feeding duration as well as with 
increasing age of pupa. During egg development, the tissue protein was transferred to the 
ovary. This type of conversion was more in those fed ad libitum than in those restricted to 
feed for shorter durations. Tissue fat and ovarian fat decreased as a function of age of pupa, 
indicating that fat served as the main source of energy for pupal metabolism. Bombyx mori 
restricted to feed for 6 h duration allocated 55",, pupal energy for reproduction while those 
fed ad libitum allocated about 67% of the pupal energy for reproduction. However, they 
allocated about 15-19% of assimilated energy for egg production in all the feeding regimes. 

Keywords. Silkworm; Bombyx mori; feeding duration; energy allocation; egg production. 



1. Introduction 

Food is a factor of paramount importance which regulates animal growth, 
reproduction and diversity of animals in nature (White 1978). Negative effects of 
diminished/restricted rations on food utilization in herbivores was perhaps first 
considered by Muthukrishnan and Delvi (1974). Subsequent publications in this area 
concentrated on the effects of restricted feeding on food utilization and growth of 
lepidopterous larvae (Mathavan and Muthukrishnan 1976; Muthukrishnan et al 
1978; Muthukrishnan 1980; Christopher 1983). Surprisingly none of the investigators 
concentrated on the effects of feeding regime on the fecundity. Egg production 
depends on accumulation of sufficient nutrient during larval period and attain- 
ment of ideal adult weight; it will facilitate for the allocation of substantial 
nutrient energy by the adult for egg production (Engelmann 1970; DeWilde and 
DeLoof 1973a,b; Slansky 1980a,b; 1982). The present paper reports on the effects of 
restriction of feeding duration on food utilization and energy allocation for egg 
production by the silkworm, Bombyx mori. 

2. Materials and methods 

Fourth instar larvae of B. mori (TNxNB 4 D 2 ) were obtained from the Sericulture 
Department, Madurai. They were brought to the laboratory, acclimated and reared 
in cellular trays. Freshly moulted final instar larvae were used for the experiment. 
The larvae were divided into 5 groups consisting of 100 larvae each. Larvae in the I 

333 



334 S Mathavan, G Santhi and B Nagaraja Sethuraman 

group were restricted to feed for 3 h/day; those in the II, III, IV and V groups were 
allowed to feed for 6, 12, 18 and 24 h/day respectively. Triplicates were maintained in 
each group. Faecal pellets were collected separately for each tray during the time of 
removal of unfed leaves, (once in a day) and were oven dried at 90C for weight 
constancy. Prior to spinning, few worms from each group were sacrificed for dry 
weight determination and the remaining worms were allowed to pupate. 

A minimum of 10 female pupae were identified from each series on every alternate 
day. The above collected pupae were weighed and dissected; the reproductive (ovary) 
and vegetative tissues (body tissue) were carefully separated. Ovary length, egg 
number and weight of the ovary were measured and dried separately. The powdered 
dry ovarian and body tissues were analysed for their chemical composition. Protein 
was estimated following the method of Lowry et al (1951) and that of Raymond et al 
(1964) was followed for lipid estimation. Calorific value was estimated using a semi 
micro bomb calorimeter (Parr Instrument Company, USA). 

The scheme for energy budget followed here is the slightly modified IBP formula 
(Petrusewicz and Macfadyen 1970) C = P+ R + F; where C the food consumed, P the 
growth, R the weight loss due to metabolism and F the undigested food defecated 
including nitrogenous excretory products. Food consumed was estimated following 
the method of Mathavan and Pandian (1974). Assimilation was estimated by 
subtracting F from C; P was estimated by subtracting the initial dry weight of the 
larvae at the commencement of the experiments from the final dry weight of the same 
at the end of the experiment. Rates of feeding (Cr), assimilation (Ar), conversion or 
production (Pr) and metabolism (Mr) were calculated by dividing the respective 
amounts of weight by the product of mid-body weight (g) of the animal and duration 
(day) required for the completion of the'life stage; the rates are expressed in terms of 
mg/g live insect/day. The gross conversion efficiency (K^ expressed the percentage of 
food converted in relation to the food consumed, while net conversion efficiency (K 2 ) 
refers to the percentage of food converted in relation to assimilated food. 

3. Results 

Larvae restricted to feed 3 h/day survived for about 7 days and subsequently died. 
Restriction of feeding duration in other groups resulted in slow growth and extended 
larval duration. Larvae fed for 24 h/day required only 7 days to complete the feeding 
period whereas those restricted to feed 12 and 6 h/day required about 8 and 1 1 days, 
to pupate successfully (table 1). However, pupal period remained the same (8 days) in 
all the groups. 

Table 1 presents the data on the mass budget of fifth instar larvae of the silkworm, 
fed on mulberry (Moms alba) at different restricted feeding durations. Food intake 
decreased from 3810mg in the group fed ad libitum to 1900 mg, when the feeding 
duration was reduced to 6 h/day. Assimilation, conversion and metabolism increased 
with increasing feeding duration. 

Rates of consumption, assimilation, conversion and metabolism showed steady 
increase, with the increase in the feeding duration (table 1). For instance, 
consumption rate was 156mg/g/day in the group fed for 6 h/day; it increased to 
339 mg/g/day in those fed ad libitum. However, net conversion efficiency of 34% in 
the 24 h feeding group has been elevated to 42% in those restricted to feed for 
6 h/day (table 1). 



Energy allocation to reproduction in B. mori 



335 



Table 1. Effect of restriction of feeding duration on energy budget of B. mori 



Feeding duration 


Parameters 


6h 


12 h 


18h 


24 h 


Instar duration (days) 


11 


8 


7 


7 


Pupal duration (days) 


8 


8 


8 


8 


Consumption (mg) 


1900 


2550 


3730 


3810 


Assimilation (mg) 


664 


955 


1396 


1432 


Conversion (mg) 


277 


318 


442 


488 


Metabolism (mg) 


387 


637 


954 


944 


Consumption. rate 










(mg/g live insect/day) 


159 


271 


333 


339 


Assimilation rate 










(mg/g live insect/day) 


56 


101 


125 


128 


Conversion rate 










(mg/g live insect/day) 


23 


34 


39 


43 


Metabolic rate 










(rng/g live insect/day) 


32 


68 


85 


84 


Net conversion efficiency (%) 


42 


33 


52 


34 



soo 



400 - 





2 6 10 
Pupal oge(day) 



4 6 e 

Pupal age (day) 



10 



Figure 1. Egg number and ovary length of B. mori as functions of feeding duration and 
pupal age. 



Restriction of feeding duration inflicted considerable change in the reproductive 
potential of B. mori. It is clear from figure 1 that with advancing age of pupa ovary 
length increased in all the groups; the ovary length doubled (12 cm) in the ad libi- 
tum group in comparison to the group fed for 6 h/day (6 cm). Corresponding to 
ovary length, egg number/female has also increased with increasing feeding duration 
(figure 1). . 

Data obtained for the egg weight of different age pupa and adult moths obtained 
from varying feeding schedules are represented in figure 2. The moths in the 24 h/day 
fed group almost doubled their ovary weight (990 mg) compared to the moths from 
the group fed for 6 h/day (480 mg). 

Figure 3 represents the data on the changes of protein and fat content in the 
egg and the pupal tissues during the different days of pupal development. Percentage 
composition of protein in the egg increased with advancing pupal age in all the 



S Mathavan, G Santhi and B Nagaraja Sethuraman 




Figure 2. Ovary length and weight (wet) of B. mori as functions of feeding duration and 
pupal age. 



40 



230 



01 

LJ 



20 



70 



8 

Q. 
CO 

S 50 



IA) 




35 



o 25 



15 



(B) 





JL_ 



o 

l_ 

a 
c 

D 



35 




2 4 6 

Pupal age (day) 



2468 
Pupal age (day) 



Figure 3. Changes in the percentage composition of protein and fat contents in the ovary 
and pupal tissues of B. mori during the course of pupal development. 



experimental groups. However, protein content was considerably low in the egg 
obtained from 6 h/day fed group compared to the ad libitum fed group. The protein 
content in the pupal tissue was maximum during the early pupal age and it 
subsequently declined with the advancing age of pupa in all the experimental groups 



Energy allocation to reproduction in B. mori 



337 



(figures 3C and D). This observation shows that the protein from the pupal tissue is 
constantly transported to the egg in the process of vitellizing the egg with protein. 

Egg and pupal tissues of ad libitum fed series contain considerably high 
concentration of fat than those obtained from restricted feeding schedules. However, 
fat content in the egg as well as pupal tissue steadily declined during the course of 
pupal development in all the experimental groups (figures 3 A and B). 

While the energy content of egg increased with the advancing development, the 
energy content of the pupal tissue declined with the advancing pupal age. For 
instance, during the course of development, egg energy increased from about 
5680 cal/g to about 6025 cal/g in the ad libitum fed group. The energy content of 
pupal tissue in the same feeding schedule declined from about 5950 cal/g to about 
5500 cal/g (figures 4 A and B). Almost a similar trend is obtained in the changes of 
the energy content of egg and pupal tissue^ of other feeding schedules. 

4. Discussion 

The following adverse effects were obtained by restricting the feeding durations 
on B. mori: (i) Increase in the feeding larval period; (ii) decrease in the 
quantity of food consumed and converted as well as the rates of consumption and 
conversion; (iii) increase in the efficiency of conversion; and (iv) reduction in the 
reproductive potential 

In the present study, the larvae fed for 3 h/day suffered 100% mortality. These 
larvae extended their feeding period to more than the ad libitum fed group and still 
failed to spin. It may be true that the larvae require a critical feeding period of more 
than 3 h/day to complete the feeding larval period. The larvae restricted to feed for 
shorter durations extended their feeding period in order to obtain the maximum 
energy prior to pupation. Mathavan and Muthukrishnan (1976) reported that when 
restricted to feed for 3 or 6 h/day, Danaus chrysippus required 18 or 14 days to 
complete the final instar as against 6 days required by the larvae fed for 24 h/day. 

Fed on decreased food abundance, the larvae compensate either by increasing in 
the feeding duration or by increase in the rate of feeding and/or efficiency of 
conversion. In the present study the B. mori larvae extended feeding duration at 
restricted feeding rations. The rates of feeding and conversion were low at the 



246 
Pupal age (day) 



, 

^ 6200 - 



&5800 - 




5000 - 



(B) 




246 
Pupal age (day) 



Figure 4. Energy content of egg (A) and body tissue (B) of B. mori as a function of feeding 
duration and pupal age. 



338 S Mathavan, G Santhi and B Nagaraja Sethuraman 

restricted feeding group compared to the ad libitum fed group. Similar results were 
reported by Mathavan and Muthukrishnan (1976) and Christopher (1983) for 
lepidopterous larvae. Critical analysis of rates of feeding and conversion reported in 
the present study showed that the B. mori larvae compensate the restricted feeding 
duration by increasing consumption rate over the limit expected of it in proportion 
to the feeding duration. The feeding duration in the 6 h fed group was 4 times less 
than ad libitum fed group. However, the reduction in the consumption rate and 
conversion rate only two times less in the 6 h fed group than the ad libitum fed group. 
Further, B. mori larvae enhanced the conversion efficiency at the restricted feeding 
duration than the ad libitum fed group. Similarly, Muthukrishnan and Delvi (1974) 
reported that the grasshopper, Poecilocerus pictus partially compensated the 
diminishing ration by increasing the conversion efficiency. Thus the B. mori larvae 
try to compensate the stress of restricted feeding duration by increasing rates of 
feeding, conversion and efficiency. 

Slansky and Scriber (1985) in the recent review on food consumption and utilization 
stated that considering reproduction, the physiological response of an insect for a 
decreased food abundance will be delayed or decreased reproduction. The oogenesis 
and oviposition, i.e. timing and rate of reproduction, number and quality of eggs 
depend on (i) the nutrient accumulated during the feeding larval period; (ii) the 
quantity and quality of the adult food; and (iii) the amount of nutrient allocation 
by the female for each egg (Engelmann 1970; DeWilde and DeLoof 1973b; Bell and 
Bohm 1975; Slansky 1980a, b; 1982; 1985). In insects in which the adult does not feed, 
reproduction depends on the success of the female attaining the ideal final body 
weight (Engelmann 1970; Hinton 1981). In the case of B. mori, the whole process of 
egg development is completed in the pupal period itself and on emergence the adults 
do not feed and they concentrate on depositing the eggs. Hence, the fecundity of 
B. mori depends on the amount of energy stored during larval period and on the 
attainment of 'adult weight. The amount of food allocated for egg production in 
B. mori fed for different feeding durations was calculated. Restricted food availability 
results on the reduced fecundity of B. mori. However, when related to the percentage of 
assimilated energy allocated for egg production, it is almost the same in all the feeding 
groups, i.e. they allocated about 15-19% of the assimilated energy for egg 
production. The data reported by Hiratsuka (1920) on the allocation of assimilated 
energy for egg was 12-5%. In general, lepidopterans allocate about 10-20% of total 
assimilated energy for egg production (Slansky and Scriber 1985). Amount of pupal 
energy allocated for egg production was about 55% in the 6 h/day fed group (figure 
5). It amounted to 67% in those fed for 24 h/day. Energy allocation for egg by the 
pupae obtained from 12 or 18 h/day fed group did not vary significantly from that of 
24 h fed group. In other words, the pupae from the 12/18/24 h fed group allocated 
energy for egg production almost with an equal efficiency. 

Both in the egg and the pupal tissue, percentage composition of fat steadily 
decreased with an advancing pupal age in all the feeding regimes. It is because fat 
served as a major source of energy for metabolism during pupal development (see 
also Crescitelli 1935; Klekowski et al 1967). It has been shown that during 
vitellogenesis, considerable amount of lipoproteins are deposited in the egg which 
originate from fat reserves of pupal tissue (Chino et al 1977; Lubzens et al 1981). 
However, the protein and energy content of egg constantly increased with advancing 
age and feeding regimes. More than 80% of the body protein was deposited into the 



Energy allocation to reproduction in B. mori 



339 



68 - 



^ 62 - 



56 - 



6 12 18 

Feeding duration f h) 



24 



Figure 5. Percentage of energy allocation for egg production in B. mori reared on different 
feeding durations. 



egg during vitellogenesis in those fed ad libitum. The incorporation of body protein 
into the egg was only about 40% in those fed for 6 h/day. It is evident from the 
results that individuals in the restricted feeding group accumulated less protein in the 
egg than the maximum fed group since the protein available for incorporation is less 
in the former than the later feeding group. 

Acknowledgement 

We thank the University Grants Commission, New Delhi for extending financial 
support. 



References 

Bell W J and Bohm M K 1975 Oosorption in insects; Biol. Rev. 50 373-396 

Chino H, Downer R G H and Takhahashi K 1977 The roll of diacylglycerol carrying lipoprotein I in lipid 

transport during insect vitellogenesis; Biochim. Biophys. Acta 487 508-516 
Christopher M S M 1983 Bioenergetics and population dynamics studies of a chosen lepidopteran: Catopsilia 

crocale cramer, Ph.D. Thesis, Madurai Kamaraj University, Madurai 
Crescitelli F 1935 Respiratory metabolism in the pupa of Galleria sp. (Lepidoptera); J. Cell. Comp. Physiol 

6 351-368 
De Wilde J and DeLoof A 1973a Reproduction; in The Physiology oflnsecta (ed) M Rockstein (New York: 

Academic Press) vol. 1, pp 11-95 
De Wilde J and DeLoof A 1973b Reproduction-endocrine control; in The Physiology of Insects (ed.) 

M Rockstein (New York: Academic Press) vol. 1, pp 97-157 

Engelmann F 1970 The physiology of insect reproduction (New York: Pergamon Press) 
Hinton H E 1981 Biology of Insect eggs (Oxford: Pergamon Press) vol. 1, pp 11-49 
Hiratsuka E 1920 Researches on the nutrition of the silkworm; Bull. Ser. Exp. Stn. Jpn. I 257-315 
Klekowski R Z, Prus T and Zyromska Radzka H 1967 Elements of energy budget of Tribolium castaneum 

(Hbst) in its developmental cycle; in Secondary productivity of terrestrial ecosystems (ed) K Petrusewicz 

(Warsaw-Cracow: Polish Academy of Sciences) vol. 2, pp 859-879 
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with Folin phenol 

reagent; J. Biol. Chem. 193 265-275 



340 S Mathavan, G Santhi and B Nagaraja Sethuraman 

Lubzens E, Tietz A, Pines M and Applebaum S W 1981 Lipid accumulation in oocyte of Locusta 

migratoria migratorioides; Insect Biochem. 11 323-339 
Mathavan S and Pandian T J 1974 Use of faecal weight as an indicator of food consumption in some 

lepidopterans; Oecologia (Berl.) 15 177-185 
Mathavan S and Muthukrishnan J 1976 Effects of ration levels and restriction of feeding durations on 

food utilization in Danaus chrysippus (Lepidoptera: Danaidae); Entomol Exp. Appl. 19 155-162 
Muthukrishnan J and Delvi M R 1974 Effect of ration levels on food utilization in the grasshopper 

Poecilocerus pictus; Oecologia (Berl.) 16 227-236 
Muthukrishnan J, Mathavan S and Navarathina Jothi V 1978 Effects of the restriction of feeding duration 

on food utilization, emergence and silk production in Bombyx mori L (Lepidoptera: Bombycidae); 

Monit. Zooi Itai 12 87-94 
Muthukrishnan J 1980 Physiological studies on chosen arthropods, Ph.D thesis, Madurai Kamaraj 

University, Madurai 
Petrusewicz M and Macfadyen K 1970 Productivity of terrestrial animals; IBP hand book No. 13 (Oxford: 

Blackwell Scientific Publications) pp 190 
Raymond J E G, Austin J and Linford E 1964 Biochemical studies on marine zooplankton. The 

biochemical compositions of Nyomysis integer, J. Du. Cons. Int. Exp. Mer. 28 354-363 
Slansky F Jr 1980a Effect of food limitation on food consumption and reproductive allocation by adult 

milkweed bugs, Oncopeltus fasciatus; J. Insect Physiol. 26 79-84 
Slansky F Jr 1980b Quantitative food utilization and reproductive allocation by adult milkweed bugs, 

Oncopeltus fasciatus; Physiol. Entomol. 5 73-86 

Slansky F Jr 1982 Insect nutrition: an adaptationists perspective; Fla. Entomol. 65 45-71 
Slansky F Jr and Scriber J M 1985 Food consumption and utilization; in Comprehensive insect physiology, 

biochemistry .anil pharmacology (eds) G A Kerkut and L I Gilbert (Oxford: Pcrgamon Press) vol. 4, 

pp 87-163 
White T C R 1978 The importance of relative shortage of food in animal ecology; Oecologia (Berl.) 33 

71-86 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 3, May 1987, pp. 341-347. 
Printed in India. 



