First Ph.d. Thesis Work On Indian Horseshoe Crabs(merostomata:xiphosura).

STUDIES ON INDIAN HORSESHOE CRABS (Merostomata:Xiphosura)

WITH SPECIAL

REFERENCE TO ITS FEEDING BEHAVIOUR

THESIS SUBMITTED FOR THE DEGREE OF DOCT OR OF PHIL OSOPHY (SCIENCE) DOCTOR PHILOSOPHY OF THE UNIVERSITY OF CAL CUTT A CALCUTT CUTTA

RAKHAL DEBN ATH DEBNA M.Sc. (Zool.), NET, M.Phil. (Env. Sci.)

DEP AR TMENT OF MARINE SCIENCE DEPAR ARTMENT UNIVERSITY OF CAL CUTT A CALCUTT CUTTA 35 , B ALL YGUNGE CIR CULAR R OAD BALL ALLY CIRCULAR RO CAL CUTT A - 700019,WEST BENGAL CALCUTT CUTTA I N D I A

JUNE , 1992

Dedica ted to My Dedicated la te late Mother & Father

*** First Ph.D. work on Indian Horseshoe Crabs(June'1992)

© Dr. R. Debnath, 6th April' 2005

Electronic Publication

All rights reserved. No part of this Thesis may be reproduced, stored or transmitted, in any form or by any means, without the permission of the author.

C O N T E N T S PAGE NO.

1. 2. 3. 4. 5. 6.

Acknowledgement.......................................4 - 5 Preamble...................................................... 6 Historical Resume.......................................7 - 8 Physiography & Hydrology........................9 - 14 Methodology................................................15 - 19 Part-1. Studies on Population Aspects A. Geographic Distribution & Habitat..................................................20 - 25 B. Morphology & Morphometry...........................................................26 - 53 C. Absolute Population Estimates.........................................................54 - 83 D. Breeding Activity, Sex-ratio & Predation.......................................84 - 104 E. Epifaunal Associates......................................................................105 -114

7. Part-II. Studies on Food,Feeding & Digestion A. Digestive System............................................................................115 - 119 B. Gut Content Analysis.....................................................................119 - 122 C. Feeding, Digestive Physiology & Trophic Level...........................122 - 133

8. Part-III. Studies on Central Nervous System A. Anatomy of The Central Nervous System.....................................134 - 136 B. Cytomorphology of CNS ..............................................................136 - 142 (with Special reference to its Neurosecretory Elements)

9. Summary ...................................................143 10. References .................................................144 - 158 11. Published Papers (not included in this electronic edition)

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AC K N OWLE D GE M E NT I express my deepest sense of gratitude to my revered teacher , Prof. Dr. Amalesh Choudhury , Department of Marine Science , University of Calcutta ,for his learned counsel ,affectionate guidance , sustained interest and constant encouragement during the course of this investigation as also in preparation of the manuscript . My sincere acknowledgements are due to the authorities of the Susama Devichoudhurani Marine Biological Research Institute, Sagar Island , West Bengal , India for the invaluable facilities which I enjoyed during the investigation of my research project . I am fortunate to acknowledge Dr. B.K.Tikadar , the then Director , Zoological Survey of India , Calcutta , for his generous suggestions making an impetus to start the present investigation in the year 1984 . I am thankful to the Head of the Department of Marine Science , University of Calcutta for providing all sorts of laboratory and library facilities during the tenure of this work. I am also thankful to the Research Scholars of this department for their cheerful help and interest in course of this dissertation work . I would also like to extend my sincere thanks to Dr. P.S.Choudhury and Dr. D.K. Nanda of the Department of Zoology , University of Calcutta for their help during the study on neuroendocrine system and preparation of the manuscript incoroporated in Part-III of the present thesis . I would like to acknowledge my indebtness to Prof. Dr. Carl N. Shuster , Jr. , Virginia Institute of Marine Science , USA , for kindly offering me the benefit of his long experience with Limulus and pains taking review of my manuscript delt with breeding behaviour of Indian horseshoe crabs . I am also greateful to Dr. M.L. Botton, Fordham University , USA , and Dr. H . Sugita , University of Tsukuba , Japan , for providing me reprints , books and valuable suggestions during the entire course of the present investigation , without which I could not present this thesis . and I greatefully acknowledge the Department of Forest , Govt. of Orissa the authorities of Paradwip Port Trust for their help and co-operation in course of the study . I am thankful to Sri Jalad Gayen , Sri Jiten Singh , Sri Sadhan Sutradhar and Sri Sisir Kr. Roy for assisting me during coastal tours and field investigations . I also acknowledge Sri Siben DebSinha for his assistance in arranging the reference column . I must acknowledge the University Grants Commission and the Department of Environment , New Delhi , for financial assistance . This is also an occasion to acknowledge the Directorate of Higher Education , permission during the Govt. of Tripura , India for sanctioning my station leave preparation of this thesis paper . It is a great pleasure to me to express my heartiest thanks to Dr.R.K. Sinha , and Mr. S. Kundu, Kundu University of Calcutta , who helped tremendously to give a final shape of this thesis . Lastly I wish to record a deep sense of gratitude to my mother and also to my beloved wife for their good wishes and encouragement which always inspire me to go ahead .

Dated, the 8 th June,1992 CAL CUTT A , INDIA CALCUTT CUTTA

sd/- Illegible _____________________ ( RAKHAL DEBNATH )

Tachypleus gigas , with a bizzare appearance and horseshoe shaped body is one of the four extant species of the present world , basically unchanged for last 200 million years . The animal had noticed the drifting of Continental Shelf and the rising mountains . It roams along the coast line of Bay of Bengal sharing the space with other closest relative , Tachypleus ( = Carcinoscorpius , n. comb.) rotundicauda. For mankind , study of the hemolymph and eye of these has led to medical breakthrough . Care should be taken immediately to protect these unique and harmless marine creatures , considered now as the " threatened " and "endangered " species from the verge of extinction .

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PREAMBLE The horseshoe crabs or "King crabs" ( an erroneous term) are regarded as one of the most primitive animals existing in today's world , dating back to the period of 200 millons years . They are basically marine animals occurring on sandy and muddy bottoms from the shore lines to a depth of 40 meters . Of the four species known from the world , two species Tachypleus gigas (Muller) and Carcinoscorpius rotundicauda (Latreille) are known from the eastern cost of the Indian region with a distribution from Bay of Bengal to Malay archipelago or upto Philippines . Thease primitive animals have retained many of their interesting characters of related but extinct invertebrates e.g. , trilobites and eurypteryds and because of their morphological similarities with the fossil , Mesolimulus walchi (Stormer, 1952) they are called "living fossil" . Although horseshoe "crabs" present the arthropodal crab-like features , they are really not at all crabs , rather a close relative of Arachnida and thereby known as a "phylogenetic relic" (Selander et. al. , 1970 ; Riska , 1981). After latter half of the paleozoic era, horseshoe crabs showed morpholical features similar to those of present day species ( Fisher,1982,1984; Sekiguchi,1988). The horseshoe crabs have been exploited commercially by animal traders and quack medical practitioners (pic.at p.19), rural people in costal areas sell them as ornamental decorative pieces and many others . As a result , these animals have decreased considerably in number and completely disappeared from some of the earlier known areas of their occurrence . Despite the wide distribution of Asian horseshoe crabs , very little is known on the biology and natural history of the animals except the works of Japanese scientists (Sekiguchi , 1988) . little work has been done on the Indian horseshoe crabs until recently (Annandale , 1909 ; Roonwal , 1991 ; Rama Rao and Surya Rao , 1972 ; Debnath ,1985 ; 1987 ; 1991 ; Debnath and Choudhury , 1985 , 1987 , 1988 a,b,c; 1992 a,b; Chatterjee et.al. , 1988 ; Choudhury et. al. , 1991 ). It is generally known that the length of distribution of L. polyphemus in North America is almost identical with the length of combined distribution of three Asiatic species (Shuster , 1982 ). Although a considerable number of local or discrete populations exist in both Tachypleinae and Limulinae , the number of L . polyphemus has been estimated as 1,000,000 or more in Delaware Bay , USA ( Finn et. al. , 1990) . But no such attempt of censusing the horseshoe crabs in Indian Subcontinent ever made until recently ( Debnath and Choudhury , 1988c) . A greater number of earlier studies have attracted the scientific world there to select these curious and mysterious animals as the study materials for morphology , visual system , circulatory system , and more recently on the biochemistry of hemolymph fluid of the specimens (Shuster , 1978 ; Cohen et . al. 1979 ; Bonaventura et. al. , 1982 ; Novitsky , 1984 and Sekiguchi , 1988). The present study is amied at providing a broad base of vital information on occurrence , habitant requirements , food and feeding behaviour and population dyanamics ultimately leading to a conservation and management plan both by way of identified protected habitat area with maxiumum natural population .