Influence of azadirachtin on insect nutrition and reproduction 

G K KARNAVAR 

Department of Zoology, University of Kerala, Kariavattom 695 581, Trivandrum, India 

Abstract. Neem extracts and pure azadirachtin influence the behaviour and physiology of 
large number of insects. These compounds affect the sensory receptors, thereby inhibit food 
intake. This anti-feedant property is exploited by using neem extracts and azadirachtin for 
plant protection. The gut motility and movement of food in the digestive tube are regulated 
by azadirachtin. Holotrichia serrata, the beetle of the 'white grub' feeds on neem leaves and 
no ill-effect of azadirachtin is reported. 

Azadirachtin and neem extracts regulate the reproductive functions of insects through 
their endocrine system. Morphogenesis, ovarian development, fecundity, egg viability and 
moulting are adversely affected by azadirachtin. Radioimmunoassays' show that the 
ecdysteroid level falls drastically on azadirachtin treatment. Juvenile hormone synthesis also 
is inhibited in Locusta on administration of azadirachtin. In Oncopeltusfasciatus, high doses 
of azadirachtin inhibit adult development resulting in permanent larvae which show oocyte 
differentiation with yolk incorporation equivalent to day 4 of adult development. 

Keywords. Azadirachtin; neem; nutrition; reproduction. 



1. Introduction 

Azadirachtin, a triterpenoid isolated from the Indian neem tree, Azadirachta indica 
A Juss, like many other plant products has varied effects on the behaviour and 
physiology of insects. Crude neem extracts and pure compounds like azadirachtin, 
salanin, meliantroil and toosendanin derived from the seed kernel were used to study 
their insecticidal properties. The biocidal action of neem products was known for 
centuries in India and leaves and seeds were used by the farmers to protect stored 
products from insect attack. More recently, neem extracts and azadirachtin are 
extensively used to study their insect repellant, anti-feedant and growth regulator 
properties. 

Azadirachtin was isolated from neem seed and its chemical composition elucidated 
by Butterworth and Morgan (1971) and Zanno et al (1975). Large number of 
investigators have studied the effects of whole and partially purified extracts, 
azadirachtin and other pure fractions on various insect species (Ruscoe 1972; 
Rembold et al 1982; Steffens and Schmutterer 1982; Garcia et al 1984; Dorn et al 
1986; Kalyanasundaram and Babu 1982; Ladd et al 1984; Prabhaker et al 1986; 
Meisner et al 1985; Zebitz 1984; Mordue et al 1985; Gujar and Mehrotra 1983; 
Hellpap and Zebitz 1986; Schmutterer 1985). 

Extensive research during the past decade has brought to light, the insect 
repellant, anti-feedant, growth disruptive and reproductive inhibitory properties of 
neem seed extract and pure azadirachtin. The cumulative effects of the neem extract 
on insects have led to the emergence of a new potential insecticide. Over 150 species 
of insects show feeding deterrence and 30 species ovipositional inhibition and over 
70, growth and reproductive inhibition, when they or the host plants are treated with 
azadirachtin or alcoholic/aqueous extracts of neem. 

341 



342 G K Karnavar 

This paper deals with the influence of azadirachtin and neem extracts of various 
grades of purification on insect nutrition and reproduction. 



1.1 Influence on nutrition 

Azadirachtin affects the total sensory activity of receptors of maxilla, thereby 
inhibiting food intake in species like Heliothis spp. and Spodoptera littoralis 
(Simmonds and Blaney 1983; cf. Mehrotra and Gujar 1986; Meisner et al 1983). 
Azadirachtin and a commercial preparation of neem seed extract (margosan-O) and 
methanolic and aqueous extracts of the leaves and seeds are effective anti-feedant on 
various insect groups (Attri 1975; Saradamma et al 1977; Adler and Uebel 1984, 
1985; Mariappan and Saxena 1983; Kareem et al 1974; Arnason et al 1985). A 
comprehensive list of insect species thus affected is given by Mehrotra and Gujar 
(1986). The cotton flea beetle, Podagrica spp. and the leaf roller, Sylepta derogata 
attacking the okra plants (Hibiscus esculentus) do not feed on the leaves when 
methanolic extracts of neem seed and leaf are sprayed at weekly intervals (Adhikary 
1984). Redfern et al (1984) also have found that the ethanolic extract from fresh neem 
seeds at 0-2 and 0-4% aqueous solutions retained the anti-feedant potency for a 
period of 21 days. However, pure azadirachtin rapidly looses 50% of its anti-feedant 
property within 7 days of exposure to sun light. By 16 days complete potency will be 
lost (Stokes and Redfern 1982). The potency could be retained for longer period by 
mixing azadirachtin with plant oils. Mixtures of seed oils of Anona squamosa and 
Azadirachta indica in different proportions show high potencies in controlling 
Nephotettix (Mariappan and Saxena 1984). 

Neem extracts, ground seed and deoiled cake are used to protect stored products 
from grain pests such as Rhizopertha dominica, Tribolium castaneum and Sitophilus 
granarius (Jilani and Helen 1983; Ambika and Mohandas 1982; Pereria and 
Wohlgemuth 1982; Malik and Mujtaba Naqvi 1984; Singh and Singh 1985). Dry 
ground neem seed mixed with cowpea and maize prevented Callosobruchus and 
Sitophilus zeamais from feeding (Ivbijaro 1983a,b). 

Neem seed kernel suspension at 0-5, 0-75 and 1% protect the tobacco crop from 
the attack of Spodoptera litura (Joshi et al 1982, 1984; Meisner et al 1983). Similarly, 
aqueous suspension of both seed and leaf extracts prevent the diamond back moth, 
Plutella xylostella from feeding the cabbage heads (Adhikary 1985). Fagoonee and* 
Lange (1981) have reported that a concentration as low as 0-001% methanolic extract 
as highly toxic to Crocidolomia binotalis. Neem oil and deoiled cakes are effectively 
used against the brown hopper, Nilaparvata lugens and the food intake by the 
nymphs was very much reduced (Saxena et al 1984; Krishnaiah and Kalode 1985; 
Chiu Shin foon et al 1983). 

Azadirachtin A and B and 7-acetyl azadirachtin, when fed to Rhodnius pwlixus 
through blood at a concentration of 25-30 ug/ml acted as anti-feedant. This effect 
could be reversed by orally administering ATP, a phagostimulant (Garcia et al 1984; 
Garcia and Rembold 1984). In the cucumber beetle, Acalymma vittatum and 
Diabrotica undecimpunctata, both azadirachtin and salanin act as anti-feedants (Reed 
et al 1982). Similar feeding inhibition is found in Phyllotreta striolata, Dysdercus 
koenigii, Spodoptera litura, Dissosteira Carolina, Ostrina nubilalis and 6 species of 



Influence of azadirachtin on insect nutrition and reproduction 343 

cockroaches (Meisner and Mitchell 1982; Arnason et al 1985; Adler and Uebel 1984, 
1985; Koul 1984, 1985). 

Azadirachtin has significant influence on the passage of food through the gut by 
affecting the gut motility in Locusta migratoria migrator ioides (Mordue et al 1985). 

It is evident from the earlier studies that neem products and pure azadirachtin 
elicit anti-feedant activity. However, in Holotrichia serrata, the adult beetles feed on 
neem leaves. These beetles emerge after a prolonged pre-emergent adult stage. An 
active feeding is followed by the maturation of oocytes in this insect (G K Karnavar, 
unpublished). No information is available on the role of azadirachtin present in the 
diet on the physiology of this insect. 

2. Influence on reproduction 

2.1 Through endocrine system 

Neem extracts and azadirachtin have physiological action on the endocrine system 
of insects affecting the moult cycle, ovarian development, fecundity, oviposition and 
egg viability (Schluter et al 1985; Redfern et al 1981; Ascher and Gsell 1981; 
Prabhaker et al 1986; Mordue et al 1985; Dorn et al 1986). A single injection of 10 jug 
azadirachtin resulted in sterilizing effect on Locusta migratoria migratorioides. The 
maturation of the terminal oocytes was arrested and there was no sign of oviposition 
in such insects. Radioimmunoassay of the hemolymph showed a low level of 
ecdysteroid (Rembold and Sieber 1981; Sieber and Rembold 1983). In the fifth instar 
nymphs of L. migratoria, Mordue et al (1986) reported that ecdysteroid level 
drastically dropped on injection of azadirachtin, consequently the air swallowing 
capacity was very much impaired and moulting inhibited. 

The hemolymph ecdysone content of the larvae of Ostriniafurnacalis is very low 
and brain, corpora cardiaca and corpora allata show histppathological changes when 
the insects are treated with 20ppm azadirachtin (Shin foon Chiu et al 1985). Anti- 
ecdysteroid activity of neem extract is also reported in the sweet potato white fly, 
Bemisia tabaci and Oncopeltus fasciatus (Coudriet et al 1985; Redfern et al 1982). In 
these two, treated larvae failed to pupate and mortality was very high. 

Rembold et al (1984) have reported a complete inhibition of juvenile hormone 
synthesis following the injection of azadirachtin in Locusta. Similar effect is found in 
Manduca larvae also (Schluter et al 1985). 

Based on the effects of azadirachtin on moulting, the doses are classified as low, 
medium and high by Dorn et al (1986). Low doses (0-00098 to 0-03123 /xg/larva) of 
azadirachtin did not prevent adult formation but ecdysis was retarded in Oncopeltus 
fasciatus. While medium doses (0-0625 to 0-25 jug/larva) suppressed ecdysis 
completely and the larvae remained permanent. At high doses, both ecdysis and 
apolysis are blocked. Garcia and Rembold (1984) have shown that the ecdysis 
inhibition in Rhodnius prolixus was linearly related to the log of azadirachtin dose. 
Ecdysis inhibition by azadirachtin is due to an interference with the effects which are 
under ecdysteroid control. Azadirachtin may induce ecdysis inhibition by blocking 
ecdysone production by the prothoracic gland (Sieber and Rembold 1983). In Musca 
autumnalis, azadirachtin inhibits both adult development and reproduction (Ibrahim 
and Hayes 1984). 



344 G K Kamavar 



2.2 Growth disruption 

Crude methanolic extract of neem and pure azadirachtin cause morphogenetic effects 
in Disdercus (Koul 1984). Mythemna seperata fifth or sixth instar larvae fed on leaves 
treated with partially purified neem extract showed high degree of mortality and the 
survived larvae developed into pre-pupae (Schmutterer et al 1983). Azadirachtin 
when fed with artificial food at the rate of 20 ppm to the third and fourth instar 
larvae of Ostriniafurnacalis, the larval period was prolonged. This inhibition is due 
to low /?-ecdysone level (Shin foon Chiu et al 1985). 

The growth disrupting property of azadirachtin on Epilachna varivestis show that 
a dose less than 0-3 jj.g could inhibit metamorphosis (Schluter 1985) and 5-10 ppm 
for 48 h sufficient for growth inhibition in Spodoptera litura (Koul 1985). Methanolic 
extract of neem kernel with tropital increases the growth disrupting effect in the 
same insect (Lange and Schmutterer 1982). 100% mortality in Leptinotarsa 
decemlineata, Pieris brassicae and Memestra brassicae was found at a level of 
100 mg/kg (Feuerhake and Schmutterer 1982). 



2.3 Fecundity and egg viability 

Egg hatchability is affected in Musca autumnalis when treated with azadirachtin 
(Ibrahim and Hayes 1984). In the case of the stored product insects also, the 
productivity is effectively reduced by neem seed extract (Pereira and Wohlgemuth 
1982; Pereira 1983; Ivbijaro 1983a). The fecundity rate of the carmine spider mite, 
Tetranychus cinnabarinus is affected by the neem seed extract (Mansour and Ascher 
1983). Growth retardation and impairment of fecundity are reported in the green rice 
hopper, Nephotettix virescens (Von Hyde et al 1985). Schistocerca gregaria treated 
with azadirachtin (2 jug/g) showed a complete inhibition of the development of the 
reproductive system. Moreover, hemolymph proteins also decreased qualitatively 
and quantitatively (Subrarnonyam and Rao 1986). 

The repellant effect of neem- extract and azadirachtin results in the feeding 
deterrence in some insects and anti-oviposition effect in others. In Liriomyza sativae, 
aqueous solutions of neem seed extract applied to leaves prevented oviposition 
(Webb et al 1983). Larval mortality after hatching was 100% in such treated plants. 
Similar reduction in egg number and viability are reported in Corcyra cephalonica 
after exposure to neem oil (Pathak and Krishna 1985). 



2.4 Oocyte development in permanent larvae 

A significant observation of the development of oocytes in the permanent larvae of 
Oncopeltus fasciatus induced by the administration of high doses of azadirachtin is 
reported by Dorn et al (1986). At a dose of 0-5 /xg/larva, 45% of the female larvae 
show globular yolk incorporation in the oocyte, which normally starts at day 4 in the 
adult females. No evidence regarding the action of azadirachtin on the endocrine 
system responsible for oocyte maturation is available in the above studies except the 
comparison of the time span between ecdysteroid peak and onset of egg maturation 
in normal and treated individuals. 



Influence of azadirachtin on insect nutrition and reproduction 345 

Acknowledgement 

I thank Ms S Chellayan for the help rendered in the preparation of this review. 

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Influence of azadirachtin on insect nutrition and reproduction 347 

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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 4, July 1987, pp. 349-360. 
Printed in India. 



Host dependency among haematophagous insects: A case study on flea- 
host association 

R S PRASAD 

Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695 581, India 

MS received 3 March 1987; revised 11 May 1987 

Abstract. Obligatory haematophagy is the end result of long standing interspecific 
associations. Present day specificities to host, blood meal and physiological stage of the host 
are all offshoots of the primitive interspecific associations. The cause/effect relationship of 
these dependencies and specificities are probably based on the route through which 
haematophagy evolved in different groups of insects. 

In the present analysis, flea-host association is taken into consideration. It is possible to 
find an array of host relationships ranging from promiscuous and catholic host associations 
to strict ones. In general 3 categories may be recognized. In fleas like Xenopsylla cheopis a 
utilizable protein in an optimum concentration gives the necessary stimuli for maturation 
(Cheopis-type), while in a second group the stimuli is provided by certain circulating 
hormones of the host (Cuniculi-type) as seen in rabbit flea Spilopsyllus cuniculi and in a third 
gr6up (Monositus-type) a priming period characterized by tissue fluid feeding and neosomy 
is necessary before whole blood diet can stimulate maturation as exemplified by Tunga spp. 

It appears that vertebrate associations of Siphonaptera initiated as adaptations to the 
nest microhabitat and haematophagy and adaptations to physical/chemical factors of 
epidermal habitat being subsequent developments. 

Keywords. Haematophagy; flea-host associations; nest microhabitat; epidermal habitat. 

1. Introduction 

Obligatory haematophagy is the end result of long standing interspecific 
associations. Such associations lead to metabolic dependency and/or dependency to 
physical and/or chemical environmental factors relating to the breeding place of the 
host and/or epidermal habitat offered by the vertebrate host. 

According to Smyth (1962) metabolic dependency with regard to endoparasites 
may be evident in one or more of the following: (i) developmental stimuli; 
(ii) nutritional material; (iii) digestive enzymes and (iv) control of maturation. Blood 
feeding triggers not only the development of ovary in the adult but also influences 
moulting in exopterygote haematophages. This influence is negotiated through the 
hormones secreted as a result of either the mechanical stimuli from proprioceptors of 
gut/abdomen or chemical stimuli from diffusible substances or digestion products 
from blood meal. In all haematophagous insects blood meal supplies nutritional 
materials necessary for reproductive and house keeping metabolic functions. 
Nematoceran and brachyceran Diptera, however, supplement their blood meal with 
phytoexudates or nectar and the carbohydrates derived from this source are mainly 
used for flight and routine metabolic functions. Fritz (1983) mentioned that as 
dextrose was present in low concentration in blood, acquisition of the necessary 
sugars for haematophagous insects like streblids which feed only on blood, was by 
frequent blood feeding. This requires experimental verification. Clements (1963), 
Engelmann (1970), Nelson et al (1975), Prasad (1987) and others deal with nutrition 

349 



350 R S Prasad 

and reproduction of haematophagous insects. Ectoparasites are not dependent on 
the host for supply of digestive enzymes, because they are adequately equipped with 
the necessary enzymes to digest the blood meal. According to Waage (1979) 
evolution of many dipteran/vertebrate associations have come about through the 
advantage gained by those species which are able to digest their host's blood cell 
proteins and use it for longevity and reproduction. Depending on the route through 
which haematophagy evolved, the group may already possess the necessary protease 
(pre-adapted) or may evolve the digestive capacity more gradually (Waage 1979). But 
it is important to remember that some of the dietary proteins control the rate of 
midgut protease synthesis and the quantity secreted. Maturation is a host dependent 
feature of haematophagous exo- and endopterygotes. Except in a few cases of 
autogenous egg production, stimulation of ovaries from resting stage at emergence 
depends on blood intake by the insect. Similarly though not spermatogenesis (in 
certain ticks even spermatogenesis is under the control of blood feeding) mating 
competency in male is induced only after blood feed. 

These points clearly indicate that haematophagous arthropods are metabolically 
dependent on the host for several functions. Waage (1979) suggested that the nature 
and intimacy of an ectoparasite/host relationship will depend on the sequence in 
which regular physical associations (habitat preference, host seeking behaviour and 
morphological adaptations promoting capacity to feed on the host, physiological 
capacity to digest host substances and behaviour associated with initiation and 
termination of feeding) occur. 

Preferences and adaptations to microenvironment of the epidermal habitat is an 
important component of host specificity. The type of integument of the host (feather 
vs hair), the temperature conditions of the epidermal habitat, thickness and texture of 
skin, the extensiveness of the peripheral circulation etc can be very decisive factors in 
host preferences. Among many insect species non-nutritional chemicals play primary 
role in determining host selection and suitability (Slansky and Scriber 1985). 
Subsequent to Hosoi's (1958) discovery of the importance of adenine nucleotides as 
phagostimulants for the mosquitoes Culex pipiens several studies have shown that 
many unrelated groups of haematophagous insects show adaptation to same 
phagostimulatory chemicals "(convergent evolutionary trend) as well as marked 
diversity in mechanism of blood recognition (Galun 1986). Galun and Rice (1971) 
found that the source of adenine nucleotide at least for Aedes aegypti and Glossina 
austeni is blood platelets. They suggested that quantitative variations of platelet ATP 
of different mammals could contribute to differences in host preferences of 
haematophagous insects. The diversity which Galun (1986) speaks about relates to 
those which recognize blood through the properties of plasma, yet others through 
some cellular fractions and a third group somewhat intermediate between these. 

Having thus outlined some generalities regarding host-haematophagous arthropod 
associations, a more specific case of flea-host association and specificities may be 
examined. 



2< ^lea-host association 

The maximum flea representatives parasitize rodents and insectivores (table 1). 
Undoubtedly the lifestyle of the host (foraging, breeding and nest/burrow 



Host dependency among haematophagous insects 



Table I. Distribution of flea families on mammals and birds. 



351 



Host record 


Flea families 


Host record Flea families 


Monotremata 


Pulicidae, Stephanocircidae 


Rodentia Ceratophyllidae, 


Marsupialia 


Hystrichopsyllidae, 


Coptopsyllidae, 




Pygiopsyllidae 


Hypsophthalmidae, 


Insectivora 


Ceratophyllidae, 


Hystrichopsyllidae, 




Chimaeropsyllidae, 


Leptopsyllidae, 




Hystrichopsyllidae, 


Macropsyllidae, 




Leptopsyllidae, 


Pulicidae, 




Pulicidae 


Pygiopsyllidae, 


Chiroptera 


Ischnopsyllidae 


Rhopalopsyllidae, 


Edentata 


Malacopsyllidae, 


Stephanocircidae, 




Tungidae 


Tungidae 


Lagomorpha 


Hystrichopsyllidae, 


Carnivora Ancistropsyllidae, 




Leptopsyllidae, 


Ceratophyllidae, 




Pulicidae 


Malacopsyllidae, 






Pulicidae 






Vermipsyllidae 






Ungulata 






Perissodactyla Vermipsyllidae 






Artiodactyla Ancistropsyllidae, 






Pulicidae, 






VermipsyHidae 






Aves Pulicidae, 






Rhopalopsyllidae, 






Tungidae 



construction behaviours) influences considerably this differential rate of 
parasitization. Both these groups of hosts live in some sort of nests made in crevices 
or burrows. Even among bird fleas, most of these are found associated with species of 
birds which breed on ground and in banks or use mud freely in the construction of 
their nests (Rothschild and Clay 1952). Those which live on hosts without regular 
nesting sites have hypertrophied anchoring devices and would be highly 
promiscuous (Hopkins 1957; Traub 1980). Speaking of host association of fleas 
Holland (1964) stated that some species of fleas will breed on only a single species or 
genus of animal (as all Amphalius, Geusibia and Ctenophyllus spp. on Ochotona spp.) 
others may live on a more-or-less related group of hosts (as Chaetopsylla setosa 
Rothschild on many large carnivora); others again apparently breed indiscriminately 
on hosts sharing a common habitat (as Ctenophthalmus pseudagyrtes Baker) on 
shrews, moles and microtine and cricetine rodents or Dasypsyllus gallinulae 
perpinnatus Baker on many species of Passeriformes and other birds. Based on the 
degree of closeness of fleas to their hosts, they may be broadly classified into nest 
ectoparasites (nest-burrow blood suckers) and host ectoparasites. While the former 
are found most of the time off the host (certain species of Coptopsylla Snd 
Ceratophyllus) the latter either remain attached to the body of the host for the whole 
life time (slow feeding intradermal parasites) such as Tunga and Echidnophaga or 
spend more time on the host in a free state moving about among the fur like 
Ceratophyllus, Leptopsyllus, Xenopsylla and others which are also fast (rapid) feeders 
taking several blood meals (Balashov 1984; Nelson et a\ 1977). The distribution and 



352 R S Prasad 

host specificities of the nest fleas are controlled by the microenvironmental 
conditions prevailing in the nest of the host animal. To a certain extent this would be 
true of the other groups also. Hopkins (1957) qualified fleas as nest specific rather 
than host specific, because the larvae are free-living and are considerably influenced 
by the nesting conditions of the host. There are several instances to show that nesting 
conditions of hosts influence the distribution and host preference/specificity of fleas, 
Sharif's (1948) studies in the Deccan Plateau showed that rat fleas Xenopsylla 
cheopis and X. brasiliensis were predominantly associated with domestic rats, 
whereas X. astia with field rats like Bandicota and Tatera. He suggested larval 
nutrition to be the most important factor in this pattern of distribution. According to 
him the larvae of X. astia were dependent on the presence of vitamin B in the diet 
whereas the other two species could grow even in its absence. In nature the profuse 
growth of fungi in the burrows of Bandicota and Tatera provide the necessary B- 
vitamins whereas; the comparatively dry nests of domestic rats do not encourage 
fungal growth and so are not suitable for X. astia. While Sharif's nutritional 
hypothesis helps to give possible explanation for the preponderance of X. astia on 
semi domestic and wild rodents like B. malabarica ( = B. indica) and T. indica in the 
places of his study, it does not explain the comparative absence or rarity of X. cheopis 
from the nests of these hosts. In spite of the actual interchange of fleas between semi 
domestic and domestic rats (Annual Reports of the Haffkine Institute; Prasad 1966) 
burrow faunistic studies of field rodents show the absence or rarity of X. cheopis 
( Joshee 1961; Kamath 1961). It would be unwise to say that X. cheopis cannot breed 
in a nest which contains nutritionally rich litter. It could be that the excess of ground 
moisture is injurious to X. cheopis larvae. X. cheopis is known to have a prediliction 
for comparatively dry situations (Pollitzer 1954), Another interesting instance is the 
distribution of X. nubica which is specific in northern Uganda to Gerbillinae of two 
genera. The preferred species of Gerbil is common in southern Uganda but here the 
flea is absent presumably because the gerbil burrows here are too damp. Burrow 
conditions similarly .appear to control the distribution of X. cheopis which in East 
Africa is restricted to Rattus rattus in the southern areas, but occurs freely on a wide 
variety of field rats in the northern areas possibly because the burrows of field rats in 
northern area offer a similar drier condition as those of R. rattus in the southern 
areas. Moisture conditions of the nesting sites seem to affect the distribution of bird 
fleas also. The hen flea Ceratophyllus gallinae is found more frequently than any 
other fleas in the nest of sparrows, starlings, sparrow-hawks, swallows etc where 
comparatively dry atmosphere prevail. The moorhen flea Dasypsyllus gallinulae 
prefer nests with damp conditions such as those of robin, warblers, finches, 
blackbirds etc. The duckflea C. garei on the other hand can survive in wet swampy 
situations which prove fatal to the other two species of fleas and this is the only flea 
found in the nest of ducks, geese and certain waders and sea birds. That the 
distribution and host preferences are not guided by the nesting conditions alone (at 
least in some cases) is interestingly illustrated by the distribution of these 3 bird fleas 
in the nests of pheasants and partridges. Nesting habits of these birds are similar and 
they often use each others nest. Yet C. gallinae, D. gallinulae and C. garei are found in 
the nest of pheasant, but C. garei is the only flea in partridge nest. 

There is very little information on the role of host odour influencing host 
association of fleas. The existing information are varied. The studies of Benton et al 
(1959) indicate preference of Sylvilagus floridanus, the natural host, by the flea 



Host dependency among haematophagous insects 353 

Cediopsylla simplex over an opossum Didelphis marsupialis in twin host choice 
experiments. Bates (1962) negated the possibility of olfaction as an important factor 
in host finding by bird fleas. Shulov and Noar (1964) found the rat fleas X. cheopis 
making a clear distinction between hosts, preferring white rat to 3 other species of 
murids Meriones tristrami, Acomys dimidiatus and Microtus guentheri. Humphries 
(1968) found olfaction playing no role in host location by the hen flea Ceratophyllus 
gallinae while Vaughan and Mead Briggs (1970) found that rabbit flea Spilopsyllus 
cuniculi was strongly attracted to the urine of the host, but showed no host 
specificity. Benton et al (1959) found Ctenophthalmus pseudogyrtes unable to 
recognise its natural host the chipmunk Tamias striatus from distances exceeding two 
inches. A static air olfactometer showed that the rat fleas X. cheopis and X. astia are 
neither able to distinguish the presence of a host nor are they able to discriminate 
between jR. rattus, R. norvegicus and Mus musculus even at short distances of 6 cm 
(K Jaya and R S Prasad, unpublished results). In these cases the host choice take 
effect after reaching it as shown by Sgonina (1939, cited by Humphries 1968) for 
certain mammal fleas. This is strengthened by the fact that there is an interruption of 
regular feeding and breeding if the antennae of the female fleas are blocked before 
releasing them on live host. Though a small percentage of female X. astia whose 
antennae were painted with nail polish and the maxillary palps ablated before 
releasing on the host, were blood fed, none laid eggs (R S Prasad, unpublished 
results). Whether some host-borne chemical signal is involved or not is not known. It 
is significant to remember that flea transfer between unrelated hosts such as prey to 
predator, dog/cat flea to human beings etc (Hunter et al 1979) are probably 
indications of want of ability to distinguish interspecific differences in host-borne 
cues. However, an air-borne kairomone emanating from the new born young rabbit 
which is also present in the urine is involved in boosting copulation and egg 
production in S. cuniculi (Rothschild and Ford 1969, 1973). 

At least in the <;ase of rat fleas it has been shown that defensive behaviour of the 
host influences not only the establishment of flea ectoparasites on the host but also 
their breeding potentials (Lila Chandy and Prasad 1987). For example mortality of 
adult fleas was high and fecundity rate low on white mouse, M. musculus albino, 
whose grooming activities are more intense than white rat R. norvegicus. Variations 
in grooming activities associated with age and sex of the host appeared to be impor- 
tant in intraspecific differences noticed in mortality (higher on adult and female rats 
compared to young and male rats respectively) and fecundity rates (higher on young 
and male rats compared to adult and female rats respectively). 

Since all the species of 'adult fleas have only blood as their food, these have to derive 
the nutrients necessary for meeting day to day metabolism as well as reproductive fun- 
ctions from blood itself. Frequent feeding is a characteristic of fleas except those such 
as Tunga spp. which are slow and continuous feeders. The question as to how far do 
variations in the blood components affect host suitability and specificity is difficult to 
answer as there is very little information on the nutritional compatibility of the 
blood from different hosts. From early 1900 there are indications of interspecific 
variations in host suitability. Bacot (1914) noticed that only when fed twice would X. 
cheopis lay eggs on human beings, even then the fecundity was very poor. Strickland 
(1914) found Ceratophyllus ( = N osopsyllus) fasciatus fed on mouse, rabbit or man did 
not lay eggs, but if the same lot was allowed to feed on rat, egg laying resulted shortly 



354 R S Prasad 

after feeding. Haas (1965) studied the comparative suitability of 4 murine rodents to 
fleas X. cheopis and X. vexabilis and showed that wild M. musculus figured as the 
most unsuitable host for both the species of Xenopsylla whereas R. exulans appeared 
to be the best host for X. vexabilis and jR. norvegicus for X. cheopis. Prasad (1969) 
based on comparative fecundity studies of X. cheopis concluded that even though 
flea mortality was high on R. rattus and M. musculus (albino) and was low on 
Bandicota bengalensis, the former two hosts proved most suitable for X. cheopis. 
Samarina et al (1968) showed that fertility value of Ceratophyllus consimilis was 
highest when fed on hamster than on rat or mouse. This was found to be true for C. 
( = N.) fasciatus and X. cheopis. Bibikova (1965) found that feeding on unspecific 
host usually takes much longer and blood of usual host digests faster. The results of 
direct feeding of fleas need not necessarily reflect nutritional influences because there 
would be several non-nutritional parameters interfering feeding and quantity of 
blood ingested. The pupal production of human strain of Pulex irritans fed directly 
on man or artificially on citrated human blood was much higher than when fed on 
dog/citrated dog blood. Fox strain of P. irritans on the contrary did not reproduce 
when fed either directly on man or artificially on citrated human blood. The pupal 
production of the same strain was high when fed directly on dog, but did not 
reproduce when fed artificially on citrated blood of dog (Hudson and Prince 1958), 
Experiments involving artificial feeding of rat fleas X. cheopis and X. astia on hepa- 
rinised blood of frog, chick, white rat, house rat, white mouse and man through 
the skin of a young white rat (the usual host for laboratory rearing of these fleas) 
showed that all the blood samples could induce yolk deposition, but a significantly 
higher percentage of X. astia fed on R. rattus blood showed yolk deposition while a 
significantly lower percentage of X. cheopis showed yolk deposition with chick 
blood. Similar experiments to assess egg laying potential when fed on heparinised 
blood of frog, hen, white rat, house rat, gerbil and man showed no significant 
difference in egg production attributable to the interspecific variations in blood 
sources, whereas with regard to X. cheopis no definite conclusions could be drawn 
due to poor gorging and erratic egg laying. However, egg laying started on the 9th 
day on blood of house rat whereas on blood from other sources egg laying started 
only around 13-16 days after the first blood meal. No such marked difference could 
be noticed with X. astia where egg laying started at the earliest by the 4th day and 
latest by the 7th day. These blood samples were offered through the skin of a young 
white rat, a host with which these fleas are familiar in the laboratory as white rat is 
used as host for laboratory rearing of these fleas (Kamala Bai and Prasad 1976, 
1981). Feeding these fleas artificially on blood fractions of white rat showed that 
blood cell fraction triggered yolk deposition but plasma alone did not. The factor 
associated with this nutritional inadequacy of blood plasma was shown to be the 
protein concentration because when plasma was fortefied with rabbit albumin, yolk 
deposition was initiated (Kamala Bai and Prasad 1976). Subsequent studies on 
artificial diet showed that both quality (fleas were not able to utilize bovine albumin 
in place of bovine haemoglobin for yolk synthesis) and quantity (twice fed fleas laid 
more eggs than those fed once artificially) of protein are important and that a diet 
balanced with salts, sugars, cholesterol and B-vitamins in addition to protein and a 
phagostimulant (ATP) was necessary for vitellogenesis. However, B-vitamins appear 
to be dispensible for X. astia (Kamala Bai and Prasad 1979, 1981). It then follows 
that the ammo acid composition of blood proteins is an important attribute in 



Host dependency among haematophagous insects 355 

conferring nutritional adequacy of the blood meal. However, attempts to feed these 
fleas on artificial diets in which protein is replaced with amino acids failed. 

Synchronization of breeding activities of the host and its flea ectoparasite has been 
demonstrated in the case of rabbit fleas S. cuniculi. Before the breeding season 
commenced female rabbits carried an extra load of fleas (Allan 1956). Following the 
observation of Allan (1956), Rothschild and Ford (1964a) discovered that the rabbit 
flda's ovaries can mature only on a doe rabbit during the last 10 days of its 
pregnancy or on a new born baby rabbit during the first 5 or 6 days of life. Theii 
further work revealed that maturation was probably initiated and maintained only 
when corticosteroids circulating in the blood reached a relatively high level. Ovarian 
maturation induced as a result of direct spraying of hydrocortisone or corticosterone 
on the flea revealed a direct action of these hormones on the flea (Rothschild and 
Ford 1964b). Lutinizing hormone and progestins were found to induce ovarian 
regression. Growth hormone secreted by anterior pituitary constituted one of the 
major copulation factors (Rothschild and Ford 1966) which accounted for the fact 
that copulation of rabbit fleas took place only on nestling rabbits. Maturation oi 
male rabbit flea S. cuniculi (defined as the stage of development at which sperm can 
be successfully transferred to the female spermatheca) is also under the influence oi 
host's hormones (Rothschild et al 1970). Rothschild and Ford (1969, 1973) showed 
that in addition to the hormones described above there are certain pheromone-like 
nestling factor (kaifomone) from the new-born young rabbits which speed up 
maturation and copulation and enhance sperm transfer. The rabbit flea S. cuniculi is 
not an isolated case of hormonal dependency. Rothschild and Ford (1972) showed 
that the breeding of the rabbit flea Cediopsylla simplex is similarly synchronized with 
the breeding cycle of the host. According to Rothschild (1965) Ischnopsyllus, the bat 
flea, could be another case where an ectoparasite respond to the sexual cycle of the 
host. These fleas crowd on female bats before they leave their winter quarters foi 
their summer breeding roosts. 