7 HISTORICAL RESUME Out of only four extant species of horseshoe crabs in the present world , one occurs off the east cost of North America and the others in the coastal weather of South east Asia . Bacause of its limited distribution , the animal was recorded in ancient Chinese literatures (two old Chinese books like Wu Du Fu between 250 and 305 A.D. and Jiang fu between 276 and 324 A.D. and others ; c.f. ; Sekiguchi , 1988) but not in the European before the discovery of the New World . It is to be noted here that no such informations are available in ancient Indian literatures so far studied . However , according to Shuster (1953) , " the horseshoe crabs was well known to North American Indians who sometimes ate them , and often used to pointed tail to arm the tips of their fish spears . When the Europeans first came to the New World they were already familiar with the living animal , since the Asiatic species had been imported earlier from the Far East , as a curiosity , along with commodities of trade ' ............ was at first mistakenly identified as a crustacean by Europeans who called it the King crab." A persual of literature survey on merostomate studies conducted by Shuster (1988) reveals the total of 911 publications from prior to 1600 A.D. upto 1951 and thereafter , average 159 publications are added each decade bringing the total ranging between 1360 to 1500 . In recent years , however , most of the works are concerned with biochemical studies on horseshoe crabs , especially on its lysate . There are hardly 500 to 800 research publications out of the totals (1360 - 1500) , i.e. , only 50% delivery with the three different aspects like i) morphology including anatomy , histology and morphometry ii) natural history studies including ecology and ethology and iii) taxonomy , palaeontology and evolution . While the rest 50% accounts for biochemical and physiological studies . The limited scope of the present thesis work permits the author only to cite the following perusal on literature survey . Anatomy and external morphology of horseshoe crabs were worked out by several workers like Koons (1883) , Benham (1885) , Gravier (1929 a,b) , Shoji (1929 a, b ; 1932) , Hoeven (1938) , Snodgrass (1952) , Shuster (1960 b,1982) , Shuster and Horrel (1966) , Eldredge (1970) , Sekiguchi et.al. , (1976 ; 1978) , Sekiguchi and Nakamura (1979) , Riska (1981) , Yamamichi et.al. , (1983) , Lafon (1984) , Hammen (1986) , Debnath and Choudhury (1988b ; 1992a) , Clare et. al. , (1990) , Debnath (1991) , Shuster et.al. , (1992) . Studies on reproductive system and developmental biology were conducted by Benham (1984) , Kingslay (1892 ; 1893) , Iwanoff (1907;1933) , Baptist (1953) , Kobayshi (1957) , Sekiguchi (1960 ;1966 ;1970a ,b ;1973) , Brown and Humphries (1971) , Bennett et. al. ,(1972) , Brown (1976) , Sekiguchi el.al. , (1976a,b;1982 ; 1988) , Sugita and Sekiguchi (1979;1982 ; 1983 ;1984), Brown and Clapper (1980 ; 1981) . Whereas the anatomy of general digestive system ,its histology , feeding and digestive physiology have been studied by Schlottke (1934a,b and 1935) , Shuster (1948) , Smith and Chin (1951) , Smith (1953) , Botton (1981 ; 1982 ;1984a;b;d and e) , Tanimoto and Kondo (1982) , Botton and Haskin (1984) , Debnath and Choudhury (1987) , Yamashaki et.al. , (1988) , Debnath et.al. , (1989) .Sharrer (1941) , Bargman (1949) , Enami (1957) , Herman and Preus (1973) , Choudhury et.al. , (1991) had worked on the central nervous system and delt with neuroendocrinilogy of horseshoe crabs. But Barber (1956) , Barber and Hayes (1963) , Kaplan et.al. , (1976) and Wyse (1971) had worked out the functions of various chemo-as well as mechanoreceptors . The natural history of horseshoe crabs specially Limulus polyphemus including its ecology , life cycle and different behavioural aspects were conducted by various workers like Shoji (1927) , Goto

8 and Hattori (1929) , Asano (1942) , Roonwal (1944) , Dunton (1953) , Shuster (1953 ,1954 , 1958 , 1960a ; 1982) , Baptist et.al. ,( 1977 , 1982 , 1984 , 1985 , 1986 and 1987) , Sekiguchi and Nakamura (1977 ;1979) , Ireland and Barlow (1978) , French et.al. , (1978) , Rudloe (1978 ; 1979a,b ; 1980, 1981, 1982 , 1983) , Gomez - Aguirre (1979) , Rudloe and Rudloe (1981) , Barlow ( 1982 , 1983) , Cohen and Brockman (1983) , Chamberlin and Barlow (1984) , Debnath and Choudhury (1985 ; 1988b,c) , Botton and Loveland ( 1987 ) , Botton et.al. , (1988) , Brockman (1990) , Finn et.al. , (1990) , Ross - Farhang et.al. ,(1990) . Molting and other physiological works on Merostomata were conducted by a number of researchers like Fraenkel (1960) , Banner and Stephens (1966) , Robertson (1970) , Jegla et.al. ,(1972) , Page (1973) , Johansen and Petersen (1977) , Neff and Gaim (1977) , Laughlin and Neff (1977) , laughin (1981;1982) , Thys (1984), Sugita et.al. ,(1985) , McManus and Ricci (1989) . Molting and ageing (growth) , these two important aspects had been worked out by Bolau (1879) , Shuster (1954 ;1982) , Ropes (1961) , Shuster and Horcell (1966) , Jegla and Costlow (1970 ;1979a,b) Jegla (1972) . While , Bang (1956;1979) , Groff and Leibovitz (1982) ,Leibovitz (1986) , Leibovitz and Lewbart (1987) , Miyata et.al. , (1989) , Kawano et.al. , (1990) , Muta et.al. , (1990) were studied on different aspects of the disease and immunity problems . The geographic distribution of horseshoe crabs was studied by a number of workers e.g. Sewell (1912) , Waterman (1953 ; 1954) , Beamont and Mansueti (1955) , Shuster ( 1955 ; 1958 ; 1979 ;1985), Teale (1957) , Sekiguchi (1978) , Sokoloff (1978) , Sekiguchi and Nakamura (1980) , Sellenschlo (1983), Botton and Haskin (1984) , Shuster and Botton (1985) , Botton and Ropes (1987) , Debnath and Choudhury (1989) . Predation on horseshoe crabs was reported by Rudloe and Rudloe (1981) , Shuster (1982) , Botton (1984c) , Debnath and Choudhury (1988a) In recent years the most promissing field of research works on hoeseshoe crab's "blood" espacially its lysate may be reflected by the benefit of human being : Dores and Herman (1980), Pearson and Weary (1980) , Bishayee et.al. , (1980) , Dorai et.al. , (1981) , Srimal et.al. , (1985) , Roa and Bhagirathi (1989) and many others . Palaeontological works including phylogeny and evolution of Xiphosura , mainly , were of lves (1891) , Stormer (1952 ; 1955) , Selander and Yang (1970) , Selander et.al. , (1979) , Barthel (1974) , Eldredge (1974) , Fisher (1975 ; 1977 , 1982 ; 1984) , Sekiguchi and Sugita (1980) , Miller (1982) , Shishikura et.al. , (1982) , Sugita et.al. , (1982) , Miyazaki et.al. , (1989) . Literatures exclusively on horseshoe crabs have been published by several workers (Cohen et.al. , 1979 ; Bonaventura et.al. , 1982 ; and Sekiguchi , 1988) which shed light on better understanding of its biomedical , physiological and environmental research and also on general biology . Number of popular articles and monograph works also appeared time to time elucidating its importance to the welfare of human being [ Nishi (1975) ; Rudloe and Rudloe , (1981) ; Novtsky (1984) etc.] In fine , regarding the taxonomic position of horseshoe crabs , Linaeus (1758) , Muller (1785) , Latreille (1802) , Leach (1819) had given a shape of taxonomic position in their respective books of classification . But the most important contributions to the modern taxonomy were van der Hoeven (1838) , Dana (1852) , Willemoes-Suhn (1883) , Heymons (1901) , Pocock (1902) . Accordingly, the existing systematic position of hoeseshoe crabs is accepted as :

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Phylum : Arthropoda Sub-phylum : Chelicerata Heymons , 1901 Class : Merostomata Dana , 1852 Sub - class : Xiphosura Latreille , 1802 Order : Xiphosura Latreille , 1802 Sub - order : Limulina Richter and Richter , 1929 Super family : Limuroidea Leach ,1819 Family : Limuroidea Leach ,1819 Sub-family : Limulinae Leach ,1819 Genus : Limulus Muller ,1785 Species : L. polyphemus (Linnaeus ,1785) Sub-family : Tachypleinae Pocock , 1902 Genus : Tachypleus Leach , 1819 Species : T. gigas (Muller ,1902) Species : T. tridentatus (Leach ,1819) Genus : Carcinosoorpius Pocock , 1902 Species : C. rotundicauda (Latreille ,1802)

But the Japanese workers (like, Sekiguchi , 1988) have proposed for a new taxonomic system in the family Limulidae considering all sorts of biological works of horseshoe crabs i.e. , comparative anatomy, hybridization (cross breeding) , biochemistry , genetic studies and others . According to them the three Asian horseshoe crabs belong to the same genus , Tachypleus . Proposal for a new taxonomic position thus stands as follows : Family Limulidae Sub-family Limulinae Leach,1819 Genus Limulus Muller,1785 Species : L. polyphemus (Linnaeus ,1785) Sub-family Tachypleinae Pocock,1902 Genus Tachypleus Leach,1819 Species T. tridentatus (Leach ,1819) Species T. gigas (Muller ,1785) Species T. rotundicauda (Latreille ,1802) . n. Comb.

A Prehistoric progeny on the exposed sand flat, whose status is in RDB and soon be on..........CANVAS ! And, its fate is then, really depend on....................US !

10 PHYSIOGRAPHY OF THE STUDY AREAS

The eastern , western and southern cost of the peninsular India is ornamented by majestic rivers having extensive and highly productive estuarine areas . India's most of the largest rivers like Ganga , Subarnarekha , Mahanadi and Godavari etc. carry a vast amount freshwater to the Bay of Bengal passing through West Bengal , Orissa and Andra Pradesh , the three south-eastern costal states respectively . The Hooghly -Matla estuarine complex , marked as an important spot on the global map for its famous deltaic Sundarbans and luxuriant mangrove vegetation , is the richest and biggest in the world .This environmental complex supports a galaxy of benthic fauna of which horseshoe crabs are most conspicuous . o

o

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The Indian part of Sundarbans is located geographically between 21 to 22 30`N latitudes and 88 to o 89 29`E longitude . Its costal zone is in the form of low , swampy terrain characterized by extensive fluvio-marine plains with network pattern of creeks and parallel beach ridges with intervening estuaries and mudflats (Mangrove Report , 1987) . The total coast line from Sundarbans to Baitarani delta (Fig. 1& 2) of about 300 km , is inturruped by large estuaries , Hugli (= Hoogly) , Matla , Saptamukhi and Gosaba in 24 parganas , Rasulpur river in Midnapue of West Bengal , Subarnarekha , Burhabalaga , Baitarani and Mahanadi rivers in coastal Orissa . The eastern coast of West Bengal is characterized by sand and mud bottoms , while the coastal plains of Midnapur in West Bengal and Balasore , Cuttack and Berhampur districts of Orissa are characterized by the sandy shores mainly . But from the Godavari river mouth towards the south-eastern part of Andra its cost is characterized mostly field areas in regard to horseshoe crab studies is given below (Fig.1b):

A) Prentice island : o

It is an elongated and narrow , slightly bent , virgin island ; situated between the latitude 21 43` to o o o 21 46`N and the longitude 88 18` to 88 19` E in Sundarbans . This mangrove island is surrounded on all sides with the river Saptamukhi that has a link with Moorigonga via the Hatania - Doania Khal dividing Namkhana into two halves on the west . On the east and south east , there are two islands : Pathar Pratima containing a National Crocodile Project (established in 1982) at Bhagabatpur and the Luthian island with a bird sanctuary , respectively . Prentice island with its rich but shorter mangrove vegetations and numerous creeks and inlets provides a model ecosystem for the study of various micro-and macro-fauna including the horseshoe crab , Carcinoscorpius rotundicauda .