One of the most fascinating flea-host association is that of the so-called sticktight 
and chigoe fleas. The former is exemplified by the notorious pest of poultr) 
Echidnophaga gallinacea while the latter by Tunga spp. In both the cases the females 
are permanent parasites on the body of their respective hosts. Continuous feeding 
mating and egg laying are accomplished while firmly anchored on the body of the 
host. However, there are some major differences in feeding of these fleas 
Echidnophaga feeds on whole blood soon after attachment whereas the work oJ 
Lavoipierre et al (1979a) show that T. monositus pass through 3 phases of feeding 
after attachment. In the first phase the major diet is tissue fluid exudate and 
neutrophil (exudate-neutrophil feeders) which lasts for about 10 days aftei 
attachment. Probably between the 10th and 14th day the flea passes through the 2nd 
phase in which the gut contents show mostly fibroblasts and collagenous materials 
(connective tissue feeders). The 3rd phase (14 days after attachment) is marked b) 
feeding on whole blood. As in the case of rabbit flea S. cuniculi there appears to be ar 
interesting correlation and synchronization of the reproductive activities of the fles 
and the feeding phases. Tissue feeding (phases 1 and 2) is associated with neosom} 
(change involving the hypertrophy of the abdomen especially the 2nd and 3rc 
abdominal segments) while the haematophagous phase initiates reproductiv< 
activities of the flea such as mating, ovarian development and egg laying. It is during 
the 2nd phase (connective tissue feeding phase) that the midgut epitheliurr 



356 R S Prasad 

hypertrophies with greater secretory activities, the condition resembling that seen in 
rabbit flea 5. cuniculi (Mead-Briggs 1964). This increased secretory activity is 
probably for efficient utilization of the blood meal for ovarian maturation and egg 
production. According to Lavoipierre el al (1979a) there appears to be a remarkable 
integration between host inflammatory and repair responses and feeding behaviour, 
growth and reproduction of the flea and this interaction between the parasite and the 
host certainly does not appear to be fortuitous. It is replicative and predictable and is 
never anarchic. These authors also found the dorsal surface of the pinna of the ear to 
be the specific site for attachment of T. monositus and attachment elsewhere results in 
premature death of the flea. A comparative study showed that feeding behaviour of 
T. monositus is similar whether the host offered M. musculus albino or Peromyscus 
maniculatus (the natural host of this flea). However, the intake of neutrophils on the 
latter host was more during the cellular exudate feeding phase and haematophagy 
commenced earlier when on Peromyscus compared to Mus. There is an increase in 
the rate of neosomy also on Peromyscus (Lavoipierre et al 1979b). 



3. Conclusions 

One of the most prominent features that may be noticed in flea-host association is 
that fleas mostly parasitize hosts which dwell in burrows/nests. This being the case 
the climatological and/or nutritional requirements of the free living larval stages 
become important factors in host preference of adult fleas. This prompted many 
workers to suggest that fleas are nest specific rather than host specific. Rothschild 
(1966) recognized mainly two life styles of fleas. The closer association with the nest 
microhabitat (primitive, if it is accepted that the progenitors of fleas were scavengers 
in the nest of vertebrates) and closer association with the microhabitat of the 
epidermis of the host, sometimes extending to very prolonged and intimate 
associations as seen in Echidnophaga and Tunga. 

But with regard to host association of adult fleas for which adaptation to 
epidermal habitat is also important, three categories may be recognized. The first is 
exemplified by a type like the rat fleas X. cheopis/X. astia (Cheopis-type). These are 
not nest fleas as they spend more time of their adult lives on the host compared to 
certain species of Coptopsylla-or Ceratophyllus. The former is found on the body of 
the host for hardly 3 h in its life time and during this period it ingests about 50-100% 
of its own body weight of blood (Balashov 1984). In contrast to this X. cheopis/X. 
astia remain on the body of the host for longer periods, feed very frequently, 
ingesting each time only very small quantity of blood (usually less than their own 
body weight). This limited capacity is probably genetical and there is considerable 
restraint in the extensibility of the abdomen which restricts the quantum of blood it 
is capable of ingesting. This coupled with the comparatively large sized eggs into 
which fairly large quantities of yolk material are to be packed and the low protein 
storage capacity of haemolymph (K G Narayana Pillai and R S Prasad, unpublished 
results) makes it obligatory on the part of these insects to remain in close contact 
with the host which would facilitate frequent feeding (Prasad 1986). 

The second category is the hormone bound fleas (Cuniculi-type) examples of 
which are the rabbit fleas and probably the bat fleas. There is no information on the 
mechanisms involved in the case of bat fleas. In rabbit fleas parasitism has advanced 



Host dependency among haematophagous insects 357 

so much that the fleas are able to breed only if fed on a pregnant host. As it is with all 
narrow adaptations, it would have its own advantages and disadvantages. It would 
be interesting to know if these fleas would respond to hormonal changes associated 
with breeding of vertebrates other than their natural hosts. This association grades 
into a third and more intimate relationship which may be qualified as Monositus- 
type, seen in fleas belonging to the genera Echidnophaga and Tunga. There is no 
known hormonal involvement as in the previous instance and Lavoipierre et al 
(1979a) state that survival of T. monositus is dependent on the host inflammatory and 
repair responses with which there appear to be remarkable integration of feeding 
behaviour, growth and reproduction. As was already explained neosomy is 
dependent on tissue fluid feeding and reproduction on haematophagy. What are the 
precise physiological mechanisms guiding these two phases? The situation finds some 
parallel with the condition in ticks. In these cases also species variations in the host 
do influence blood feeding, rate of blood digestion etc. Are the reproductive 
functions of the first and the third groups influenced by common factors? Are these 
dependencies simply for dietary proteins? or are there any specificities for blood 
proteins or their constituent amino acids?. Milk fed X. cheopis and X. astia did not 
develop oocytes indicating that they are not able to utilize casein for ovarian 
maturation. Among blood proteins, bovine albumin in place of bovine haemoglobin 
did not trigger maturation (Kamala Bai and Prasad 1976, 1979). Does it mean that 
these fleas have evolved certain specificities in amino acid requirements? So far there 
are no experimental results which unequivocally declare that blood from certain 
species of hosts are nutritionally inferior or certain others superior. 

There are indications of preferences to certain sites on the epidermal habitat. For 
example rat fleas X. cheopis seem to prefer the posterior half of the body of white rat 
especially the base of the tail, while X. astia prefer anterior half mainly the neck 
region. The areas preferred by X. astia are warmer than those preferred by X. 
cheopis. If there is any other factor influencing this preference is not clear (Prasad 
1972). Rabbit fleas S. cuniculi choose the rabbit's muzzle and cheeks during the early 
stages after emergence and later move on to the ears where they become well 
anchored. Nosopsyllus fasciatus preferred the hind quarters of the host while 
Leptopsylla segnis the fore quarters. T. monositus show very strict preference for the 
dorsal part of the pinna of the ear. 

From the present analysis it would become clear that nutritional status of the 
blood is only one of the many factors deciding suitability of a particular vertebrate as 
a host. Perfect adaptation to a host thus would depend on a proper coordination of 
factors influencing larval and adult environments. There are still wide lacunae in our 
understanding of host association of this group of insects. 

Are haematophagy and vertebrate host association (specificities) the effect of 
primary adaptation to the nesting conditions of the host (cause) or vice versa? If the 
assumption that progenitors of Siphonaptera [probably a boreus-like Mecopteran 
(Hinton 1958; Rothschild 1975; Schlein 1980)] were primarily associated with the lair 
or nest of vertebrates (James and Harwood 1969) it might then follow that 
development of haematophagy and the associated factors like adaptation to host- 
borne chemical emanations and epidermal habitat were subsequent developments. 
As mentioned by Waage (1979) the invasion of a nest microhabitat by predator 
ancestor and the evolution of increasingly intimate host association is a probable 
scenario for the evolution of Siphonaptera. A nest/burrow habitat would offer 3 



358 R S Prasad 

major food sources: (i) the detritous (for scavengers); (ii) the arthropod body fluid 
(prey-predator relationship) and (iii) the vertebrate blood (host-parasite relation- 
ship). Fleas have exploited the first and third sources of food offered by the nest/ 
burrow habitat thereby optimizing on the available nutrient source and increasing 
fitness in the habitat. If this exploitation was directed by the cues correlated with the 
energy content of the food (theories of optimal diet/optimal foraging) is not known. 
Except for social insects, the fitness of an insect will be a function of individual 
survivorship and fecundity (Mitchell 1981). As it is evident from the foregoing 
account, survivorship of fleas is not influenced by nutrition alone. At least 3 factors 
in a nest/burrow habitat control survivorship: moisture content affecting mostly the 
larval stages; nutrient content of the nest/burrow detritus affecting the larval stages 
and behavioural resistance of host affecting the adult fleas. Fecundity on the other 
hand is controlled by nutrition. Proteinaceous meal is a must for ovarian maturation 
and egg production. About 16 (chicken)-23% (human) of protein content, vertebrate 
blood is nutritionally superior to either arthropod haemolymph [insect haemolymph 
has about 1-10% of protein (Wyatt 1975)] or plant sap which has practically no 
proteins. Vertebrate blood diet would thus offer definite advantage in reproductive 
functions to those which switched over to this diet and a selection at this level would 
be to the advantage of the organism. Such switching over could be possible under 
situations of indiscriminate feeding by the insect concerned. According to Emlen 
(1968) predators will tend to specialize in their diet (not necessarily on usually 
superior food) when food is abundant and to feed more and more indiscriminately as 
food becomes scarce. The feed back between the food's use and its value leads 
towards increased specialization. Change over from predation on arthropods to 
parasitism on vertebrates has other advantages also. For example, a vertebrate being 
larger, a single animal can support a large population of the parasite. 

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Printed in India. 



Effect of okra fruit blocks, seeds and pericarp on post-embryonic 
development of Farias vittella (Fab.) in relation to some phytochemicals 
of selected okra genotypes 

RAM SINGH* 

Division of Entomology, Indian Agricultural Research Institute, New Delhi 110 012, India 
* Present address: Department of Entomology, Haryana Agricultural University, Hisar 
125004, India 

MS received 30 December 1986; revised 3 March 1987 

Abstract. Post-embryonic development of the spotted bollworm, Earias vittella (Fab.) 
studied on pericarp, seeds and fruit blocks of 8 okra genotypes, revealed significant diffe- 
rences in larval survival, larval period, pupation and adult emergence among genotypes as 
well as fruit components. First instar larval survival on fruit components was in the order of 
fruit blocks > seeds > pericarp > axil. Mean total developmental period was 24-20, 21-33 and 
21-09 days on pericarp, seeds and fruit blocks, respectively. Likewise pupation was 25-30, 
65-42 and 71-58% on pericarp, seeds and fruit blocks, respectively. Primary phytochemicals, 
like protein, free amino acids, total sugars, non-reducing sugars and moisture content in 
pericarps were in higher concentrations in comparison to seeds and whole fruits. These 
compounds also differed significantly among genotypes. There were positive correlations 
between thejDest survival and these nutritional compounds. Tannins were 0-35, 0-23 and 
0-29% in pericarp, seeds and whole fruits, respectively. Tannin content was significantly 
higher in pericarps of tolerant genotypes and showed significant negative correlation 
(r= -0-81) with fruit borer survival. 

Keywords. Okra; Earias; phytochemicals; pericarp; post-embryonic; genotype; antibiosis; 
nutrition. 



1. Introduction 

Okra (Abelmoschus esculentus (L.) Moench), an important vegetable grown in India, 
is attacked by over 37 insect species (Nayar et al 1976). Among these, shoot and 
fruit borer (Earias vittella (Fab.)) also popularly known as spotted bollworm of 
cotton is the most ubiquitous, causing damage to okra fruits to the extent of 90% 
(Krishnaiah et al 1976). Okra fruit components like epicarp, seeds, cut pieces and 
whole fruit have been reported to affect developmental behaviour and reproductive 
potential of this pest (Vishwapremi and Krishna 1974a,b). The poor reproductive 
potential of this fruit borer on epicarp was attributed to less number of free amino 
acids and lower concentration of water soluble proteins in epicarp when compared 
to seeds (Mani et al 1986). The present study report the influence of different okra 
fruit components on survival and development of E. vittella in relation to some 
phytochemicals of susceptible and tolerant okra genotypes. 

2. Materials and methods 

The okra genotypes, viz KS-305 and Line 14-78 which had tolerance and the geno- 
types, viz Pusa Sawani, Lam hybrid, AC-302, AC-333, Vashalivadu and Sel-2 which 

361 



362 Ram Singh 

had the susceptible reactions to the attack of this borer under field conditions (Singh 
1985) were selected to carryout the following studies. 

2.1 Insect survival and development 

Survival of first instar larvae was studied on 4 different components viz pericarp, 
seeds, axil (placenta) and fruit blocks (2 cm long) of 4-5 day old fruits in separate 
aerated plastic containers (10 x 5 cm) in 3 replications. Freshly hatched 10 larvae per 
container were released and kept in BOD incubator at 301C. The food was 
changed on alternate days and observations on surviving larvae were recorded after 
4 days of their release. In another set, similarly rearing of first instar larvae was 
carried out till pupation. Data were then recorded on larval, pupal, total (larval 4- 
pupal) developmental periods, pupation and adult emergence. Growth indices for 
larval and total developmental periods were calculated by the following formulae 
(Sharma et al 1982). 

T i ,u - A Pupation (%) 

Larval growth index = - 



Mean larval period (days) 

Total developmental Adult emergence (%) 

growth index = Mean total developmental period (days) 

2.2 Phytochemical estimations 

Okra fruits, 4-5 days old, were sampled during last week of August at peak period of 
insect incidence. Along with whole fruits, pericarp and seeds were oven dried sepa- 
rately at 60C for 48 h. These were ground in a Willey grinding mill to pass them 
through 20 mesh seive. Axil portion of the fruit was not collected for phytochemical 
estimations, because it constitutes less than 10% of the total fruit weight (Singh 1985) 
and is also of less importance in influencing insect incidence. Moisture content was 
estimated by drying the fresh fruits and their components in an oven at 70C for 48 h. 
Total sugars were extracted in distilled water by following the method of Srinivasan 
and Bhatia (1953) and estimated according to the method of Yemm and Willis (1954). 
Reducing sugars were estimated by the method of Somogyi (1952). Free amino acids 
were extracted according to Barnett and Naylor (1969) and estimated by Yemm and 
Cocking (1955). For proteins estimation, initially total nitrogen was determined by 
nicro-Kjeldahrs method (AOAC 1970) and nitrogen percentage was multiplied by 
5-25. Tannin content was estimated according to the method of Burns (1971). These 
phytochemicals were expressed as per cent of dry weight of the sample taken. The 
iata were analysed and simple correlations of phytochemicals were worked out with 
Dest survival. 

J. Results 

5.1 Insect survival and development 

fable 1 shows that mean survival of first instar larvae was maximum (67-49%) on 
>kra fruit blocks followed by seeds (59-58%), pericarp (28-33%) and axil (1042%). 



Okra fruit phytochemicals in relation to spotted bollworm 

Table 1. Survival of first instar larvae of bollworm on different fruit parts of okra 
genotypes. 



363 



Larval survival (%) 



Genotype 



Pericarp 



Axil 



Seeds 



Fruit block 



Pusa sawani 


26-67(30-94) 


13-33(21-14) 


63-33 


66-66 


Lam hybrid 


63-33(52-78) 


26-67(30-94) 


46-66 


76-66 


AC-302 


36-67(37-22) 


0-00(3-63) 


76-66 


86-66 


AC-333 


33-33(35-22) 


23-33(28-78) 


66-66 


76-66 


Sel-2 


33-33(35-22) 


6-67(13-49) 


66-66 


73-33 


Vashalivadu 


26-67(30-99) 


13-39(21-14) 


70-00 


70-00 


KS-305 


6-67(12-29) 


0-00(3-63) 


43-33 


46-66 


Line 14-78 


0-00(3-63) 


0-00(3-63) 


43-33 


43-33 


Average 


28-33 


10-42 


59-58 


67-49 


SEM 


(2-23) 


(2-46) 


3-12 


3-24 


CD at 5% 


(6-78) 


(7-47) 


9-46 


9-84 



Figures in parantheses are angular values. 

Hence axil portion was least suitable in all genotypes with no larval survival on KS- 
305, Line 14-78 and AC-302. Larval survival on pericarpic region of tolerant geno- 
types was specifically poor where it ranged from 0-0-6-67% only. Pericarpic region of 
Lam hybrid was most suitable, where larval survival was upto 63-33%. Seed portion 
of all genotypes supported 43-33-76-66% larvae to survive. However, best survival of 
the larvae was recorded on fruit blocks. 