B) Sagar island : The largest delta in the western sector of Sundarbans , is situated nearly 85 km south of Calcutta in o o o o deltaic West Bengal . The island extends from 21 38` to 21 57`N latitude and from 88 2.35` to 88 11` E longitude and covers an area of about 235 km2 . This is a tide-dominated island where the tidal range varies from 5 to 6 mt. and the island is only 6.5 mts. above the sea level (Mukherjee , 1983). It is sorrounded by a large bodies of water , river Hoogly in the north and western side and river Mooriganga in the eastern side . The southern part of the island faces the open sea , Bay of Bangal . It has 12 tidal creeks amongst which the Chemaguri creek has a bound mud-flat enriched with artificial mangroves that support the lixuriant population of C . rotundicauda as the breeding ground .

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C) Digha : A prominent tourist place of Kanthi coastal plain of Midnapur district directly facing the Bay of Bengal . The micro-physiographic features of Digha are i) beach and runnel (channel) topography in the foreshore regions , ii) dunes and sand flats and, iii) tridal creeks , silt flat and marshy lands of east and west . The diurnal type of tridal action (two hige tides and low tides) wash the beach everyday with a cyclic of erosion and deposition . During the high tides the rolling waves transport maximum sediments and deposited in the back shore areas . The long and wide sea beach (slope measured 1.0 - 1.50 ) is interrupted by small and narrow tridal runnels by which tridal water enters the dune flats . Dunes are migrating in nature and to check the northern migration of the dunes the Government organaizations like Department of Irrigation and Forest planted Casuarina equisetifolia in rows , on the dune furface . Near the Subarnarekha delta and in the mouth of Talshari Khal , tridal creeks , marshy lands , mud and salt flats , and terraces are the important morphological features . The growing need of tourism causes serve human interference in to the beach and overfishing all among the shore line prevents the migration of Tachypleus gigas to its breeding ground situated at the shore and at present the natural population is so reduced that for collection of fewer horseshoe crabs one has to depend on the fishing net .

D) Chandipur : A recently growing se resort , situated in Balasore district , about 10 kms. away from the township . The surface of the Balasore costal plain is more or less terraced , the lowest terrace among the sea margin being impregnated with salt where the present Chandipur is situated . Probably the sea beach of Chandipur is almost flat one and widest (about 4 kms.) in the India with only 0.4 to 0.50 slope . Foredunes are developed on the margin of highest high water level . Water mass remains on the beach behind the sand bars even after receding of water during ebb tide . To the north-east of Chandipur , a short and narrow estuary , called the Burhabalange , is situated (Fig.2) . the estuary gradually becoming narrower the shore line was straightened and the river bank shifted south-west ; the successive positions of shift are marked by sand ridges of indefinite origin and small scraps . The mouth is marked by huge sand bars . Chandipur sesshore harbours a considerable population of Tachypleus gigas .

E) Dhamra : This estuary is an unique mangrove ecosystem , situated at the junctional complex between rivers Baitarani and Brahmani in costal Balasore of Orissa . The study area was at the north-eastern part of the main estuary . Wide mangrove land and mud-flat extended upto 5000 mts. down to the littoral zone . Slope of the beach was found 0.90 at every 1000 mts. (measured by Clinometer) i.e. the total slope , thus , stands 4.5 to 50 for the entire beach extended upto the lowest tide mark . Between infra- and supra-littoral zones , comparatively shorter mangrove plants , grasses etc. grow . This is the only place that that harbours both C. rotundicauda and T. gigas in the inlet creeks or on the mud flat . About 280 km. long coast line of Balasore , Midnapur and 24 Parganas (deltaic Sundarbans) are being eroded in different rates in different seasons and the rate of erosion is estimated to be higher in Digha , Junput , Sagar Island and Bakkhali than in the Balasore coast line (Paul , 1986)

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HYDROLOGY The aquatic environment of estuarine Sundarbans appeared to be more or less stable during January to June , when the salinity was highly fluctuating ranged from 8.2 to 28%0 as compared to the water temperature 20 to 340C ( Mangrove Report , 1987) . On the other hand , the sea shores of Digha and Chandipur , if compared hydrologically , featured an almost simillar aquatic environment where the salinity ranged from 19.0 to 31.0%0 with the temperature ranging from 24 to 350C . But in estuarine Dhamra and Mahanadi delta near Paradip port the salinity ranged between 10.5 to 29.0 %0 with the range of water temperature varied from 23 to 340c (Source : Marine Survey Deptt., Paradip Port Trust , 1985) ( Fig.1a.)

Fig.1a. 50

Hydroclimatograph Envelopes of the extremes in the salinity / temperature environment during the months of January through June at three coastal areas of India: o

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1. Estuarine Sundarbans (Mangrove Reports,1987) : 8.2 - 28.0 ppt & 20 - 34 C 2. Etuarine Dhamra and the Mahanadi Delta near o Paradip Port ( MarineSurveyDeptt.PPT,1985 ) :10.6 - 29.0 ppt & 23 - 34 C o 3. Sea shores of Digha & Chandipur :19.0 - 31.0 ppt & 24 - 35 C. 123456789012345678901234567890121 123456789012345678901234567890121

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Fig . 1b. Shows the coastal West Bengal (the Kanthi coastal plain west to the River Hugli = Hoogly and the estuarine Surdarbans to the east) with three study areas (A, B & C) : A. Prentice island , dots represent collecting sites, B. Chemaguri creek mouth (Sagar Island) and C. Digha sea shore .

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Fig . 2.

Shows the coastal Orissa with two study sites,where sampling was done ( A & B). A. The Chandipur sea shore (District Balasore) and B. Koith-kola mud flat at estuarine Dhamra (District Cuttack,).

15

MATERIALS AND METHODS:

PART I : STUDIES ON POPULATION ASPECTS A. Geographic distribution and habitat : 1. Areas surveyed : In search of horseshoe crabs during breeding season in the maritime states of Orissa specially at the coastal districts like Puri , Cuttack and Balasore , regorous field trips were conducted during the period , February to March , 1985 . Two more short surveys were conducted in May (at Chandipur , district Balasore) and June and July (at digha , Junput and Subarnarekha river delta) . Besides those , local tours were also conducted at every fullmoon tides at Sagar Island , Frazerganj , Canning and Gosaba regions of Hoogly-Matla estuarine complex . Every time , the author with field assistants visited lonely coasts , fishing villages , harbours or ports and tried to get reliable informations . In the next year (1986) , half yearly tours were also made to Prentice island (W.B.) successively from March , 1986 to August 1987 . The survey was also made at Dhamra (Koithkhola , district Balasore) twice in May ,1987 . 2. Methods of survey : Horseshoe crabs were found to breed on the littoral sand or mud beaches indiscriminately as discrete and isolated populations . Thus it was very tough to establish a particular population located during survey . However , two or three such specific fields were established where the horseshoe crabs found to venture regularly at spawning or breeding season . Local men called the "crabs", Ram-Lakhsman or simply Laxsmania in costal Orissa , while in West Bengal these bizzare animals popularly termed as Rajkankra (a Bengali word meaning "King-crab") . Living or dead specimens were collected and tried to confirm by asking or consulting local people , for the situation , if the specimens had actually inhabited there or had come from other places accidentally . When it was ascertained that horseshoe crabs were actually living in the area , and effort had been made to find eggs deposited on the sea shore by digging the mud or sand . B. Morphology and Morphometry : Total of 150 T. gigas (100 male and 50 female ) and 75 C. rotundicauda (39 male and 36 female) were collected from various field stations of costal Orissa and West Bengal for studying the comparative body weight , size and shape . eleven parameters including body weight , such as inter-ocular length (IOL) , dorsal prosoma breadth (DPB) , dorsal body length ( DBL) ,excluding the telson , ventral prosoma breadth (VPB) , telson length (TL) , its breadth (Tb) and heigth (FMH) , length of the moveable marginal spines (MSL) were considered for the study . All morphometric variables were expressed in nearest mm. except body weight (gm) (Fig. 17B) In a separate attempt , 23 mated pairs of T. gigas were collected on 8th March , 1985 from Chandipur sea-shore during the fullmoon high tide , between 10 AM to 12 N. The animals were readily segregated and the aforesaid parameters of individual males and females were recorded within three hours of collection . In this study , nine paired characters ( e.g. IOL , DBP, DBL , VPB , TL , CH , frontal length i.e. FL , FMH and RL as the distance between median tip of the prosoma and the base of chilaria) and two unpaired characters , e.g. PL (male clasper length) and Sl (the distance between dorsal opisthosomatic margin of female and prosomatic margin of male ) were considered for biometric analysis . Besisde the characters mentioned above , the total body weight and total egg -mass (EM) were also taken into consideration . In this context , it should be mentioned that random sampling