The results of elaborate studies on the post-embryonic development of the pest are 
given in table 2. Feeding of larvae on pericarp of tolerant genotype KS-305 pro- 
longed total developmental period to the extent of 5 days in comparison to 
Vashalivadu. None of the larvae survived on pericarp of Line 14-78. The per cent 
pupation (40) and adult emergence (95) were recorded to be the highest on Lam 
hybrid. As a result, this genotype also manifested highest total growth index (4-15). 
Poorest growth index (1-87) was observed on tolerant KS-305. Total developmental 
period on seeds of different genotypes ranged from 19-15-22-86 days. Tolerant 
genotypes showed slightly longer developmental period than susceptible ones. 
Pupation ranged from 20-90-65%, adult emergence 52-50-96-0% and total growth 
index 1-78-4-93 in different genotypes. Total developmental period on fruit blocks of 
test genotypes varied between 18-25 (Lam hybrid) and 25-25 (Line 14-78) days. 
Pupation (30-35%), adult emergence (30-00%) and total growth index (1-00) were also 
lowest on tolerant genotype Line 14-78. Highest adult emergence (100%) and total 
growth index (5-48) were recorded on Lam hybrid. 



3.2 Phytochemical variations 

Data on biochemical analysis are given in table 3. The concentrations of phyto- 
chemicals in pericarps varied significantly except moisture level, where proteins 
ranged from 13-85-17-90, free amino acids 2-52-4-05, total sugars 5-00-10-75, 
reducing sugars 1-75-3-75, non-reducing sugars 3-25-7-08, moisture content 90-85- 
92*60 and tannins 0-20-0-61% in tested genotypes. In seeds, proteins varied from 
13-25-18-37, free amino acids 1-92-3-04, total sugars 4-35-10-50, reducing sugars 



.364 Ram Singh 

Table 2. Effect of feeding on okra fruit components on post-embryonic development of 
spotted bollworm in okra genotypes. 



Developmental period 


(days) 


Pupation 
(%) 


Larval 
growth 
index 


Adult 
emerg- 
ence 
(%) 


Total 
growth 
index 


Fruit 
component 


Genotype 


Larval 
XSE 


Pupal 

XSE 


Total 

XSE 




Pusa sawani 


12-42 04 


11-23 1-6 


22-79 2-1 


10-70 


0-86 


90-50 


3-80 




Lam hybrid 


11 -33 0-7 


10-67 0-8 


22-90 1-4 


40-00 


3-53 


95-00 


4-15 


Pericarp 


Vashalivadu 


11 -24 0-3 


10-63 0-4 


22-00 1-6 


30-00 


2-67 


70-80 


3-22 




AC-302 


11 -44 0-6 


12-50l-2 


24-74 1-4 


35-50 


3-10 


90-50 


3-66 




AC-333 


12-860-6 


ll-330-7 


24-38 1-4 


20-50 


1-54 


60-60 


2-49 




Sel-2 


13-79 0-2 


12-67 1-1 


25-79 2-4 


15-40 


1-12 


80-00 


3-10 




KS-305 


13-63 0-2 


12-50 0-4 


26-80 0-9 


25-00 


1-83 


50-00 


1-87 




Line 14-78 


0-00 


0-00 


0-00 


0-00 


0-00 


0-00 


0-00 


Average 




12-39 


11-65 


24-20 


25-30 


2-09 


76-77 


3-18 




Pusa sawani 


9-25 0-2 


10- 14 0-6 


19-75 1-2 


65-50 


7-00 


95-30 


4-82 




Lam hybrid 


9-45 0-3 


12-35 0-8 


22-70 1-4 


70-40 


7-45 


90-50 


3-98 


Seeds 


Vashalivadu 


8-50 0-2 


10-32 0-4 


19-15l-3 


85-50 


10-06 


80-00 


4-18 




AC-302 


8-20 0-4 


10-45 0-3 


19-45 1-4 


90-00 


10-98 


96-00 


4-93 




AC-333 


10-900-2 


11-50 0-2 


22-79 1-6 


75-35 


6-91 


70-40 


3-09 




Sel-2 


10-33 0-8 


10-41 0-4 


21-25 2-1 


90-65 


8-78 


94-00 


4-42 




KS-305 


10-55 0-7 


12-65 0-6 


22-86 1-3 


25-50 


2-42 


40-60 


1-78 




Line 14-78 


9-90 0-2 


12-70 0-8 


22-70 1-8 


20-50 


2-07 


52-50 


2-31 


Average 




9-63 


11-31 


21-33 


65-42 


6-96 


7741 


3-69 




Pusa sawani 


9-75 0-6 


12-24 0-7 


21-75 2-7 


80-25 


7-13 


80-00 


3-33 




Lam hybrid 


8-50 0-5 


9-50 0-2 


18-25 1-3 


84-65 


9-96 


100-00 


548 


Fruit 


Vashalivadu 


8-50 0-2 


9-55 0-2 


18-55 1-3 


65-40 


7-69 


96-40 


5-20 


blocks 


AC-302 


9-25 0-2 


12-20 0-9 


22-20 2-6 


90-45 


9-78 


94-00 


4-23 




AC-333 


8-20 0-2 


9-60 0-8 


18-63 1-4 


85-50 


1043 


100-00 


5-37 




Sel-2 


9-100-8 


1<M507 


20-10 1-7 


70-50 


7-75 


98-50 


4-90 




KS-305 


11-250-1 


12-25 0-8 


24-00 2-4 


65-60 


6-73 


85-50 


3-93 




Line 14-78 


12-50 0-3 


13-150-6 


25-25 3-2 


30-35 


2-43 


30-00 


1-00 


Average 




9-63 


11-08 


21-09 


71-58 


7-74 


85-55 


4-18 



1-28-5-12, moisture 87-63-9143 and tannins 0-12-039% with significant differences 
among genotypes. However, in seeds, moisture level did not differ significantly. With 
regard to biochemical analysis of whole fruits, proteins ranged from 13-35-17-25, free 
amino acids 2-00-3-40, total sugars 446-10-31, reducing sugars 1-29-4-92, moisture 
88-00-91-80 and tannin content 0-17-049% in different genotypes. 



4. Discussion 

Pericarpic region of okra fruit is very important from the angle of fruit borer attack, 
because it directly comes into contact with insect eggs, larvae and even adults. 
Female moths prefer to lay more eggs on hairy surface of pericarp (Singh 1985). 
Larvae also initially feed and penetrate through this portion. Irrespective of the 
genotypes, feeding of larvae exclusively on pericarp resulted in poor survival, longer 
larval period, less pupation and poor larval growth index in comparison to seeds and 
fruit blocks. These observations are in accordance with that of Vishwapremi and 
Krishna (1974a,b) who also reported longer larval period and poor reproductive 



Okra fruit phytochemicals in relation to spotted bollworm 



365 



Table 3. Quantitative variations in biochemicals of okra fruit components in different 
genotypes. 



Fruit 


Protein 


Free amino 
acids 


Non- 
Total Reducing reducing 
sugars sugars sugars 


Moisture 


Tannin 


component 


Genotype 


(%) 


(%) 


(%) 


(%) 


(%) 


(%) 


(%) 




Pusa sawani 


16-12 


2-71 


8-50 


2-50 


6-00 


92-60 


0-24 




Lam hybrid 


17-90 


3-12 


8-20 


2-65 


5-55 


91-19 


0-24 


Pericarp 


Vashalivadu 


16-25 


4-04 


5-00 


1-75 


3-25 


91-58 


0-26 




AC-302 


16-00 


2-64 


9-50 


2-42 


7-08 


91-01 


0-20 




AC-333 


16-50 


2-74 


8-50 


2-55 


5-95 


90-90 


0-20 




Sel-2 


14-65 


2-84 


10-75 


3-75 


7-00 


91-75 


0-42 




KS-305 


15-25 


2-61 


7-50 


248 


5-02 


90-85 


0-60 




Line 14-78 


13-85 


2-52 


6-50 


2-45 


4-05 


91-79 


0-61 


Average 




15-81 


2-90 


8-06 


2-57 


5-49 


9146 


0-35 


SEM 




0-14 


0-05 


0-12 


0-06 


0-14 


N.S. 


0-01 


C D at 5% 




0-40 


0-16 


0-35 


0-18 


0-56 





0-04 




Pusa sawani 


1540 


1-92 


6-50 


2-85 


3-65 


9143 


0-14 




Lam hybrid 


14-65 


2-46 


7-00 


2-55 


4-45 


89-85 


0-20 


Seeds 


Vashalivadu 


18-37 


3-04 


4-35 


1-28 


3-07 


90-88 


0-12 




AC-302 


16-25 


2-06 


6-35 


2-75 


3-60 


87-63 


0-19 




AC-333 


17-25 


2-28 


6-25 


2-84 


341 


89-23 


0-13 




Sel-2 


13-25 


2-12 


10-50 


5-.12 


5-38 


90-32 


0-31 




KS-305 


13-75 


2-35 


6-25 


2-75 


3-50 


89-00 


0-39 




Line 14-78 


14-50 


2-01 


5-25 


2-25 


3-00 


89-15 


0-35 


Average 




1543 


2-27 


6-56 


2-80 


3-76 


89-69 


0-23 


SEM 




0-13 


, 0-06 


0-10 


0-08 


0-11 


N.S. 


0-01 


C D at 5% 




0-39 


0-21 


0-30 


0-21 


0-36 





0-03 




Pusa sawani 


15-54 


2-00 


7-01 


2-67 


4-34 


90-70 


0-17 




Lam hybrid 


15-64 


2-65 


7-28 


2-80 


448 


88-00 


0-21 


Whole 


Vashalivadu 


17-25 


3-40 


4-46 


1-29 


3-17 


91-30 


0-17 


fruits 


AC-302 


16-53 


2-10 


7-08 


2-60 


4-48 


88-10 


0-20 




AC-333 


16-78 


2-44 


6-79 


2-74 


4-05 


90-40 


0-17 




Sel-2 


13-79 


2-46 


10-31 


4-92 


5-39 


91-80 


0-40 




KS-305 


14-56 


2-43 


6-60 


2-66 


3-94 


88-24 


0-48 




Line 14-78 


13-35 


2-32 


5-53 


2-38 


3-15 


88-90 


0-49 


Average 




1543 


2-47 


6-88 


2-76 


4-12 


89-68 


0-29 


SEM 




0-15 


0-04 


0-11 


0-03 


0-12 


N.S. 


0-04 


-CD at 5% 




0-43 


0-12 


0-34 


0-08 


0-35 





0-04 




Table 4. Simple 


correlation between 


phytochemicals of okra fruit 


components and spotted 


bollworm survival. 


Correlation coefficient (r) 




Phytochemical 




Pericarp 


Seeds 


Fruit blocks 




Moisture 




0-60 


0-58 


0-74* 










Total sugars 




0-45 


0-54 


0-35 










Reducing sugars 




0-35 


0-39 


0-25 








Non-reducing sugars 


0-55 


0-41 


.046 










Protein 




0-42 


0-38 


0-36 










Free ammo acids 




0-15 


0-11 


0-01 










Tannin 




-0-81* 


-046 


-0-56 









""Significant at 5% level. 



366 Ram Singh 

potential due to larval feeding on pericarp. To support the normal growth of insect, 
primary plant chemicals (Hsiao 1974) like proteins, ammo acids, sugars and moisture 
contents appeared to be in sufficient amounts in pericarpic region of okra fruits. In 
addition, concentrations of total sugars, non-reducing sugars and amino acids were 
higher in pericarp than seeds and whole fruits which indicated better phagostimu- 
latory activity for the pest. Such facts are in accordance with the views of 
Thorsteinson (1958). But qualitatively pericarp has been reported to be inferior to 
okra seeds with respect to free amino acids and water soluble proteins (Mani et al 
1986). However; there is need to investigate quantitative differences among different 
essential and non-essential amino acids present in pericarp and seeds. Similar to 
present studies from sugars point of view, Mani et al (1986) also did not find pericarp 
inferior to seeds. Under utilization of nutritional compounds may also be due to the 
presence of some antinutritional factors in higher amounts in pericarpic portion. 
Tannin was found to be higher in pericarp (0-35%) than seeds (0-23%) and whole fruit 
(0-29%). Antibiosis property of tannins have been reported against a number of pests 
(Bennett 1965; Maxwell et al 1967; Feeny 1968). Tannins react with digestive, 
enzymes and other proteins in insects thereby reducing the nutritive value of the 
ingested foods (Chan et al 1978). They may also act as feeding inhibitors (Sharma 
and Agarwal 1981). This antibiotic compound was further significantly higher in 
pericarps of tolerant genotypes in comparison to susceptible ones. However, in 
present studies, tannin content in okra fruits did not exceed 0-49% which may not be 
sufficient to induce complete antibiosis. Chan et al (1978) reported 3-4% tannin in 
cotton squares of Heliothis armigera F. resistant genotypes. Against E. vittella, 
Sharma et al (1982) estimated 1-96% tannin in bolls of least suitable genotype. Hence 
tannin effects may be supplemented through the inferior quality of free amino acids 
and water soluble proteins in epicarp as reported by Mani et al (1986). 

With regard to developmental parameters on seeds and fruit blocks, the overall 
differences between larval period, pupal and total developmental period and growth 
indices were negligible. However, pupation on fruit blocks (71-58%) was better than 
on seeds (65-42%). It ,was probably due to better pupation site or thigmotactic 
stimulus offered by fruit blocks. Vishwapremi and Krishna (1974a) also reported 
shortest pupal period and heaviest pupal weight for the larvae reared on fruit cut 
pieces in comparison to whole fruit, seeds and epicarp. Contrary to this, okra seeds 
showed better reproductive potential (Vishwapremi and Krishna 1974b; Ambegaonkar 
and Bilapate 1982). 

Moisture content, total sugars, reducing sugars, non-reducing sugars, proteins and 
free amino acids of pericarp, seeds and whole fruits of okra genotypes were positively 
correlated with pest survival (table 4). Among these, only moisture content of whole 
fruit had positive and significant (r = 0-74) correlation with fruit borer survival. 
Tannins of pericarp, seeds and whole fruits showed negative correlation with pest 
survival. 



Acknowledgements 

Author thanks Dr R A Agarwal for guidance and to Indian Agricultural Research 
Institute, New Delhi for providing a fellowship. Author is also thankful to Dr S KVerma 
for going through the manuscript critically. 



Okra fruit phytochemicals in relation to spotted bollworm 367 

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Yemm E W and Cocking E C 1955 The determination of ammo acids with ninhydrin; Analyst 80 209-213 
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96', No. 4, July 1987, pp. 369-381 
i('.j Printed in India. 



In vitro studies on the involvement of brain and gonad in the functional 
differentiation of prostate gland in Cryptozona belangeri (Deshaves) 
(Mollusca: Gastropoda) 

UMA NATARAJAN and VIJAYAM SRIRAMULU 

Zoology Department, Annamalai University, Annamalainagar 608 002, India 
MS received 1 January 1986; revised 27 February 1987 

Abstract. Prostate glands from mature and juvenile Cryptozona hvhmgeri were cultured in 
vitro for 8 days in single or double association with cerebral ganglia, visceral ganglionic 
complex and gonads obtained from adult snails. The histology of the glands was studied 
and compared with those of normal glands and with glands cultured in vitro without any 
organ association. Functional differentiation was not evident in glands cultured in isolation 
or in single association with gonad or brain. In double associations, involving cerebral 
ganglia, visceral ganglionic complex and gonad response from the gland was obtained only 
when the gonad was involved. The cerebral ganglia and visceral ganglionic complex acting 
through the gonad elicit differential responses from the prostate gland. It was concluded 
from the in vitro studies that the prostate gland may be dependent on a gonadal hormone 
for functional differentiation, which in turn is under the control of trophic factors released 
from the brain. 

Keywords. Crypiozona belam/eri; in vitro studies; functional differentiation; prostate; 
cerebral ganglia; visceral ganglionic complex; gonad. 

1. Introduction 

Regulation of reproductive activities in pulmonates has remained a perplexing 
problem. Various explanations have been proposed by different workers to account 
for the reproductive process in molluscs but as pointed out by Golding (1974), the 
differences in the techniques employed, the diversity of regulative mechanisms and 
the disarray relating to species variations serve to stress the enormity of the problem. 
In the absence of well defined endocrine organs in molluscs, the gonad and the 
neurosecretory cells located in the brain and the optic tentacles are known to be 
implicated in the control of reproductive activities. 

In the brain, the cerebral ganglia influences differentiation of the male germ cells 
(Guyard 1971), vitellogenesis (Streiff 1967; Choquet 1971) and reproductive tracts 
(Boer and Joosse 1975). The involvement of the visceral' ganglionic complex in 
reproductive activities has been poorly investigated. 

The status of the gonad as an endocrine gland has remained controversial. Histo- 
logically there is no evidence to account for the production of hormones in the gonad 
(Boer and Joosse 1975). However experimental studies embracing castration, trans- 
plantations and organ culture methods have shown that the gonad has some effect 
on the reproductive tract, albumen gland, prostate and female glands (Abeloos 1943; 
La Violette 1954; Bailey 1973; Runham et a! 1973). 

The major drawback in investigations involving the brain and the gonad is the 
heavy mortality rate resulting from surgery or castration in shelled pulmonates 
(Goddard 1 960). Removal of the shell or even a sizeable part of it is lethal to the 



370 



Uma Natarajan and Vijayam Sriramulu 



animal. Further, the gonad is closely apposed to the hepatopancreas, which is 
invariably damaged during surgery. To overcome this difficulty a recent trend is the 
usage of in vitro methods. 

The present study has been undertaken because of the paucity of data on regula- 
tive measures of the accessory glands in terrestrial pulmonates and also taking into 
consideration the value and advantages of using in vitro techniques. The experiment 
is designed to study the influence of the gonad and brain on the functional differen- 
tiation of the prostate gland in the terrestrial pulmonate snail Cryptozona belangeri 
(Deshayes). 