16 were done for measuring egg-diameter and calculating total egg-numbers . The length-weight relationship study was based on the data obtained from the total of 45 living C. rotundicauda comprising 28 males and 17 females collected from Prentice island during the period from February to September , 1986 . For the similar study , total 104 live horseshoe crabs belonging to the species T. gigas comprising 85 males and 19 females were collected from Digha sea shore , during the period of June to July , 1985 . In all the cases , total body length of the animals was measured from the tip of the cephalothorax over the mid-dorsal line down to the opisthosomatic end and the telson was excluded (expressed in cm.). All measurements were taken with standard scale or measuring tape , while the weights were taken with an automatic pan balance (Yamato , Japan) . Collections were restricted only to the breeding population which came ashore to spawn in the spring and summer months , ranging from March to July of 1985 to 1987 . Statistical methods like simple and partial correlations , ANCOVA , 't'-test , Chi-square test etc. used for morphology and biometric analyses were based on Bailey (1959) , Lewis (1966) and Snedecar and Cochran (1967) . The length-weight study was based on the basic principle of Le Cren (1951). C. Absolute population estimates : 1. Study areas : Chandipur (district Balasore , Orissa) and Digha (district Midnapur , West Bengal) ,two well known sea shores are recognised for horseshoe crabs' breeding and abundance (Roonwal , 1944 ; Panikkar ,1951 ; RamaRao and Surya Rao , 1972 and Debnath , 1985) . These places were selected for making-recapture studies . The two shores are regularly inundated by the high saline water (19.0 to 31.0 ppt.) of Bay of Bangal and regarded as the spawning ground for one of the two horseshoe crabs i.e. , T. gigas .Both these sea resorts receive fresh water from the river , Burabalanga at Chandipur and Talsheri Khal (Digha Khal) , and Subarnarekha at Digha (Fig. 1,2) . Horseshoe crabs were found to come at shores during fullmoon high tide of spring and summer months at Chandipur . But their inshore migration was interfered by 10-15 long tow nets each handled by 30-40 fishermen everyday at Digha coast . 'Crabs' collected from the nets were usually considered for making the present study . 2. Individual marking : Individual marking by mechanical means was done rather than the use of levels , tags , paints or dyes . The animals captured in pair or solitary were gently marked with steel-needle on their carapaces (dorso-lateral surface) as code numbers like 7/3-1 , 7/3-2, 7/3-3, ......... 26/5-1 etc. (date/month-sl.no.) . Both the male and female of the same mating pair were marked with similar codes (Fig. 19A, C). 3. Handling and release : Handling of horseshoe crabs is very much difficult; and precautionary measure was taken by wearing gloves in the hand . At Digha , total net collections of the day were taken and marked with code numbers on the dorsal-lateral surface of the animal carapaces . At Chandipur handling of horseshoe crabs was rather more difficult . Sufficient care was taken at the time of capture and making so that the horseshoe crabs felt minimum disturbance and they were released at the end of each operation during low tide at discrete locations . The horseshoe crabs damaged by predator crows , Corvus splendens L. ( Debnath and Choudhury , 1988a) or heavily infested with various of epifauna were removed from the primary capture-stock and these were rejected .

17 4. Line-transect method for population estimation : Number of T. gigas was counted in eight random transects measuring 10 X 10 sq. mt. within an area of 1 sq. km. at Chandipur sea shore (Table 18). Such studies were conducted from 20th to 31st March of 1986 and the total population was calculated following usual line transect method (Andrewartha , 1970) and the figures were expressed in two ways : i) in terms of days and ii) in terms of area (toa) . 5. Population estimation by Regression methods : The results of estimated population (N) , number ofcaptured (C) and recaptured (R) animals derived from Jackson's method (1939)for Chandipur sea shore (March to July , 1985) and Jolly-Seber's method (1965) for Digha sea shore were transformed into logarithms . Finally , the transformed values of N , C and R were correlated and regressed by the following statistical methods of Lewis (1966) and Bailey (1959) . D. Breeding activity ; sex-ratio and predation : Breeding activity was observed in the feilds . Mechanism of pairing (making amplexus i.e. , grasping the famale's opisthosomatic marginal spines with the modified clasper legs of the male ) , movement , burrowing and dispersal activities were noticed in both C. rotundicauda and T. gigas at five selected field stations of Prentice island , Chemaguri mud-flat (Sagar Island) , Digha sea shore , Chandipur sea shore and Koith-kola (Dhamra mangroves). Total males and females were counted to determine the sex-rations . Marking with steel-needle and tagging with colour rubber bands (in the telson-trunk) were done in both species to demonstrate the spawning movement along the Bay shores . Active predation of the horseshoe crabs by crows, Corvus splendens was an accidental observation during the study to the breeding activities of T.gigas . In order to study the effect of predation on the sex-balance , capture-recapture method was employed at Chandipur (in addition to get the population estimate) . On many occasions dead or moribund 'crabs' , especially the females , were found long after the days of release . After the capture - recapture method has been performed the author visted the beach again and found a huge number of dry carapaces (females mainly) in which some of them bore the previous needle markings . From the whole data the approximate population size and sex-ratio were obtained . At Digha , horseshoe crab population did not come across the tidal edge to lay their eggs due to human interference . They were caught during fishing and the fishermen threw them on sand believing them as disturbing elements . Crows , thus , got access to the crabs very easily at the exposed beach . Day to day collection was sorted out to estimate the sex-ratio of the crabs , and at the same time dead crabs (dry shells) were also collected at random to determine the sex-ratio . E. Epifaunal Associates : Ecto-commensals or epifaunal organisms were collected from dorsal as well as ventral surface of T. gigas and C. rotundicauda in spring and summer months ( from 1985 to 1988 ) at five coastal stations viz. Prentice island , Sagar Island and Digha of West Bengal ; and Chandipur and Dhamra of Orissa . Among the ecto-commensals barnacles constituted the major part . Only noticeable epifaunal organisms (at least a colony of barnacles) were taken into consideration to certify a horseshoe crab as infested . The ectocommensals were scrapped off the shells of 50 highly infested horseshoe crabs (32 T. gigas and C. rotundicauda ) and transferred in 70% alcohol for further population study (

18

PART II : STUDIES ON FEEDING BEHAVIOUR ; FOOD , FEEDING AND DIGESTION A. Gut content analysis : Seventy two adult horseshoe crabs ( T. gigas) comprising 42 solitary males , 15 mated males and 15 mated females from net collections at Digha sea shore were considered for gut content analysis during May 1985 . The 'crabs' were primarily anasthesized within half and hour after collection , by injecting 10 % formalin in the cephalothorax . By next 30 minutes the animals were sacrificed to remove the entire guts (gastro-intestinal tracts) from the body , preserved in 3.7% sea water formalin for 12 hours (as the preservative makes the gut hardened) and then transferred to 70% alcohol . Later in the laboratory, the guts were measured (length , diameter and weight) , and dissected out the gut contents . The cleared guts after the removal of contents were again weighed . The contents were preserved in small glass-vials containing 70% alcohol . Samples of gut contents were examined under a stereoscope (Olympus) . Counting of prey items and grouping them upto the lowest possible taxa were done following the Botton's technique (Botton , 1984a) . The food matters , whether partly or fully recovered , were sketched and drawn after camera lucida for determination of probable size and shape according to scale (mm.) In similar way C. rotundicauda obtained from Chemaguri mud-flat , Prentice island and Dhamra , and T. gigas obtained from Chandipur and Dhamra of Orissa were also sacrified to identify the food-matters (Fig. 32A) . B. Digestive physiology in context to prey items : For quantitative estimation of food ingested , ten mating pairs of T. gigas were considered . The amplexed pairs were obtained from Digha sea shore in May 1987 and they were readily segregated and primary preservation in the feild was done by injecting 10% formalin in the ventral part of cephalothorax . As described above each of the 20 samples (10 male + 10 female) was examined following the procedure developed by Botton (1984a) . Another batch of 10 pairs of T. gigas of almost similar size were chosen for enzymatic study , sacrificed after capture , in the laboratory . The gut was flushed with distilled water and three regions of the digestive tract dissected out-oesophagus , gizzard and stomach intestine , together with the digestive gland (hepatopanereas). Crude enzyme homogenates were prepared with glass distilled water , in mechanical homogenizer at 40c . The resultant supernatant was selected as crude enzyme extract for carrying out biochemical studies . Proteolysis (both acidic and alkaline) were measured following Ichishima (1970) using 2% casein as substrate at pH 2.5 and 8.0, respectively ; esterase action on 0.01 % beta naphthyl laurate at pH 7.4 seen according to Seligman and Nachles (1963) ; cellulolysis using 1% carboxymethyl cellulose at pH 5.5 as substrate by the method of Kesler and Tulou (1980) ; invertase action on 2.5% sucrose at pH 5.5 following Pal et. al. , (1980) and amylolysis following Bernfeld (1955) using 1% amylum at pH 6.9 .Total water soluble protein content of extract was measured according to Lowry el. al.,(1951) .

19

PART III : STUDIES ON ANATOMY AND CYTOMORPHOLOGY OF THE CNS WITH SPECIAL REFERENCE TO ITS NEUROSECRETORY ELEMENTS Both sexes of adult horseshoe crabs , C. rotundicauda , were collected from Chemaguri mud-flat of Sagar Island (West Bengal) during the months of May to June 1988 . Fifteen animals having carapace width of about 152.0 to 210.0 mm. for 8 males (weigth ranged between 147.0 to 378.0 gm.) and 182.0 to 225.0 mm. for 7 females (weigth ranged between 305.0 to 629.0 gm.) were sacrificed following the procedure of Lochhead (1950) for both anatomical and cytomorphic studies on the Central Nervous System (CNS) . Different parts CNS were fixed in aqueous Bouin's fluid . The tissues after dehydration were embeded in paraffin (56-580C) and serial frontal sections (15 mm.) were treated with Haeidanhain's Azan (Gurr , 1959) , Chromealum haematoxylin pholixin (modified by Bargmann , 1949) and Masson's trichrome staining techniques (Gurr , 1959) .

Fig.2a. Selling boiled juice with mustard oil extracted from horseshoe crabs has been a common practice in Bengal..........is not this e ild-life against T he W ild-lif

( Protection ) Act !!!

20

PART - I STUDIES ON POPULATION ASPECTS

21

A . Geographic distribution and habitat

INTRODUCTION Of the 4 extant species , the American horseshoe crab , Limuluspolyphemus occur in the Atlantic coast of North America , widely distributed from Maine to Yucatan (Shuster , 1953 , 1954 , 1957 , 1979) . The other three Asiatic species , Tachypleus tridentatus , T.gigas and Carcinoscorpius rotundicauda occur in the Indo-Pacific coasts . T.tridentatus is found in western and southern Japan , Taiwan , Philippines and north Borneo (Pocock , 1902) . T. gigas and C . rotundicauda are found on the coast of India , Bangladesh , Myanmar , Thailand , Malaysia , Philippines and Borneo (Annandala , 1909 ; Choudhury & Hafizuddin , 1980 ; Pocock , 1902 ; Smedly , 1929 ; Sewell , 1912) . T. gigas was also recorded from Torres Straits (Pocock , 1902) . An extensive survey report of Sekiguchi and Nakamura (1980) confirms that 1) all the 3 species are found sympatrically in the north-western coast of Borneo , 2) two species , T. gigas and C.rotundicauda , are found along the southern coast of Borneo , the northern coast of Sumatra , the coast of either sides of Malay peninsula and that of West Bengal (digha , Canning , Kkdwip , Freserganj and Junput) and 3) a combined distribution of T. tridentatus & C. rotundicauda was found along the southwestern coast of Philippins & Palwan Islands . Thus the area of distribution of the three Asiatic species is surrounded by 4 lines drawn from the western part of Japan to West Bengal , from West Bengal to Java , from Java to Torres Strait , and from Torres Strait to the western part of Japan (Sekiguchi & Nakamura , 1980) . However , no precise information about the distribution of Indian horseshoe crabs , T. gigas and C.rotundicauda is available ( Sekighchi et. al. , 1976 , 1978 , 1979 , 1980) excepting some scattered reports (Annandale , 1909 , 1922 ; Panikkar , 1951 ; Pocock 1902 ; Rama Rao & Surya Rao , 1972 ; Roonwal , 1944) . The present chapter of the dissertation deals with a detailed survey to record the geographic distribution of each of the two Indian species in their own territory i.e. in the coasts of Bay of Bangal (Debnath & Choudhury , 1989 ).

22 Table 1a Number of animals observed in different lacalities during the period February to August , 1985 . Areas surveyed Couple

* Chilka

1 5 12 7 320 19 5 1 -

Astarang Paradip Jambu Dhamra Chandipur Digha Sagar Island Frazergaunj Canning Gosaba Total

374

T. gigas Male Female 2 1 4 3 6 13 112 2 -

1 -

153

1

Dead carapaces 8 2 4 154 78 3 249

Carcinoscorpius rotundicauda Couple Male Female

Dead carapaces

2 1 1 1 3 7 21

1 1 3 1 2 1 9

-

4 1 2 1 2 3

37

18

-

13

* Reported and collected by a Telegu fisherman of a deep sea fishing trawler at Paradip port .

Table 1b Number of animals observed in three lacalities during the period March 1986 to May 1987 .

Areas surveyed

Tachypleus Couple

0Œ

gigas

Õ +

Carcinoscorpius rotundicauda Dead Carcs.

Couple

0Œ

Õ +

Dead Carcs.

Koithkola (Dhamra,Orissa)

44

27

5

35

50

16

1

34

Digha(Kanthi,W.B.)

5

75

3

35

-

2

-

-

Prentice Island (South 24 Parganas, West Bengal)

2

-

-

-

10

18

1

3

51

102

8

70

60

2

37

+ Total

36

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C. rotundicauda (

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ASTARANG

COMMENT If one walked or were able to walk the entire coast line, from Astarng to to around the Sundarbans, obviously, there would be an appreciable number of horseshoe crabs, at other sites too. ( Courtsey: Prof. Carl N. Shuster,Jr. 1993)

Fig . 3.

The map showing the geographic distribution of Indian horseshoe crabs , Tachypleus gigas and Carcinoscorpius rotundicauda in coastal Orissa and West Bengal , India .

E

24 RESULTS AND DISCUSSION: Localities of animal distribution and species occurrence are shown in fig.3. The investigator has shown the sympatric distribution of the two Indian species , T. gigas and C. rotundicauda in the Indian coastal region in Table 1. The coastal area of Orissa is discontinuously populated with horseshoe crabs , the most dominating species being T. gigas and C. rotundicauda , on the other hand , the 2nd species dominates the Sundarbans and Dhamra estuaries . Like Limulus polyphemus , the Indian horseshoe crabs are also "intermittently distributed" i.e. they are not evenly distributed. Here aslso they occur in or near the major estuarine river mouths situated at coast of Bay of Bengal. The occurrence of horseshoe crabs along the coast of Chilka-lake (Puri district) was confirmed by the information and specimens obtained from a fishing trawler at Paradip port . Except this , everywhere in coastal Orissa the investigator had encountered horseshoe crabs at field spot. For instancce, the author discovered a mating couple of C. rotundicauda , 8 to 10 inch. below the mud flat of Mahanadi river mouth at Paradip (northern side) on 28th February 1985 . Again two couples of T. gigas were recovered from the Sandy beach of Mahanadi river mouth (southern part) the following night (not shown in the map). At Chandipur (district Balasore) more than 300 mating couples were captured in an area of 2 X 3 sq. km. , a flat stretch of sand bottom . Similarly at Digha , Junput , Sagar Island , Frazerganj , Prentice island , Gosaba and in and around port Canning the author came across a number of horseshoe crabs C. rotundicauda , in the mixed population. It was found to predominate both Sundarbans of West Bengal and Dhamra estuary of Orissa (Table 1) . Horseshoe crabs are purely marine dwellers periodically venturing the estuaries (Shuster , 1960a). The notion of Limulus as a marine species has been superceded by further observations (Shuster, 1979) Furthermore, "while limuli are most often found in the more saline portions of (and on the continental shelf),, they have euryhaline tendencies ..........." (Shuster,1982, p.33). It would seem that C. rotundicauda and Limulus may have a different pattern of distribution in relation to a salinity gradient than do the two species of Tachypleus ( see. the Clmatographs or Climographs of fig.1a as was constructed and provided by Prof. C.N. Shuster,Jr. The climograph envelopes can be further refined into salinity / temperature polygons(Shuster,1990).All the 4 extant species live in the shallow sea bed or a mouth of river with bottom of muddy sands. C. rotundicauda are found not only in the sea shore but also in the brackish water zone of the upper stream of a river (Sekiguchi & Nakamura , 1979) . Annandale (1909) recorded C. rotundicauda in the river Ganga near Calcutta 90 miles north from Bay of Bengal . Sekiguchi and Nakamura (1979) collected the same from a river , about 4 km. away from the sea in Thailand . During the present survey along the coastal Orissa , a single male specimen of C. rotundicauda was collected at Chandbali , river Baitarani , about 60 km. away from the sea . Usually the fishermen catch hundreds of specimens of C. rotundicauda about 50-100 km. interior from the estuarine Sundarbans (port Canning) and sell them to the quaks ( Rama Rao & Surya Rao , 1972 ; Debnath 1987, Fig.2a) . T. gigas, in most cases , was obtained from the hard sandy beach facing directly to the sea . There is a question whether T. tridentatus shares the habitat with T. gigas or C. rotundicauda . It has been found that T. tridentatus shares the habitat with T. gigas in northern Borneo (Pocock , 1902) , but no record in sharing with C. rotundicauda . Sekiguchi & Nakamura (1979 , 1980) showed the western limit of the distribution for T. gigas and C. rotundicauda in the Indian coast , but that of T. tridentatus was not very clear . Our survey report of 1985 to 1987 revealed that T. tridentatus actually did not occur in Indian coast (Debnath and Choudhury , 1989) . There is a report that this species was collected in Vietnam (Waterman , 1953) . The present studies confirms that the western limit of distribution of the two horseshoe crabs upto the coastal zone of West Bengal and Orissa through

25

Bangladesh (Choudhury & Hafizuddin , 1980 , Debnath & Choudhury 1989 , Sekiguchi & Nakamura , 1978 , 1980) .The sympatric occurrence and wide range of distribution of three Asiatic species in the Indo-pacific seas will provide an important clue to the evolution of these three species (Sekiguchi and Nakamura , 1980) . Ives (1891) first compared the distribution of 4 extant species , along the Atlantic coast of North America and Indo-Pacific coastal water , with the fossil specimens of Limulidae in the Triassic , Jurassic , Cretaceous and Oligocene beds of Europe and Syria and suggested that the Mesozoic waters of Europe were the probable centre of dispersal of the ancestors of the present species . Distribution of the fossil and living species suggests that Limulidae have always been tropicotemperate species , restricted almost entirely to the Northern hemisphere and then L. polyphemus migrating westward and T. tridentatus , T. gigas and C. rotundicauda eastward . Thus none of the 4 extent species were derived from the other , but from a common European ancestor or separate ancestral species (shuster, 1957 ,1960b,1982). In addition to geographic ranges of comparable extent , the 4 species have so similar in ecology , morphology and serology that they probably could be accommodated in one genus (Shuster , 1962 , 1982) . From ecological point of view , the present day distribution and extent of horseshoe crabs can be defined chiefly by the water temperatures , salinities and depths of habitat . Temperature appear to be the limiting factor for the northern ranges of L. polyphemus and T. tridentatus . In the south , temperature seems less likely as the limiting factor , at least for T. gigas and C. rotundicauda (Shuster , 1982) . Amongst the three Asiatic species , only C. rotundicauda is found more adapted to a wide range of salinity than T. tridentatus and T. gigas . This is evidenced by the occurrence of T. gigas mostly at the sea shores directly facing the high saline sea-water regime and C. rotundicauda in the brackish water estuaries and river mouths of Subarnarekha , Mahanadi and Baitarani .