2. Material and methods 

2.1 In vitro studies 

The millipore filter organ culture technique devised by Grobstein (1955) and 
Auerbach (1960) was adopted in cultivating the prostate gland in vitro. The proce- 
dure of Ball and Auerbach (1961) was followed for the preparation of the millipore 
assembly. A plexiglass strip (32 x 6 x 2 mm) possesing a central hole of 3 mm dia- 
meter was pasted with a round disc of millipore filter paper to obtain an open well of 
3 mm diameter and 1 mm depth. The plexiglass strip was lowered on to the depre- 
ssion of the tissue culture dish of Grobstein (1955) type (figure 1). 

2.2 Culture medium 

With the exception of a few modifications indicated below, the medium was similar 
to that used by Burch and Caudros (1965) for Helix aspersa. The molluscan tissue 
extract in the culture medium was replaced by chick embryo extract to remove the 
possible interference of homologous proteins in the molluscan tissue extract (Rajan 
and Sriramulu 1979). Hedon-Fleig saline (Lockwood 1961) was used for the survival 
of the explants. Tricine was employed as a buffer (Bailey 1973). Table 1 gives the 
composition of the culture medium used in the present study. 



CDC 




CM 



PG 



Figure 1. Sectional view of the millipore culture set up. (CD, Culture dish; CDC, culture 
dish cover; CM, culture medium; MF, millipore filter paper; PG, plexiglas; T, explant tissue). 



Endocrine control of prostate in snail 371 



Table 1. Medium adopted for culture of prostate in C. belangeri. 





(ml) 


Medium 199 + 0-5% peptone 


500 


*Brain heart infusion (Difco) 


20 


S.L. Broth 


20 


M.9 stock solution A 


8 


M.9 stock solution B 


8 


*Salt solution C 


100 


*Sodium bicarbonate (4 g/1) 


100 


Calf-foetal serum (Difco) 


40 


*Chick-embryo extract (Difco) 


40 


Double distilled water 


403 


*Tricine (1-25 molar aqueous solution) 


25 


Antibiotic mixture 


8 


S.L. Broth 


(g) 


Casein hydrolysate 


10 


Yeast extract 


5 


K 2 H PO 4 


6 


Diammonium hydrogen citrate 


2 


Glucose 


- 20 


Sodium acetate (hydrated) 


25 


Glacial acetic acid 


1-32 ml 


Salt solution 


5ml 


Tween 80 


U 


Made up to 100 ml with double distilled water 




Stock solution A 


(g) 


Na 2 H PO 4 (anhydrous) 


75-0 


KH-,PO 4 (anhydrous) 


37-5 



Made up to 1000ml with double distilled water 

Stock solution B 

NaCl . 50-0 

MgSO 4 20-0 

NH 4 C1 100-0 

Made up to 1000ml with double distilled water 

Salt solution C 

NaCl 36-0 

KC1 1-0 

MgS0 4 2-0 

Made up to 1000 ml with double distilled water 

Salt solution for SL broth 

MgSO 4 -7H 2 O 11-5 

MnSO 4 -H 2 6 2-86 

Made up to 1000 ml with double distilled water 

Fungizonc 50 mg 

Penicillin 1,000,000 IU 

Streptomycin 1-0/1000 ml 

*Modifications introduced in the culture medium adopted by Burch and Ca 
(1965). 



372 Uma Natarajan and Vijayam Sriramulu 

23 Preparation of explains 

Healthy snails of weight groups of 2-3 g (juvenile) and 6-8 g (mature) were washed in 
running water to remove dirt and soil. The shell was removed with sterilized scissors 
and the body wiped with moistened sterilized cotton and 70 alcohol. The unana- 
esthetized snails were then dissected in Hedon-Fleig saline for the removal of the 
prostate gland. It was severed into small bits measuring 2x2x1 mm each. Each 
piece was transferred to an unit of the millipore filter assembly. The nutritive culture 
medium was introduced into the depression in the culture dish. 

2.4 The associated organs in the culture 

The associated organs were obtained from mature snails. The brain was detached 
from the surrounding tissues in Hedon-Fleig saline. The brain was divided into two 
halves, one comprising the dorsal cerebral ganglia and the other the ventral visceral 
ganglionic complex (pleural, parietal, pedal and visceral). The gonad was also 
removed and separated from the associated hepatopancreas. The components of 
brain and gonad were then used either singly or in combination for association with 
the prostate gland in vitro (tables 2 and 3). 

2.5 Maintenance of culture 

The cultures were maintained for 8 days at 25C in a BOD incubator in saturated 
humid condition with a gas phase of 95% O 2 and 5% GO 2 . The medium and gas 
phase were changed on alternate days. Frequent observations through microscope 
were made to detect any sign of contamination and to note the viability of the tissue. 



TaJble 2. Prostate explant of C. belantjeri and the associated organs maintained in 
culture. 

Organs cultured in association 
Explant . with the explant 



Heterologous association involving 



single associated organ 
Juvenile prostate . Control 

Juvenile prostate Mature gonad 

Juvenile prostate Mature cerebral ganglia 

Juvenile prostate Mature visceral .ganglionic 

complex 

Heterologous association involving 

double associated organs 
Juvenile prostate Mature gonad and cerebral 

ganglia 
Juvenile prostate . Mature gonad and visceral 

ganglionic complex 
Juvenile prostate Mature cerebral ganglia and 

visceral ganglionic complex 



Endocrine control of prostate in snail 373 

Table 3. Prostate explant of C. belanyeri and the associated organs maintained in 
culture. 

Organs cultured in association 
Explant with the explant 

Homologous association involving 

single associated organ 

Mature prostate Control 

Mature prostate Mature gonad 

Mature prostate Mature cerebral ganglia 

Mature prostate Mature visceral ganglionic 

complex 

Homologous association involving 

double associated organs 
Mature prostate Mature gonad and cerebral 

ganglia 
Mature prostate Mature gonad and visceral 

ganglionic complex 
Mature prostate Mature cerebral ganglia and 

visceral ganglionic complex 



.6 Histology 

lie tissues maintained in culture were fixed in aqueous Benin's dehydrated in 
scending grades of alcoholic series, cleared in xylol and embedded in paraffin wax 
58-60C). Sections were cut at 6 /i thickness and these were deparaffinised .and 
tained in Heidenhain's iron haematoxylin, counterstained with aqueous eosin and 
lounted in Canada balsam. 



. Observations 

. 1 Gross morphology of the prostate gland 

n an immature or reproductively inactive snail, the prostate appears as an indistinct, 
>alc white, thread like structure, running along the dorsal surface of the sperm- 
jviduct. In a mature snail the prostate attains its maximum growth in size, appearing 
.s a separate elongated organ, slightly yellow and closely apposed to the sperm- 
>viduct. This organization of the prostate is retained till the end of the reproduc- 
ively active period and thereafter the gland involutes and reverts back to its 
uvenilc state. 



.2 Histology of the prostate gland 

.2a Juvenile: The small prostate gland cells occur in abundance and lie scattered 
miformly throughout the tissue in the juvenile snail. The small spherical nuclei 
ppear distinct. They are centrally placed within the cells and are surrounded by a 
lomogenous thin layer of cytoplasm, with no trace of secretory products (figure 2). 



374 



Uma Natarajan and Vijayam Sriramulu 




Figures 2-6. 2. Section through the prostate gland of juvenile snail showing the onset of 
differentiation of the tubules of the gland ( x 80). 3. Section through the prostate gland of 
adult snail showing maximum growth of the gland. Note the compact organization of the 
gland, its shape and its regional differentiation into an outer light area (C) and an inner 
deeply staining area (M). A thin connective tissue layer surrounds the gland ( x 25). 4. Sec- 
tion through the prostate gland of juvenile snail cultured in isolation. Note the poor orga- 
nization of tissue ( x 80). 5. Section through the adult prostate gland cultured in isolation. 
Note necrotic patches towards periphery and deterioration in organization compared to 
figure 4 ( x 25). 6. Enlarged view of a portion of figure 5 showing disrupted tubules ( x 80). 



3.2b Adult: Based on the histoarchitecture of the adult prostate gland 3 distinct 
regions can be made out, viz (i) a thin connective tissue layer enclosing the entire 
gland; (ii) glandular cells of the prostatic tubules and (iii) the lumen enclosed by the 
glandular cells lined by epithelium. 

Based on staining properties, two zones can be made out in the gland: (i) an outer 
light zone in which tubules are loosely arranged with lightly staining cells, and (ii) a 



Endocrine control of prostate in snail 375 

deeply staining central region in which the tubules are compactly arranged. In these, 
the cells appear pearshaped, and the broad distal ends of the cells face outwards 
while the narrow proximal ends face lumen of the tubule. The nuclei are oval, located 
in the broader end of the cells. The chromatin material stains intensely with haema- 
toxylin and the cell cytoplasm is filled with secretory granules which tend to distend 
the cell walls (figure 3). 



3.3 Prostate gland cultured in isolation 

3.3a Juvenile: Prostate glands cultured in isolation appear much reduced in their 
size and many of the cells, particularly towards the peripheral region of the gland, 
appear empty with shrunken cytoplasm (figure 4). 

3.3b Adult: Disruption in the general organization is evident. Patches of necrotic 
tissue are visible. Nuclei lie scattered in the disintegrated cytoplasm. Tubular organi- 
zation is noticed only within the region nearer to the lumen. The tubules are, 
however, small. The sparse secretory products stain very lightly (figures 5 and 6). 



3.4 Prostate gland in association with gonad 

3.4a Juvenile: The gland shows a tendency towards degeneration. The central 
region is the worst affected, where there is a marked reduction in the number of cells. 
The nuclei appear pycnotic and the cytoplasm is devoid of secretory products (figure 7). 

3.4b Adult: Deterioration is evident and the characteristic organization of the 
gland remains disrupted with no trace of tubules. Cell boundaries appear broken 
resulting in the cytoplasmic content being dispersed among the tissues. Large gaps 
and spaces are interspersed between the cells (figures 8 and 9). 



3.5 Prostate gland in association with cerebral ganglia 

3.5a Juvenile: The cytomorphology of the juvenile gland closely approximates that 
of glands cultured in isolation. Dead tissue areas with unhealthy cells testify to the 
inability of ganglia to maintain the normal organisation of the juvenile prostate 
gland. 

3.5b Adult: Tubular organisation is absent and the central tissue appear vacuo- 
lated. A distinct deviation from the normal histology of the gland persists although a 
slight improvement over the glands cultured in isolation is evident (figure 10). 



3.6 Prostate gland in association with visceral ganglionic complex 

3.6a Juvenile: The glandular cells show an increase in their volume. The cell limits 
are clearly delineated and the cells exhibit a tendency to be grouped together rather 
than to remain dispersed. 



376 



Uma Natarajan and Vijayam Sriramulu 




Figures 7-12. 7. Section through juvenile prostate gland cultured with gonad. Note atro- 
phy of the gland with poor cytological features ( x 25). 8. Section through adult prostate 
gland cultured with gonad. Note absence of tubules and disrupted histology ( x 25). 9. 
Enlarged view of figure 8 showing the disposition of the disorganized tubules ( x 80). 10. 
Section through adult prostate gland cultured with cerebral ganglia. Note vacuolated cells 
towards the centre ( x 80). 11. Section through adult prostate gland cultured with visceral 
ganglionic complex. Note peripheral tubules with hypertrophied cells. The secretory 
products of central tubular cells are apparent in the luminae. Cells are not distinct ( x 80). 
12. Section through juvenile prostate gland cultured with gonad and cerebral ganglia. Note 
transformation of juvenile histology of gland to adult histology. Tubules have cells with 
abundant secretions surrounding central lumen ( x 80). 



3.6b Adult: A marked change is evident in cells comprising the peripheral tubules 
which show hypertrophy with secretory products concentrated more towards the 
narrower ends facing the lumen. Secretory granules are visible in the large luminae 
(figure 11). 



Endocrine control of prostate in snail 377 

3.7 Prostate gland in association with gonad and cerebral ganglia 

3.7a Juvenile: A complete transformation of the juvenile to adult tissue organisa- 
tion is encountered when juvenile prostate gland is cultured with gonad and cerebral 
ganglia. The tubules resemble those found in adult sections taken in vivo. The cells 
are filled with secretory products and are arranged around a central lumen. Peri- 
pheral nuclei are distinct (figures 12 and 13). 

3.7b Adult: In a major part of the gland, a tubular arrangement is visible and the 
cytoplasm contains secretory products which stain deeply. The histology of the gland 
closely approximates with that of normal adult glands (figures 14 and 15). 

3.8 Prostate gland in association with gonad and visceral ganglionic complex 

3.8a Juvenile: A precocious response of the juvenile gland is met with when asso- 
ciated with adult gonad and visceral ganglionic complex. The gland cells aggregate 
to form tubules but still retain their spherical shapes. Further, the cytoplasm shows 
intense secretory activity and the elaborated product considerably increases the size 
of the cytoplasm. The stress is on the functional activity rather than on structural 
organization (figure 16). 

3.8b Adult: Regional differentiation in the histomorphology of the gland is seen. A 
major portion of the gland away from the duct has hypertrophied glandular cells, 
which stain deeply and are arranged in ill-defined tubules. Towards the duct, the cell 
membranes of the cells are disrupted showing released material in the lumen. A few 
cells have pale and vacuolated cytoplasm with few granules (figures 17 and 18). 

4. Discussion 

The prostate gland in C. belangeri shows close correlation with growth, maturation 
and reproductive activity of the snail. The functional differentiation of the gland can 
be deduced from the cytomorphological changes that take place within its glandular 
cells. The observations recorded in the present study are the result of associations 
carried out in vitro of the juvenile and mature prostate glands with the gonad, 
cerebral ganglia and visceral ganglionic complex in single or double combinations. 
This implies a direct action of the associated organs on the gland and also as the 
active substance emitted by the associated organs acts by diffusing into the culture 
milieu and there is no actual physical contact between the associated organ and the 
prostate gland. The histology of the glands thus associated, when compared to those 
of normal glands and with those glands cultured in the absence of any organ asso- 
ciation, reveals striking facts. 

The prostate gland in C. belangeri, in the absence of any hormonal support, shows 
poor degree of organization in cultures. This applies to both juvenile and adult 
glands. This incapacity to function and differentiate under anhormonal conditions is 
not restricted to the prostate gland alone but has been reported for the dart sac in 
C. belangeri (Megdalene 1981), the male genital tract in Calyptrae sinensis (Streiff 
1966), penis in Crepidula fornicata (Le Gall 1981; Le Gall and Feral 1982) and 



378 Uma Natarajan and Vijayam Sriramulu 




Figures 13-18. 13. Enlarged view of figure 12 showing arrangement of cells within tubules. 
14 and 15. Sections through adult prostate gland cultured with gonad and cerebral ganglia. 
Note regions of actively secretory cells in contrast to areas of tissue with no activity. 16. Sec- 
tion through juvenile prostate gland cultured with gonad and visceral ganglionic complex 
showing similarity in organization to figure 13. However, note the increase in cytoplasmic 
volume in the spherical cells ( x 80). 17 and 18. Sections through adult prostate gland, 
cultured with gonad and visceral ganglionic complex showing regional differentiation in 
cytomorphology. Note the active hypertrophied cells arranged within tubules (figure 17) 
and vacuolated cells near the duct (figure 18) ( x 80). 

Note: Section referred to in figures 2-18: Bouin's fluid, 6 \L Heidenhain's iron haema- 
toxylin counter stained with aqueous eosin. 

common duct in Agriolimax reliculatus (Bailey 1973). The present result only reflects 
the view of Highnam and Hill (1979) that development of the reproductive tract is 
controlled by hormones. 

The prostate gland, cultured with gonad from adult snails, showed rapid deterio- 
ration in its organization and its functional activity was disrupted. This would 



Endocrine control of prostate in snail 379 

indicate that the gonad inhibits the structural organization and functional integrity 
of the gland. These results are in contrast to those of castration, transplantation and 
organ culture experiments, carried out by various workers. Castration in limacid and 
arionid slugs resulted in negative effects in the albumen and hermaphrodite duct 
(Abeloos 1943; La Violette 1954). The effects of castration on the activity of the 
multifid gland in Helix aspersa was studied and the role of gonad in maintaining its 
activity was established (Gomot 1976). In A. reticulatus, undifferentiated tracts when 
transplanted into male phase slugs, showed differentiation of prostate glands, while 
oviductal and albumen glands remain undifferentiated. However, transplantation of 
undifferentiated tracts into the haemocoel of egg-laying animals led to the enlarge- 
ment of the female parts of the tract, while the prostate remained undeveloped. 
Castration of young specimens resulted in a cessation of development of both the 
prostate and oviductal glands. This suggests that the production of the male and 
female hormones had been impeded (Runham et al 1973). Immature penial com- 
plexes of A. reticulatus implanted into mature slugs, displayed growth and differen- 
tiation dependant on the stage of the host (Wijdenes 1981). Implants never developed 
in castrated animals. Bailey (1973) using in vitro techniques, has also suggested the 
implication of the gonad in the control of the prostate in the same slug. However, in 
Basommatophora, castration experiments have indicated that the development and 
activity of the accessory sex organs are not under endocrine control of the gonad 
(Brisson 1971; Brink et al 1979; Boer et al 1976). In the face of the overwhelming 
evidences supporting the presence of a gonadal factor in stylommatophoran pulmo- 
nates, the absence of such a gonadal factor cannot merely be deduced by direct 
association of the prostate with the gonad in culture. The possibility, that the poor 
histological picture of the gland, in single association with the gonad, could be due to 
the lack of a trophic factor, cannot be ruled out. In fact, in an animal which is stro- 
ngly dependent on external conditions of temperature and moisture for its reproduc- 
tive activity, the mediation of the nervous system in the seasonal activity of its repro- 
ductive system is strongly indicated. 