26

B. Morphology and morphometry

INTRODUCTION

Merostomate morphology has long been a fascinating subject for the zoologists and paleontologists . Most of the morphological works on extant horseshoe crabs , particularly , Limulus polyphemus , have already been done (Lockwood , 1870 ; Koons , 1883 ; Pomerat , 1933 , Shuster , 1955/ 58 ; Cohen and Brockman , 1983 ; Brockman , 1990 and Shuster et.al. 1992) . Among the three Asiatic species of horseshoe crabs , Tachypleus gigas was studied well (Pocock , 1902 ; Smedley , 1929 ; Sekiguchi et.al. 1976) . Morphological variation in Carcinoscorpius rotundicauda has also been worked out as was observed in T. gigas (Sekiguchi et.al. , 1978) . Therefore , a perusal of literature survey reveales that except for a few scattered reports (Annandale , 1909 , Roonwal , 1944; and Rama Rao and Sury Rao , 1972) no morphological work has been done on the Indian horseshoe crabs untill 1980s (Debnath , 1991 ; Debnath and Choudhury , 1988b , 1992a ; Chatterjee et.al. , 1988) . The present chapter deals , moderately , with the morphology , sexual dimorphism and biometry of breeding populations including length-weight relationship of the two species , T. gigas and C. rotundicauda .

T. gigas in dorsal view

C. rotundicauda in ventral view

27 OBSERVATION AND RESULTS (a) General organization : In reneral body plan , the structure is covered by a tough chitinous exoskeleton . The figures show the following divisions of the body : 1. a prosoma or cephalothorax , bearing the appendages around the mouth (Fig. 16B , D and 17A) 2. a mesosoma or pre-abdomen , bearing the genital operculum and the gill flaps ; 3. a metasoma or post abdomen , which is devoid of appendages ; and 4. a telson , which is long and spine like ( Fig. 4&5) . The mesosoma (2) and metasoma (3) are fused together into an opisthosoma . Six movable spines on each side of opisthosoma mark the lateral margins of the somites of the mesosoma . The opisthosoma is jointed both with the prosoma and with the telson . The body is concave below and the whole carapace is covex dorsally giving a horseshoe shape . [ The mouth is mid ventral in the prosoma , between the bases of the walking legs (five)]. there are two groups of ventral appendages : a) Six pairs of tubular prosomal appendages - the first pair is chelicera and the rest five pairs of walking legs , representing the appendages of 1st to VIth somites , and b) Six lamellate plate like abdominal appendages , the first one is comparatively tough , lacks , gills , forming a gential operculum bearing gonopores on its posterior wall , while the rest five pairs are connected by a median membrane and each support about 150 gill lamellae . These are simply gill lamellae forming book-gills and comparable with the book lungs of arachnids . Thus , all those 6 abdominal appendages represent the fused segments of VIII to XIII . The rudimentary chilaria (of segment VII) resemble the prosomal coxae . The pre-oral chelicerae have three segments , the distal one chelate . The spined gnathobases of the 5 pairs of walking legs are arranged almost radially around the mouth (Fig. 5 , 17A) . (b) Species identity : T. gig as : Both male and female appeared deep greenish in dorsal shell colour , with a gigas

triangular and dorsally crested telson (Fig. 18C) . In both the sexes the opisthosomatic lateral spines are 6 in number , but all are long and more or less equal in males , while in females the first three are long and the rest three are short . A movable spur was present on the 4th segment of the sixth prosomatic appendage which might be for burrowing purpose (Fig. 18B , D) . Inner branches of the gential operculum were not extended distal to the tips of outer branches (Fig. 18C) . Dorsal surface of either sexes were found more spiny than C. Rotundicauda. Note that no juvenile females have movable marginal spines on the opisthosoma, if present more or less of equal size. C. rrotundicauda otundicauda : Animals of eigher sexes looked brownish black or deep greenish black in appearence , with a rounded non crested (i.e. smooth) telson (Fig. 16B) . Lateral opisthosomatic spines were all short and 6 in number (Fig. 16D) . No movable spur was present on the 4th segment of the sixth prosomatic appendage (Fig. 18C) . Inner branches of the genital opercullum were extended distally upto the tip (Fig. 18C) . Dorsal surface of both sexes were less spiny than T. gigas (Fig. 16C) . (c) Sexual dimorphism : T. gigas : The male was comparatively smaller than the females . All the 6 opisthosomatic spines were long in males , while the first 3 long and the rest three were short in females , fourth and fifth were being used by the male for clasping . The 2nd and 3rd prosomatic appendages were modified as clasper organs in males , that is , the terminal portion (tarsus) of the prododite was atrophied and

28 lost, and only the dactylopodite was present . Thus it looked nonchelate and hook-like (Fig. 18B) . But in females these two appendages were chelated and looked like normal walking legs (Fig. 18B) . C. rotundicauda : The male was smaller than the female in comparatively body size . All the 6 opisthosomatic marginal spines were short with the second and third spines slightly longer than the rest . The 2nd and 3rd prosomal appendages of the male showed well developed terminal portions which were atrophied in Limulus and Tachypleus . This terminal portion , together with dactylopodite , formed a chela which looked stouter than the chelae of other appendages (Fig. 18D) . These clasping organs were conspicuous and black in colour . (d) Statistical morphology : Table 2 shows the comparative body weight (wt) , size and shape comprising five major morphometric measurements e.g. IOL , DPB , DBL , VPB and TL of T. gigas collected from Digha and Chandipur and C. rotundicauda collected from Prentice and Sagar Islands . Data include population number (N) , mean value (X) and standard deviation (SD) for each measurement . Table 3 gives the pool data comprising 100 T. gigas and C. rotundicauda in a comparative manner . Excluding the body weight , in this table 3 morphometric variables were considered , with only the range and mean value of respective characteristic . The proportionate means values (i.e. , the male to female mean ratio are presented in table 4 which will determine the relative size of male to female . Simple and partial correlation coefficients were worked out considering only four major morphometric variables e.g. 1 (IOL) , 2 (DPB) , 3 (DBL) and 4 (VPB) , These four measurements were taken from 90 males plus 19 females of T. gigas and 28 males plus 21 females of C. rotundicauda (Table 5) . The results of simple and partial correlation computation are presented in table 5 . (e) Biometry of mating in T. gigas : A summary of nine paired and two unpaired morphometric measurements , body weight and weight of the egg mass for the twenty three mated pairs of T. gigas have been presented in Table 6. In every morphometric consideration the female was considerably larger and in no case did the measurement of the male appear greater than or even closely coincident with a similar female measurement . Fig. 8 shows graphically that there was some overlapping in the ranges of only two measurements : RL and FMG . Most of the characters , however , were discrete e.g. body weight IOL , DPB , VPB , DBL and FL. Only the lower end of the range of measurements for females began at the top end of the male range so for tail length (TL) and central hight (CH) are concerned . It was also characteristic that the range of VPB , FL and RL seemed to be equal size , while the range of male TL was greater than the female range . Greatest variation in range and distance were being displayed by the Wt. class : the female range was very much higher than the male range . None of the males of the body dimensions of the sexes overlapped or occurred at the same point . Further , in no instance did the mean coincides with the mode and all comparisons between male to female means showed ( t-test) high difference in significant at 0.001 level . Proportion of male mean to female mean showed 0.720 to 0.821 (average 0.758) , while for weight alone it was 0.349 . Correlation and regression relationship were computed for mine paired characters (Table7). In addition to those relationship between weight , DPB (Male) to SL (Female) and DPB (Male) to EM (Female) also were computed . Amongst these all , only CH , FMH and RL showed significant correlation coefficients . In the regression analysis the female measurements assigned along the abcissa (X) while the male components were placed on the ordinate (Y) . High regression coefficients were

29 obtained only in the case of FMH (tanè = 25.260, b = 0.472) and RL ( tan è = 33.660, b = 0.662). Since dorsal prosoma breadth (DPB) and dorsal body length (DBL) have been found to be the most highly correlated morphometric features in all Indian horseshoe crabs (Debnath 1991) , these two were selected along with body weight (Et.) to arrange the mated animals in tabular forms for chi-square testing (Table 8) . Theoritical frequencies are shown in parentheses , while the actual ones are on their left . Since the expected frequencies (in parenthesis) in most cases were found to be less than 5, the 2 x 2 contin2 gency X -test could not be done (Baily , 1959) . This was due to a seemingly smaller population (only 23 pairs of T. gigas ) considered for the study . Otherwise the results should bear some significance . (f) Length - weight relationship : The study of length-weight relationship provides a mathematical correlation between length and weight may be converted , one to the other, in future cases where only one of the parameters is known .The method has been found to be of value in measuring variations in the expected weight or length of an animal, particularly fish . In fish, such correlations have been used in examining indications of fatness, gonad development, general well-being and other parameters (Le Cren , 1951) . McMahon ( 1973,1980) has pointed out that this unique morphometric relationship may provide an important biological information and Peters (1983) suggests that it could led to the formulation of a prospective biological theory .The application of this method to the Indian horsehoe crabs is explained below ( see also Fig.7 ) . Parabolic Equations T . gigas: : W = 0.03748 L 3.08992

Log W = -1.42611 + 3.08992 Log L .

L 2.52090

Log W = -0.63230 + 2.52090 Log L .

C. rotundicauda: Males : W = 0.4116 7 L 3.09554 Females : W = 0.43376 L 2.35312

Log W = -1.38546 + 3. 09554 Log L . Log W = -0.36275 + 2.35312 Log L .

Males

Females : W = 0.23317

Logarithmic Equations

Analysis of covariance (ANCOVA) revealed no significant difference (Table -9) between regression lines of both sexes . So a common regression line for the two sexes obtained from the pooled data as below : T. gigas C. rotundicauda

: Log W = -1.66218 + 3.29953 Log L . : Log W = -1.72704 + 3.39719 Log L .