The direct association of the prostate gland with the cerebral ganglia appears to 
have no impact on the histology of the gland in G belangeri. A number of evidences 
can be cited wherein the cerebral ganglia influence reproductive activity, but in the 
majority of these, the effects (of the principles) are usually related in some way with 
those of the gonad or the tentacle. Direct action of the principles of cerebral ganglia 
has been suggested in prosobranchs (Joosse 1979) and in snails (Geraerts and 
Bohlken 1976; Goudsmit 1975, 1978). The principles of cerebral ganglia in all these 
molluscs appears to affect either gametogenesis or the female organs. A direct action 
of cerebral ganglia on male accessory organs has not been reported till now. 

A distinct change in the appearance of the gland is evident when cultured with 
visceral ganglionic complex. This is particularly noticeable in the adult glands, in 
which the glandular cells appear vacuolated and devoid of granules. Probably some 
visceral ganglionic principle is responsible for the unconditional liberation of secre- 
tory granules from these cells, giving them an exhausted appearance. In the imma- 
ture glands, apart from a slight increase in cytoplasmic volume no remarkable change is 
evident. 

Since associations of the prostate gland with the brain or the gonad failed to 
produce positive results, interactions between these two endocrine components were 
investigated. Recent literature on pulmonate regulative mechanisms strengthens this 



380 Uma Natarajan and Vijayam Sriramulu 

view of trophic control. Runham et al (1973) concluded from results on Agriolimax 
that male and female parts of the reproductive tract are under separate hormonal 
control. Probably the sources of the two hormones are the dorsal bodies and the 
neurosecretory cells in the cerebral ganglia (Geraerts and Algera 1972), and these 
hormones influence the reproductive tract via the gonad (Boer and Joosse 1975). A 
relation between brain-gonad axis and photoperiodically stimulated sexual matura- 
tion in the slug Limax maximus has been studied (McCrone and Sokolove 1979; 
McCrone et al 1981). In the marine pulmonate Onchidium verruculatum, it has been 
shown that a male phase hormone and a female phase hormone are secreted by the 
cerebral ganglia during different phases of the protandric reproductive cycle 
(Nagabhushanam and Deshpande 1981). These hormones have been observed to 
exert their effects on development and functioning of the -male accessory sex organs 
via the gonad. An androgenic influence of the cerebral ganglia on the gonad has also 
been found in Helix aspersa (Guyard 1971). In a review of pulmonate endocrinology, 
Boer and Joosse (1975) concluded that two gonadal factors, perhaps operate sepa- 
rately in the control of male and female lines. Further, it was thought that the dorsal 
body hormone possibly controls the female factor of the gonad and a neurohormone 
from neurosecretory cells in the cerebral ganglia controls the male factor. 

The results obtained in C. belangeri, by the culture of the prostate gland with 
cerebral ganglia and gonad, is in conformity with the scheme proposed by Boer and 
Joosse (1975). The cerebral ganglia of adult snails, in conjoint action with the gonad, 
completely transforms the juvenile appearance of the gland into an organized adult 
form. This high degree of organisation is in contrast to that of juvenile glands, with 
cerebral ganglia and gonad in single association. In the adult gland the degree of 
organization and functional activity appears to be of a high order. These results 
suggests that the cerebral ganglia has a trophic effect on the gonad which is probably 
induced to produce a hormone responsible for the growth and edification of the 
glandular cells. 

When cultured with the gonad and visceral ganglionic complex, the juvenile pro- 
state gland in C. belangeri shows a precocious response in its secretory activity, but 
apparently does not alter the shape of the cells. In the adult glands subjected to a 
similar association, the secretory material appears to spill over into the lumen, and in 
some cases, empty vacuolated cells which have released almost all their materials 
into the lumen, can be seen. Apparently, the visceral ganglionic complex appears 
capable of inducing the gonad to release its hormone; in addition its effect on the 
prostate gland also seems to be direct. This effect, as previously stated, would be to 
cause exocytosis of the secretory material and could account for the differences 
encountered, in associations involving gonad and cerebral ganglia and that of gonad 
and visceral ganglionic complex. 

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chez Crepidula fornicata L. (Mesogasteropode hermaphrodite protandre); Int. J. Invert. Reprod. 5 

31-42 
.ockwood A P M 1961 Ringer solutions and some notes on the physiological basis of their ionic 

composition; Comp. Biochem. Physiol 2 241-289 
vlcCrone E J and Sokolove G 1979 Brain-Gonad axis and photoperiodically stimulated sexual 

maturation in the Slug, Limax maximus; J. Comp. Physiol. 133 117-123 
vlcCrone E J, Jan Van Minnen and Phillip G S 1981 Slug Reproductive Maturation Hormone: In vivo 

Evidence for Long-Day Stimulation of Secretion from Brains and Cerebral Ganglia; J. Comp. Physiol. 

143311-315 
vlegdalene T 1981 Influence of the optic tentacular principle on the functional differentiation of the dart sac 

in Cryptozona belangeri (Deshayes) (Mollusca: Gastropoda), M.Phil. Dissertation, Annamalai 

University, Annamalainagar 
^agabhushanam H and Deshpande V D 1981 Reproductive Endocrinology of marine pulmonate, 

Onchidium verruculatum; The First Indo-Pacific Symposium on Invertebrate Reproduction 

Abstract November, Marathwada University, Aurangabad 



382 Uma Natarajan and Vijayam Sriramulu 

Rajan R K and Sriramulu V 1979 In vitro studies on the influence of optic tentacular principle in main- 
tenance of male germ cell line in the snail Cryptozona belangeri; Indian J. Exp. Biol 17 701-703 

Runham N W, Bailey T G and Laryea A A 1973 Studies on the endocrine control of the reproductive tract 
of the grey field slug, Agriolimax reticulatus; Malacologia 14 135-142 

Streiff W 1966 Etude endocrinologique du determinisme du cycle sexuel chez un mollusque herma- 
phrodite protandre Calyptraea sinensis (L) I. Mise en evidence par culture in vitro de facteurs 
hormonaux conditionnant 1'evolution du tractus genitale male; Ann. Endocrinol. 27 385-400 

Streiff W 1967 Etude endocrinologique du determinisme du cycle sexuel chez mollusque hermaphrodite 
protandre Calyptraea sinensis (L.) III. Mise en evidence par culture in vitro de facteurs hormonaux 
conditionnant 1* evolution de la gonade; Ann. Endocrinol. 28 641-656 

Wijdenes J 1981 Experiments on the endocrine control of penial complex maturation in the slug 
Agriolimax reticulatus (Pulmonata Limacidae); Proc. K. Ned. Akad. Wet. 84 107-1 14 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 4, July 1987, pp. 383-387. 
.'('; Printed in India. 



Life and fertility tables of Achaea Janata Linnaeus on castor 

A N BYALE and G G BILAPATE 

Department of Entomology, Maruthwada Agricultural University, Parbhani 431 402, India 

MS received 7 September 1985; revised 8 January 1987 

Abstract. The life fecundity tables were constructed when Achaea Janata Linnaeus reared 
on castor leaves in the laboratory. The net reproductive rate (R a ) was 467-57 females/female/ 
generation in a generation time of 37-99 days. The intrinsic rate of natural increase (r m ) was 
0-1618 female/female/day. In the stable age-distribution, 99% contribution was made by 
immature stages. 

Keywords. Life and fertility tables; Achaea Janata. 



1. Introduction 

The castor semilooper, Achaea Janata Linnaeus is one of the important pests of 
castor crop in many states of India. The intrinsic rate of increase is a basic parameter 
which an ecologist^ may wish to establish for an insect population. Birch (1948) 
defined it as actual rate of increase of population under specified constant 
environmental conditions in which space and food are unlimited when there are no 
mortality factors other than physiological ones. According to Howe (1953), a life- 
table can be constructed by following the life history of a group of insects from their 
birth (egg laying) to emergence of adults and recording all deaths as they occur 
together with sex of those which die in as adult. Therefore, an attempt was made to 
study life fecundity tables for A. Janata when reared on castor leaves. 

2. Materials and methods 

The method used in the present study was similar to those used for Heliothis 
armiyera (Hubner) when reared on pigeonpea (Bilapate et al 1983). To construct the 
life fecundity tables, 200 eggs were placed in 10 plastic boxes in batches of 20 each. 
After hatching, all the larvae were reared individually on castor leaves. Fresh food 
was supplied daily. Observations on hatching, larval and pupal development, 
successful adult emergence, fecundity and age specific mortality in eggs, larvae, pupae 
and adults were made daily. For determining the specific fecundity, all the adults 
emerged on a particular day were transferred to a separate cage for egg laying. The 
healthy castor twig was kept in the cages as oviposition site. Considering the sex 
ratio 1 : 1 (Southwood 1968), the number of eggs laid per female were divided by two 
to get the number of female births (m x ). 

The following column headings proposed by Birch (1948), elaborated by Howe 
(1953) and Atwal and Bains (1974) were used for the construction of life-fecundity 
tables under laboratory conditions: x pivotal age in days; l x survival of females 
at age x; m x age schedule for female births at age x. 

The values of x, l x and m x were calculated from the data on life tables. The sum of 



384 A N Byale and G G Bilapate 

the products l x m x is the net reproductive rate (R ). R is the rate of multiplication of 
the population in each generation measured in terms of females produced per 
generation. The number of times a population would multiply per generation was 
calculated by the formula, 



3. Results and discussion 

The results in table 1 indicate that survival of A. Janata was 184, 177 and 170 eggs, 
larvae and pupae in a cohort of 200 eggs. A cohort of 200 eggs resulted in the 
emergence of 89 females and 81 males. 

The life-table and age specific-fecundity for A. Janata indicated that the survival of 
immature stages was 0-85 (based on one individual) individual within a pivotal age of 
32 days (table 2). The pre-oviposition period was 3 days i.e. from 33-35 days of 
pivotal age. The number of eggs laid per female were divided by two to get the 
number of female births (mj. The female births was highest (129*75 female offspring) 
on the first day of oviposition of the female (36th day of pivotal age) and decreased 
slowly thereafter. The first female mortality occurred on 7th day (/ X = 0-8Q) after 
emergence of adult when the culture was in the 41st day of pivotal age. The mortality 
rate of female slowly declined thereafter. Thus, the females oviposited for 9 days. The 
net reproductive rate (R ) of female was 467-57 female/female/generation. The results 
on the mean length of generation, intrinsic rate of increase in numbers and finite rate 
of increase in numbers of A. Janata are presented in table 3. The approximate 
generation time (T c ) was found to be 38-38 days. The arbitrary value for intrinsic rate 
of natural increase (r c ) was 0-1602 female/female/day. The precise generation time 
was 37-99 days while the finite rate of increase in numbers was 1-18 female/female/ 
day. The corrected innate capacity for increase in numbers (r m ) was calculated by 
using the formula, I<? 7 -' X 'A = 1096-6. 

The two trial values of 1 157-12 and 792-96 were plotted on horizontal axis against 
their respective arbitrary r c values, 0-16 and 0-17 on vertical axis and the corrected r m 
was calculated as 0-1618 female/female/day by interpolation method. The stable age- 

Table 1. The survival of life stages of A. Janata on castor leaves. 





Surviving No. 








Egg stage 


Larval stage 


Pupal stage 


Sex 








(0-3 days) 


(4-20 days) 


(2 1-32 days) 


Female 


Male 


18 


17 


16 


10 


6 


17 


17 


17 


10 


7 


19 


19 


18 


8 


10 


20 


18 


17 


7 


10 


18 


18 


18 


10 


8 


17 


17 


17 


10 


7 


18 


17 


17 


11 


6 


19 


17 


15 


7 


8 


19 


19 


18 


8 


10 


19 


18 


17 


8 


9 



Total 184 177 170 89 81 



Life and fertility tables of A. Janata 

Table 2. Life-tables (for females) are age specific fecundity for 
A. Janata on castor leaves. 



385 



Pivotal 


Life table 




Age schedule 


age 


for female 




for female 


(days) 


births 




births 


W 


(L x ) 


K) 


(L x m x ) (L x m x x) 


0-32 


0-85 




Immature stages 


33-35 


0-85 




pre-oviposition period 


36 


0-85 


129-75 


110-28 3970-35 


37 


0-85 


88-70 


75-39 3789-61 


38 


0-85 


89-75 


76-28 2898-92 


39 


0-85 


78-90 


67-06 2615-53 


40 


0-85 


66-40 


56-44 2257-60 


41 


0-80 


53-05 


42-44 1740-04 


42 


0-72 


40-93 


29-46 1237-72 


43 


0-56 


18-25 


10-22 349-46 


44 


0-42 


0-0 


0-00 0-0 



L x m x = L x m x x 

K = 467-57 ==17,949-23 



Table 3. Mean length of generation, innate capacity and finite rate of increase in numbers 
of A. Janata on castor leaves. 

Population growth statistics 
Mean length of generation 
I,L x m x x 17949-23 



' R 467-57 
Innate capacity for increase in numbers 
log c /? 6-147491 



T c 



38-38 



Arbitrary r c =0-16 and 0-17 

corrected r n , Ie 1 ~ rm x L x m x = 1 096-6 

Corrected generation time 
Jog e jR "6-1475491 
i~ 0-1618 

Finite rate of increase in numbers 
y = anti log t ,r w 

Weekly multiplication (1-18) 7 



Doubling time log e 



0-6931471 
0-1618 



38-38 days 

0-1602$/?/day 
0-1618 ?/?/day 

37-99 days 

3-18 
4-28/day 



386 A N By ale and G G BUapate 

distribution of A. Janata was worked out when reared on castor leaves (table 4). On 
reaching stable age-distribution, the population in its various stages viz the egg, 
larva, pupa and adult contributed to the tune of 49-06, 48-06, 2-50 and 0-34% 

Table 4. Calculated stable age distribution of A. Janata on castor (r m =0-1618). 



Pivotal 
age 
(x) 


L, 


e~r m x(x+l) 


Lxe'r m {x+\\ 


Percentage 
distribution 


Percentage 
contribution 





1 


0-8506 


0-8506 


15-38 




1 


1 


0-7235 


0-7235 


13-08 




2 


1 


0-6154 


0-6154 


11-13 


49-06 eggs 


3 


1 


0-5235 


0-5235 


9.47 




4 


1-00 


0-4453 


0-4453 


8-05 




5 


0-98 


0-3787 


0-3712 


6-72 




6 


0-96 


0-3222 


0-3093 


5-59 




7 


0-96 


0-2741 


0-2631 


4-76 




8 


0-95 


0-2331 


0-2215 


4-00 




9 


0-94 


0-1983 


0-1864 


3-37 




10 


0-94 


0-1687 


0-1585 


2-87 




11 


0-94 


0-1435 


0-1349 


2-44 




12 


0-93 


0-1220 


0-1135 


2-05 


48-06 larvae 


13 


0-92 


0-1038 


0-0955 


1-73 




14 


0-91 


0-0883 


0-0803 


1-45 




15 


0-90 


0-0751 


0-0675 


1-22 




16 


0-90 


0-0639 


0-0575 


1-04 




17 


0-10 


0-0543 


0-0490 - 


0-89 




18 


0-89 


0-0462 


0-0411 


0-74 




19 


0-88 


0-0393 


0-0346 


0-62 




20 


0-87 


0-0334 


0-0291 


0-53 




21 


0-87 


0-0284 


0-0244 


0-44 




22 


0-85 


0-0244 


0-0205 


0-37 




23 


0-85 


0-0205 


0-0175 


0-32 




24 


0-85 


0-0175 


0-0149 


0-27 




25 


0-85 


0-0149 


0-0127 


0-23 




26 


0-85 


0-0127 


0-0108 


0-19 




27 


0-85 


0-0108 


0-0091 


0-16 


2-50 Pupae 


28 


0-85 


0-0092 


0-0078 


0-14 




29 


0-85 


0-0078 


0-0066 


0-12 




30 


0-85 


0-0066 


0-0056 


0-10 




31 


0-85 


0-0056 


0-0048 


0-09 




32 


0-85 


0-0048 


0-0041 


0-07 




33 


0-85 


0-0041 


0-0035 


0-0633 




34 


0-85 


0-0035 


0-0029 


0-0524 




35 


0-85 


0-0029 


0-0025 


0-0452 




36 


0-85 


0-0025 


0-0021 


0-0379 




37 


0-85 


0-0021 


0-0018 


0-0325 




38 


0-85 


0-0018 


0-0015 


0-0271 


0-34 Adult 


39 


0-85 


0-0015 


0-0014 


0-0253 




40 


0-82 


0-0013 


0-0011 


0-0198 




41 


0-76 


0-0011 


0-0008 


0-0144 




42 


0-64 


0-0009 


0-0006 


0-0108 




43 


0-49 


0-0008 


0-0004 


0-0072 




44 


042 


0-0007 


0-0003 


0-0054 





Life and fertility tables of A. Janata 387 

respectively. The type of life-tables developed by Birch (1948) can be used to obtain 
growth statistics that can be used for both qualitative evaluation of rearing 
procedures and to estimate the projected potential increase of insects reared on 
different food commodities. The innate capacity of increase has not been quantified 
for many insects (Wellik and Pedigo 1978). 