The above common logarithmic equations have got justified values i.e. F = 0.57839 ; df. 1,100 , P>0.05 for T.gigas F = 1.23589 ; df. 1,41 , P>0.05 for C. rotundicauda (Table -10) Therefore , the common parabolic equations for respective species are found as T. gigas : W = 0.02176 L 3.29953 C. rotundicauda : W = 0.01874 L 3.39719 Table-11 and Table-12 represent the t-test of regression lines and t' -test between regression coefficients of either sexes , respectively .

WT. 1. IOL 2. DPB 3. DBL 4. VPB 5. TL

175.56 72.81 165.72 152.18 144.75 126.39

29.95 2.75 6.18 6.45 6.09 10.01

SD

DIGHA

19 19 19 19 19 19

475.26 98.24 215.28 205.62 178.13 172.45

Female N X 58.34 5.43 9.82 8.63 5.80 11.45

SD 30 32 32 32 30 30 178.63 72.16 162.92 150.18 143.65 128.31 31.39 3.92 6.64 5.08 6.48 9.62

CHANDIPUR Male N X SD 30 23 23 23 30 26 511.33 96.71 219.82 204.65 179.35 173.18

Female N X 57.88 5.62 11.84 13.92 8.21 14.66

SD 28 28 28 28 28 24

238.75 73.81 172.35 163.05 147.84 154.68

37.73 2 1 4.82 21 14.81 2 1 9.12 21 7.83 21 11.64 2 0

446.71 88.729 194.51 186.15 169.05 173.70

PRENTICE ISLAND Male Female N X SD N X

92.99 8.14 9.34 12.64 11.20 11.18

SD

18 18 18 18 18 17

235.22 75.68 179.15 168.34 153.74 153.48

34.57 2.78 11.42 7.43 9.68 9.34

12 12 12 12 12 12

N stands for the number of individuals measured; IOL = interocular distance ( not length ) and DPB = width or breadth of prosoma measured along the curvature of the carapace, but not "dorsal prosoma breadth"-----------as suggested by Prof. Carl N. Shuster,Jr. ( 1993 )

Note:

456.48 88.42 200.18 191.42 171.67 176.74

85.34 3.85 9.83 11.34 10.91 9.54

SAGAR ISLAND Male Female N X SD N X SD

Carcinoscorpius rotundicauda

IOL , inter-ocular length ; DPB , dorsal prosoma breadth ; DBL , doesal body length ; VPB , ventral prosoma breadth and TL , telson length .

90 90 90 90 90 28

N

Male X

Tachypleus gigas

and standard deviation ( + S.D.) , collected from 4 stations .

Table 2 : Showing weight (gm) and five morphometric variables (mm) of the two species of Indian horseshoe crabs , with population (N) , mean value (X)

30

31

Table -3 . Showing the comparative body size , shape and weight of two species of Indian horse-

shoe crabs (Except weight in gm all parameters are expressed in mm. Sl. Variables

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Wt. (gm.) IOL DPB DBL VPB TL Tb Th CH FMH MSL a) Long 20mm> b) Short 20mm<

Tachypleus gigas ( N = 100 ) Male (50) Female (50)

Carcinoscorpius rotundicauda ( N = 75 ) Male (39) Female (36)

Range

Mean

Range

Mean

Range

Mean

Range

Mean

120.0 - 225.0 67.2 - 81.5 155.4 - 181.7 137.3 - 170.1 134.8 - 155.5 126.5 - 141.8 5.5 - 6.8 6.2 - 7.4 32.4 - 46.5 10.2 - 15.4

176.36 71.84 163.85 153.24 145.72 128.38 6.14 6.77 35.82 12.55

335.0 - 588.0 89.3 - 108.5 199.2 - 233.4 190.1 - 218.3 168.3 - 189.7 154.5 - 190.2 6.0 - 7.1 6.8 - 7.6 39.1 - 55.6 12.2 - 21.6

478.56 98.75 216.23 206.41 177.15 173.12 6.53 7.21 46.52 18.53

150.0 - 378.0 61.6 - 82.9 151.2 - 210.6 141.2 - 191.4 133.4 - 162.7 138.5 - 172.3 5.2 - 6.1 5.6 - 6.8 29.2 - 53.1 14.2 - 19.4

235.85 74.24 175.42 163.52 148.95 155.20 5.62 6.18 37.17 13.78

305.0 - 629.0 81.6 -105.2 182.6 - 224.5 174.3 - 209.6 155.7 -192.2 140.5 - 181.3

452.21 88.75 201.45 188.35 170.88 175.84

35.4 - 58.6 13.3 - 22.2

46.1 17.55

20.0 - 23.2 7.2 - 19.2

21.81 16.5

20.1 - 26.3 8.7 - 18.9

22.7 12.15

5.2 - 11.5

9.32

6.5 - 15.3

11.22

IOL = interocular length , DPB = dorsal prosoma breadth , VPB = ventral prosoma breadth , DBL = dorsal body length , TL = tail length , Tb & Th = breadth & hieght of tail, respectively , CH = central body height , FMH = frontal marginal height , MSL = marginal spine length . Note: Long MSL means greater than 20 mm. and Short MSL means shorter than 20 mm.

32

Table 4.Showing the ratios of mean values of male to female of two species of Indian horseshoe crabs .

Sl. Parmeters

Tachypleus Male : Female 176.36/478.56 71.84 /98.75 163.85/216.23 153.24/206.41 145.72/177.15 128.38/173.12 6.14 / 6.53 6.77 / 7.21 35.82 / 46.52 12.55 / 22.07

Wt(gm) 1. IOL 2. DPB 3. DBL 4. VPB 5. TL 6. T.b. 7. T.h. 8. CH 9. FMH 10. MSL i) long 21.81 / 22.07 ii)Short 16.05 / 12.15

Except Wt & MSL:

X SD

gigas Ratio 0.3685 0.7274 0.7577 0.7424 0.8225 0.7415 0.9402 0.9389 0.7699 0.5686 0.9882 1.3209

= =

0.7787 0.1138

Carcinoscorpius rotundicauda Male : Female Ratio 235.85 /452.21 0.5215 74.24 /88.75 0.8365 175.42/201.45 0.8707 163.52/188.35 0.8681 148.95/170.88 0.8716 155.20/175.84 0.8826 37.17 / 46.01 0.8078 13.78 / 17.55 0.7851 9.32 / 11.22

X SD

= =

+

0.8306

0.8466 0.0373

NOTE: IOL = interocular distance ( not length ) and DPB = width or breadth of prosoma measured along the curvature of the carapace, but not "dorsal prosoma breadth"---------as suggested by Prof. Carl N. Shuster,Jr. ( 1993 )

33

Table 5.

Simple and partial correlation coefficients between four morphometric variables, 1(IOL),2(DPB),3(DBL) and 4(VPB) of two species of Indian horseshoe crabs

Tachypleus gigas Male(df, 89) Female(df, 18)

Carcinoscorpius rotundicauda Male(df, 27) Female(df, 20)

Simple correlation coefficient r

12

r

13

r

14

*r

23

r

24

r

34

0.7185

a

0.7218

a

0.6601

a

0.8560

a

0.7028

a

0.7162

a

0.7583

a

0.7944

a

0.3750

a

0.6925

a

0.6575

a

0.9167

a

0.8531

a

0.8299

a

0.8878

a

0.8700

a

0.3505

a

0.7253

a

0.7023

a

0.8544

a

0.3152

a

0.7637

a

0.6228

a

0.81

1st r 12.3 r 12.4 r 13.2 r 13.4

r14.2 r14.3 *r 23.1 *r 23.4 r 24.1 r 24.3 r 34.1 r 34.2

step partial correlationcoefficient

0.3203

b

0.3272

-0.0439

0.5506

0.6761

a

0.4421

0.4237

0.2475

b

0.3035

0.2125

0.6645 0.1892 0.2275

a

0.4020 0.3546 0.3229

0.5917 0.3625 0.3631

a

0.2156 0.6901 0.7665

0.7037

a

0.6479

a

0.7908

a

0.6031

0.8355

a

0.6211

a

0.8091

a

0.5816

0.1257

0.4514

b

0.5017

a

0.2601

0.1651

0.2338

0.4148

0.5146b

0.0780

0.5318 b

0.5148 a

0.3437

0.0327

0.4213

0.5917a

0.2668

a

0.3506 0.1948

2nd step partial correlation coefficient r 12.34 0.2960 b r 34.12 -0.0155 *r 23.14 0.7016 a r 14.23 0.1868 r 13.24 0.2449 b r 24.13 0.0999

0.2670

-0.1162

0.2835

0.3531

-0.0878

0.1864

0.5398

0.7647

a

0.5330

b

0.2971

0.4195

b

0.6750

a

0.1816

0.4676

a

0.0172

0.1656

0.4628

a

0.3220

where, a ,

b

P< 0.01

and

b , P< 0.05

34

Table 6. Summary of statistical analysis of body weight (wt /gm) and eleven body mesurements of twenty three mated pairs of T . gigas

Measurements

Sex

Range

X

0

0

n-1

SE

Mode

n

M

120.0 - 220.0

167.93 25.72

25.15

5.36

150

F

355.0 - 585.0

480.87 59.48

58.17

12.40

440

M

67.2 - 78.4

73.26

3.01

2.94

0.62

72

F

89.0 - 107.0

98.46

5.44

5.29

1.12

95

M

155.0 - 181.2

166.03 5.67

5.55

1.18

165

F

199.3 - 232.0

215.29 9.95

9.24

1.97

212

M

144.5 - 161.3

151.50 4.24

4.15

0.88

150

F

190.0 - 217.0

204.93 8.57

8.38

1.78

210

M

135.0 - 157.0

146.11 5.01

4.89

1.04

150

F

168.2 - 189.0

178.4

6.23

6.09

1.29

175

M

*99.5 - 145.0

128.76 9.92

9.71

2.06

130

F

149.0 - 181.0

165.58 9.91

9.69

2.06

172

M

27.4 - 29.1

34.18

2.71

0.57

33

Wt

IOL

DPB

DBL

VPB

TL

2.77

Mean Proportion

t value

P*

0.349

153.703

p

0.744

129.896

p

0.771

195.289

p

0.739

177.508

p

0.821

127.211

p

0.777

83.681

p

CH

0.735 136.511 p F 40.0 - 55.0 46.47 4.07 3.98 0.84 45 ________________________________________________________________________________________________________________________ M 28.2 - 35.3 32.31 1.59 1.55 0.33 34 FL 0.733 136.511 p F 39.0 - 47.3 44.05 2.27 2.22 0.47 45 M