According to Bilapate (1987), the net reproductive rate of A. Janata on castor 
leaves was 524-78, 382-52 and 242-70 female/female during I, II and III generation 
under laboratory conditions. The innate capacity for increase in numbers and mean 
generation time was 0-1692 and 37-01 days during first generation. The growth 
parameters namely survival of immature stages, contribution towards the female 
births and net reproductive rate yielded lower value in the present investigations 
during the rearing of A. Janata on castor leaves. This might have attributed to the 
effect of temperature during the period of investigations. 

Acknowledgement 

Authors are thankful to the authorities for providing necessary facilities. 

References 

Atwal A S and Bains S S 1974 Applied animal ecology, (Ludhiana: Kalyani Publishers) pp 128-135 
Bilapate G G 1987 Studies on the growth of population of Achaea Janata F. on castor; J. Maharashtra 

ayric. Univ. (in press) 
Bilapate G G, Raodeo A K and Pawar V M 1983 Investigation on Heliothis armiyera (Hubner) in 

Marathwada-I. The rate of increase of American cotton bollworm on pigeonpea; Indian J. EntomoL 45 

275-281 

Birch L C 1948 The intrinsic rate of natural increase of an insect population; J. Anim. Ecol. 17 15-26 
Howe R W 1953 The rapid determination of intrinsic rate of increase of an insect population; Ann. AppL 

B/0/.40 134-155 

South wood T R E 1968 Ecological methods, (London: Methuan and Co. Ltd.) p 391 
Wellik M J and Pedigo L P 1978 The innate capacity for increase and ovipositiorial pattern of the green 

colverworm; Environ. EntomoL 7 ill-Ill 



Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 4, July 1987, pp. 389-393. 
Printed in India. 



Cadmium induced vertebral deformities in an estuarine fish, Ambassis 
commersoni Cuvier 

V PRAGATHEESWARAN, B LOGANATHAN*, R NATARAJAN and 
V K VENUGOPALAN 

CAS in Marine Biology, Porto Novo 608 502, India 

* Department of Environmental Conservation, Ehime University, Tarumi 3-5-7, Matsuyama, 

790, Japan 

MS received 12 December 1986; revised 13 April 1987 

Abstract. Amhassis commersoni was treated with 3 different concentrations (0-1, 0-25 and 
0-5 ppm) of cadmium upto 120 days. Fishes treated in higher concentrations developed 
vertebral deformity and the deformation was prominent after 89th and 115th day. The 
levels of the vertebral minerals such as calcium, magnesium and phosphorus lowered 
iirall exposure concentrations and minerals lost were directly proportional to the toxic 
concentrations. Nearly 2/3 of skeletal mineral composition was eliminated in the deformed 
fish. Vertebral fracture was attributed mainly to the large amount of vertebral components 
eliminated from the vertebrae. Vertebral cadmium accumulation was also linear in relation 
to the experimental concentrations. 

Keywords. Cadmium; caudal curvature; vertebral deformities; calcium; phosphorus; 
magnesium; elimination. 

1. Introduction 

The toxic effects of cadmium (Cd) on fishes are known (Eisler 1971; Selvakumar 
1981) and it has been identified as a causative factor for the outbreak of Ttai-itai' 
disease in Japan. Pathological changes (Gardner and Yevich 1970) and depression of 
carbohydrate metabolism (Larsson 1975; Larsson and Haux 1982) and oxygen 
consumption (Selvakumar 1981) by Cd have also been reported. Exposure to Cd for 
longer durations are also known to induce vertebral deformities in fishes (Bengtsson 
et al 1975). Reports on skeletal deformities induced by chemical agents are very 
limited. Weis and Weis (1976) and Couch et al (1977, 1979) recorded pesticide- 
induced vertebral deformities in Cyprinodon variegatus. Linden (1976) noted that 
crude oil also caused vertebral malformation in Clupea harengus membras within a 
short period of exposure. 

Bengtsson (1974) and Holcombe et al (1976) noted vertebral malformations in 
zinc-treated Phoxinus phoxinus and lead treated Salvelinus fontinalis after 4 months 
and 67 weeks of exposure respectively. 

The present investigation was carried out on an estuarine fish Ambassis 
commersoni Cuvier by exposing them to 3 different sublethal Cd concentrations for 
120 days. At the close of the experimental period, the Cd content and the mineral 
composition of the Cd treated fish vertebrae were also estimated. 

2. Materials and methods 

Specimens of A. commersoni were collected from the marine zone of the Vellar 
estuary. The length of the fishes used was 6-3 0-2 cm and the weight 2-5 0-5 g. The 

389 



390 V Pragalheeswaran et al 

fishes were kept in acclimatisation tanks for a week in the laboratory. The salinity of 
the test water ranged between 27 and 28%o. Fishes were fed regularly with chopped 
clam meat. Healthy fishes were selected and exposed to Cd concentrations of 0-1, 
0-25 and 0-5 ppm (each set consisted of 12 specimens). These exposure concentrations 
were prepared on the basis of 96 h LC 50 value (5-20 ppm). Cd solution was prepared 
from Analar CdCl 2 -H 2 O salt. The experimental fishes were fed every day during all 
the 120 days. 

After 120 days of experimental exposure period, the fishes were anaesthetized with 
MS 222 and the vertebral damages if any were located by X-ray photograph using a 
Siemen-160 (West Germany) X-ray equipment. The vertebral Cd concentration was 
estimated by the method of Smith and Windom (1972) using a Hilger- Watts 
Atmospek H 1550 AAS. 

The vertebral column of the untreated and treated fishes were dried at 60C for 
24 h in a -oven. The dried samples were ashed by heating them for 12 h at 500C in a 
muffle furnace. The ashes were then dissolved in 2: 1 of concentrated HNO 3 + con- 
centrated HC1O 4 and made upto 100ml with double distilled water. The vertebral 
calcium (Ca) and magnesium (Mg) levels were estimated following the method of 
Katz and Narone (1964). Phosphorus (P) was estimated by the procedure of 
Strickland and Parsons (1972) and concentrations were noted in a Hitachi double 
beam UV spectrophotometer Model 220. 



3. Observations 

The concentration of Cd as well as those of Ca, Mg and P in the vertebrae of both 
untreated and treated fishes were estimated (figure 1). 

Two fishes were observed with vertebral abnormalities out of 12 fishes exposed to 
0-5 ppm Cd concentration. Caudal vertebral curvature (figure 2B) was clearly 
observed in one fish after 89 days of Cd exposure. After 115 days, another fish in the 
same experimental tank with abnormal swimming movement was noted. The 
damage (figure 2C) was located in the vertebral column through x-ray photograph in 
this fish with abnormal swimming movement. 

The Cd content of the vertebrae in the control fishes revealed no traces of Cd. 
However in fishes exposed to Cd, its accumulation in vertebrae was seen. After 120 
days of exposure the vertebral Cd concentrations were 8-32, 10-91 and 12-86 jug/g in 
respect of the 3 exposure concentrations, viz 0-1, 0-25 and 0-5 ppm (figure 1). 

In the untreated fishes the vertebral .mineral levels were found to be in the 
following order: (i) Ca 132-8 1 mg/g, (ii) P 52-97 mg/g and <iii) Mg 3-27 mg/g. After 
the experimental period the mineral levels were low in all Cd treated fishes and 
showed the following pattern: (i) Ca 120-72, 116-36 and 106-41 mg/g, (ii) P 46-59, 
46-50 and 34-86 mg/g and (iii) Mg 3-08, 2-82 and 2-16 mg/g respectively in the 0-1, 
0-25 and 0-5 ppm of Cd concentrations to which they were exposed. It is evident from 
the results that the loss of minerals from vertebrae was dose dependent (figure 4). 

The Cd content (18-97 /jg/g) in vertebrae of abnormal fish was higher than that in 
other treated fishes. Nearly 58% of Ca, 56% of P and 67% of Mg were lost from the 
vertebrae of the deformed fish. The skeletal mineral composition was found in the 
following order: (i) Ca 56- 1 3 mg/g, (ii) P 23:49 mg/g and (iii) Mg 1-09 mg/g. 



Cd induced vertebral deformities in A. commersoni 



391 



60 




/ 7 




Phosphorous 
( mg/g) 


Magnesium (mg/g } 




40 






2 










4 








^ 










/~7 


__ 













/ 












I x cf? 


_ __ 




in 










. 


'"~7] 






/ 






| 20 


H 




/ 




' 


1 






^ 


/~r 




2 


B 












1 






^ 








"c 













m 






/. 




frmy\ 




CD 












w/ 














u 












\\\y 














c 
o 




x 


x 


/ 




W 




/ 


y 


J 


Illll^ 





o 








20' 


Cadmium (pg/g) 

x 


'-ft 


Calcium (mg/g) 




c 




m 






'! 16 


_ 







160 


o 


y a 


', 


X 71 






| 12 


nTm 


1 


/ 




i 


fllff 


^y 1 


- 


120 


> 8 




f! 






^ 






X 

x 







80 






^ 






/ 






X 








4 


- 


x 
^ 


i 




xj 






^ 




~ 


40 







x 






X 

V 




^ 


x 
x 


j 




n 



Figure 

treated 



C 0-1 0-25 0-5 DF C 0-1 0-25 0-5 DF 

C - Control Exposure concentration (ppm) 

DF-Deformed fish 

1. Concentrations of Cd, Ca, Mg and P in the vertebrae of both untreated and 
fishes. 



3. Discussion 

In the present investigation vertebral deformities were observed in treated fishes only 
after the 89th day (caudal curvature) and 115th day of exposures (with vertebral 
damage) at a Cd concentration of 0-5 ppm (figure 2). Eaton (1974), Bengtsson et al 
(1975) and Muramoto (198 la, b) also recorded similar vertebral anomalies in Cd 
treated Lepomis macrochirus, Phoxinus phoxinus and Cyprinus carpio L. respectively. 
From the results obtained in this study it is apparent that more than 2/3 of the 
vertebral Ca, Mg and P were found to be eliminated in deformed fishes. These were 
lowered to a considerable extent in all Cd treated normal fishes. The results of the 
present study corroborate the findings of Muramoto (1981a,b) on Cyprinus carpio L. 

The caudal curvature and vertebral damages in A. commersoni might have 
been influenced by the loss of Ca, P and Mg from the vertebral column. The work of 
Matsunaga et al (1962) provides evidence to show the occurrence of as many as 72 
fractures in 'Itai-itai' patients affected by Cd food poisoning, which led to large scale 
elimination of minerals from the bones. Koyama and Itazawa (1977a,b) and Itokawa 
et al (1978) also noted vertebral deformation, vertebral mineral elimination and 
skeletal lesions in Cd exposed Cyprinus carpio L. and rat. In the present study, 
abnormal swimming behaviour, altered movement and flexibility of body of fish 
might have resulted from excess elimination of skeletal mineral like Ca, P and Mg. 

Larsson (1975) and Larsson et al (1981) have suggested that the vertebral 
demineralization in Cd-treated fishes might be mainly due to imbalance in Ca 



392 V Pragatheeswaran et al 






'- CM 

Figure 2. A. Normal fish. B. Vertebral anomalies with caudal curvature. C. Vertebral 
deformed fish. 

metabolism controlled by endocrine glands. In the present study, during Cd 
exposure, the excess (body-accumulated) Cd ions might have affected the Ca and P 
metabolism of thyroid and parathyroid functions which in turn led to vertebral 
mineral elimination. This aspect needs further study. 

Acknowledgements 

The authors are thankful to Dr C Ganeshprabu, Assistant Surgeon, Tanjore 



Cd induced vertebral deformities in A. commersoni 393 

Medical College, Tanjore for his assistance in x-ray photograph, to the Director, 
CAS in Marine Biology and to Annamalai University authorities for facilities 
provided. 

References 

Bengtsson B E 1974 Vertebral damage to minnows, Phoxinus phoxinus exposed to zinc; Oikos 25 134-139 
Bengtsson B E, Carlin C H, Larsson A and Savanberg O 1975 Vertebral damage in minnows, Phoxipus 

phoxinus L. exposed to cadmium; Ambio 4 166-168 
Couch J A, Winstead J T.and Goodman L R 1977 Kepone induced scoliosis and its. histological 

consequences in fish; Science 197 585-587 
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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 96, No. 4, July 1987, pp. 395-402. 
Printed in India. 



Distribution and population dynamics of soil nematodes in a tropical 
forest ecosystem from Sambalpur, India 

G B PRADHAN* and M C DASH 

School of Life Sciences, Sambalpur University, Burla 768 017, India 

* Present address: Department of Zoology, GM College, Sambalpur 768 004, India 

MS received 3 January 1986; revised 20 April 1986 

Abstract. An ecological study on soil nematodes was made in a tropical deciduous forest 
ecosystem. Seventeen species of nematodes were identified of which Rotylenchus sp. was the 
dominant plant parasitic form and Acrobeloides sp. was the dominant microbivore species. 
The mean annual importance value for the miscellaneous feeders, plant parasites, 
microbivores and predators were 57-5, 22-5, 12-6 and 7-4 respectively. The Shannon index of 
general diversity was maximum during February (1-73) and minimum during June (1-41). 
Nematodes were distributed in clusters resulting in so called pocket effect. Of the total 
nematodes 88-4% occurred in the top 10 cm soil during the peak period of density and the 
microbivores were more frequent in the top 5 cm soil due to the litter layer. Total nematode 
density ranged from 15-1 x 10 4 /rn 2 (May) to 66-1 x 10 4 /m 2 (November). Monthly mean 
nematode biomass was 18-86 8-36 mg dry wt/m 2 . Temperature, soil organic carbon and 
soil total nitrogen apparently played an important role in regulating the nematode population. 

Keywords. Soil nematode; forest ecosystem; importance value; diversity; organic carbon; 
nitrogen. 



1. Introduction 

Nematodes are an important consumer group in the decomposer subsystem of many 
ecosystems. Wasilewska (1970, 1971) in Poland, Yeates (1972, 1973) in Denmark and 
Johnson et al (1972, 1973, 1974) in USA have studied the soil nematodes in the forest 
ecosystems. However, knowledge on the ecology of soil nematodes from the forest 
ecosystems of India is non-existent. The results presented here on the diversity, 
distribution, population dynamics and biomass of soil nematodes of a tropical forest 
ecosystem in relation with climatic and soil factors are the first of its nature from 
India. 

2. Study site 

The study site forms part of Ushakothi National Wild Life Sanctuary located 60 km 
north-east of Sambalpur University Campus. The dominant tree in the forest site was 
Shorea robusta Gaertn. and during the rainy season there was a thick growth of 
seasonal plants like Croton sparsiflorus Morung., Euphorbia hirta L. and Hygropilla 
spinosa T. Anders etc. The soil was sandy clay loam and the pH was 5-82 in the top 
10 cm soil. 

The area experiences 3 seasons (rainy, winter and summer). The annual rainfall 
being 1290mm of which 88% rain fell during the rainy season. The minimum air 
temperature varied from 10C (December) to 45C (May). The average relative 

395 



396 G B Pradhan and M C Dash 

humidity was minimum during April (46%) and maximum during August (84%). The 
soil moisture ranged from 3*5% (May) to 18-8% (August). 

3. Methods 

At the study site a plot of 121 m 2 in area was taken. Mostly samples were collected 
from December 1980 to March 1982. Each month 7 sample units were taken and 
divided into 5 subsamples: 0-5, 5-10, 1.0-15, 15-20 and 20-30 cm soil layers. The 
samples, each 10cm 2 in area x 30 cm deep, were selected at random on a 1 x 1 m 
grid. 

For extraction of soil nematodes, about 250 cm 3 of soil for each subsample was 
processed by Cobb's (1918) wet screening and then by modified Baermann funnel 
technique (Southey 1970). Senapati and Dash (1976) had observed that 90% of the 
total soil nematodes were recovered by this method. Mishra and Dash (1981) also 
had followed this method while studying the soil nematodes of the rice field and 
pasture of Sambalpur. Samples were kept for 24 h on the funnel. Nematodes recove- 
red were classified into 4 ecological feeding groups (Banage 1963): (i) Plant parasites 
(PP), (ii) microbivores (MR), (iii) miscellaneous feeders (MS) comprising 
Dorylaimida-food varied and largely unknown, and (iv) predators (PR) mostly 
Mononchidae. Each month, afte'r counting, the nematodes were fixed with hot FA 
4:10. For the identification and biomass estimation permanent mounts were made 
from the fixed nematodes by glycerol ethanol method (Southey 1970). Total numbers 
of all the trophic groups/m 2 were calculated by taking all the subsamples of each 
layer into account. Maximum body width and length Were measured for the biomass 
calculation as per Andrassy (1956). To assess the relationship of soil nematodes 
with soil nutrients, the total organic matter and total nitrogen content of all the soil 
samples were estimated respectively by muffle furnace method (Paine 1971; Reiners 
and Reiners 1972) and micro-kjeldahl method (Jackson 1973). 

4. Results 

4.1 Diversity 

Table 1 shows the species diversity of the study site. Rotylenchus sp. was the domi- 
nant plant parasitic form found in every sample. Hoplolaimus seinhorsti Luc and 
Hemicycliophora oostenbrinki Luc were sometimes found in deeper soil layers in the 
rainy and winter seasons. Other plant parasitic species were seen occasionally. 

The microbivores were very large in number and Acrobeloides sp. was dominant 
occurring in every sample decreasing with depth. Other microbivore species were 
mainly found in the upper soil layers. The miscellaneous feeders were abundant 
throughout the year and 6 species were identified. The predators were very few in 
number. 

The importance value (IV) of the nematode trophic groups were calculated by the 
formula; 



Population dynamics of soil nematodes 397 

Table 1. Species diversity in the study site. 
Nematode trophic group 

Plant parasitic forms Rotylenchus sp. 

Hemicycliophora oostenbrinki Luc 

Hemicriconemoides cocophilus (L