10.5 - 15.2

12.62

1.61

1.57

0.33

34

F

12.2 - 21.6

17.52

3.17

3.10

0.66

16

M

51.0 - 81.0

70.01

7.37

7.21

1.53

68

F

73.0 - 104.0

88.13

8.69

8.50

1.80

82

M

20.0 - 30.0

25.43

2.48

2.42

0.57

23

F

-

-

-

-

-

-

M

-

-

-

-

-

-

F

12.0 - 35.0

27.61

6.27

6.14

1.30

30

F

55.0 - 195.0

133.56 38.13

37.29

7.95

135

FMH

RL

Pl

SL

EM

0.720

43.959

p

0.789

50.590

p

-

-

-

-

-

-

* P < 0.001 IOL ,interocular length ; DPB , dorsal prosoma breadth ; DBL,dorsal body length ; VPB , ventral prosoma breadth ;TL , tail length ; CH ,central height ; FL ,frontal length ; FMH ,frontal marginal height ; RL ,radial length ; PL,clasper length ; SL , median free part of opisthosoma and EM , weight (gm) of egg-mass .

NOTE: IOL = interocular distance ( not length ) and DPB = width or breadth of prosoma measured along the curvature of the carapace, but not "dorsal prosoma breadth"---------as suggested by Prof. Carl N. Shuster,Jr. ( 1993 )

1.233 0.05

0.151 8.58 0

0.629 0.05

0.061 3.49 0

t-value p(level of significance) b* tan0

0.260

0.136

IOL

r-value

Wt

0.230 12.95 0

1.802 0.05

0.366

DPB

0.167 9.48 0

0.930 0.05

0.199

VPB

0.128 7.29 0

0.567 0.05

0.123

TL b

0.321 17.79 0

2.315 0.05

0.451

CH

0.161 9.14 0

1.038 0.05

-0.221

FL

a & b , Significant at 1% and 5% level , respectively ; b* , Regress Coefficient of female (X) on male (Y) .

0.066 3.77 0

0.596 0.05

0.129

DBL

Table 7. Correlation and regression values between Male and Female parameters .

0.472 25.26 0

9.041 0.001

a

0.892

FMH

a

0.662 33.66 0

5.132 0.001

0.746

RL

0.132 7.52 0

0.648 0.05

0.140

DPB(M) SL(F)

0.035 2.01 0

1.053 0.05

-0.224

DPB(M) EM(F)

35

36 Table 8. Chi-Square arrangement of size (A&B) and weight (C) of mated pairs (expected frequencies within parentheses)

MALE

A . Dorsal Prosoma Beadth (DBP) Measurement FEMALE

Small 199-210

Medium 211-222

Large 223-232

TOTAL

Large 173-181 Medium 164-172 Small TOTAL

0.(078)

2(0.86)

0.(0.34)

2

5(4.69)

4(5.21)

3(2.08)

12

4(3.52) 9

4(3.91) 10

1(1.56) 4

9 23

MALE

B . Dorsal Body Length (DBL) Measurement . FEMALE

Small 190-198

Medium 199-208

Large 209-217

TOTAL

Large 157-161 Medium 151-156 Small 156-150 TOTAL

0.(0.78)

0(0.91)

3(1.30)

3

2(2.61)

3(3.04)

5(4.34)

10

4(2.61)

4(3.04)

2(4.34)

10

6

7

10

23

MALE

C. Body Weight (Wt in gm) FEMALE

Less 355-430

Medium 431-505

Heavy 506-585

TOTAL

Heavy 186-220 Meduim 156-185 Less 120-155 TOTAL

1(1.82)

2(2.08)

3(2.08)

6

1(1.82)

3(2.08)

2(2.08)

6

5(3.34)

3(3.82)

3(3.82)

11

7

8

8

23

37

Table 9. Statistics (log-trans formed) of the Length Weigth Relationship of Males and Females of two Indian horseshoe crabs . SPECIES

SEX

N

SX

SY

SX2

T. gigas

MALE FEMALE

85 19

100.42927 24.94337

190.00707 50.79693

118.68307 32.75238

SY2 425.23588 135.86436

SXY 224.57082 66.70305

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C. rotundicauda

MALE FEMALE

28 17

33.90302 21.58707

66.15546 44.63033

41.08804 27.43822

156.86390 117.39868

SX2 , SY2 and SXY , sum of squares and products ; SX and SY , sum of logarithmic values of length and weight , respectively .

80.21862 56.73484

df

T. gigas 1 103

C. rotundicauda

Sy

2

0.78873

0.55871 0.23002

= 0.57843

3.74522 4.30122 adjusted

0.55600 slopes

0.49839 0.05761

F=

0.00854 = 1.23589 0.00691

102 1

100 1

83 17

df

2.78950

3.09554 2.35312

( df = 1,41) , p > 0.05

43 1

41 42 1

26 15

0.01036 0.76802

0.00287 0.00166

0.57843 P > 0.05

F

regressions

0.00326 0.00096

MS

from

0.06444

0.00691 0.00694 0.00854

0.00766 0.00560

P > 0.05

1.23589

no significant difference

0.35633 0.06444

0.28335 0.29189 0.00854

0.19923 0.08412

no significant difference

1.05725 0.76802

0.28757 0.00166

0.27124 0.01633

SS

Deviations

(df = 1,100) , P > 0.05

3.29952 means

2.96764

3.08995 2.52090

b

1 0.03683 0.16392 0.72955 44 0.10068 0.34203 1.51828 3.397719 Difference between adjusted means

slopes

0.17811

0.6385

Difference between

0.11612 0.06199

0.03751 0.02634

0.00164 0.00287

0.26768 0.89327 0.29797 0.98316 Difference between

Comparison of slopes :

Between T&P (T)Total

Within MALE 27 FEMALE 16 (P)POOLED (M+F) 43

0.07351 0.01638

Sxy

0.03029 0.08989 Difference between

0.02379 0.00650

Sx

Comparison of slopes : F =

Between T&P (T) Total

MALE 84 FEMALE 18 (P) POOLED (M+F) 102

Within

2

Table - 10. Comparison of Regression Lines of Weight on Length of adult male and female of Indian Horseshoe Crabs .

38

39

Table

t-test

11.

of regression lines of Males and Females of two Indian Horseshoe Crabs .

SPECIES SEX(df) SS T. gigas MALE(83) 0.27124

MS 0.00326

MS 0.05716

Sb

t

0.37063

8.33702

p 0.05

FEMALE(17)0.01633 0.00096 0.00309 0.38430 6.55971 0.05 -------------------------------------------------------------------------------C. rotundicauda MALE(26) 0.19923 0.00766 0.08752 0.45189 6.85020 0.05 FEMALE(15) 0.08412

0.00560

0.07483

0.46109

5.10335

0.05

SS , sum of squares of deviations from regression ; MS , mean square deviations from regression ; MS , sample standard deviation from regression ; Sb , sample standard deviation of the regression coefficient .

83

MALE

2

0.02379

SX

0.00326

MS

3.08995

b Estimated t'

df

p = 0.05 t

W

Expected t'

C. rotundi cauda

T.gigas

26

FEMALE 15

MALE

0.02634

0.03751

0.00560

0.00766

2.35312

3.09554

1.14990 P 0.05

16

27

2.120

2.052

0.0003294

0.0002735

2.0891

84 1.987 0.0003835 1.46214 2.05118 FEMALE 17 0.00650 0.00096 2.52090 P 0.05 18 2.101 0.00050521 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

df

SEX

Table 12. t'-test between regression coefficients of Males and Females of two Indian Horseshoe Crabs

40

41

Flg PROSOMA Doc Opr Ecr Prs Lcy Ops Ldr Prk Cdr pos Pcs Ptr

Ocs

Sos Adm Pop Atr Acf Arp

Msa

Enp Klo Ldr Psa Lms

Tvn

OPISTHOSOMA Sms Dly Ana Dlp

TELSON Dls

30 mm

Tachypleus gigas ( Hand drawn Dorsal View )

Fig.4 Body structure of the horseshoe crab,T. gigas (female):External anatomy dorsal view (after Carl N. Shuster,1982 and K. Sekiguchi,1988) Acf,arcuate furrow; Adm,arthrodial membrane; Ana,anal angle ; Arp,auriculate process ; Atr, anterior ridge; Cdr, cardiac region; Dlp, dorsal process; Dls, dorsal serration ; Dly, dorsal yoke; Doc, dorsal ocelli ; Ecr, extra cardiac region ; Enp, entapophyseal pit; Flg, flange ; Klo, opisthosomatic keel; Lcy , lateral compound eye;Ldr, longitudinal furrow; Msa, muscular attachment; Ocs , Ocellar spine; Opr,opthalmic ridge; Ops, opthalmic spine; Pcs, Post-cardiac spine; Pos,Post-opthalmic spine; Prk, prosomatic keel; Prs, pre - opthalmic spine; Psa, posterior angle; Ptr, posterior ridge; Sms, short moveable spine ; Sos, sub-opthalmic region; Tvn, transverse nodule .

42 Gnv Flg Dbr

Sfa Mls Vso Chl Ept Mth Pms Chi

Wkl

Prp

Flb Evs Ivl Spo Gol Mrp Pts Anl Trm

Bra Mvs

Tlt Tvr Alr

Vlf

Fig ig.. 5. Body structure of the horseshoe crab , T. gigas (female) : External anatomy / ventral view (after Prof. Carl N. Shuster,Jr. 1982 and K . Sekigchi , 1988) . Alr , axial area ; Anl , anal slit ; Bra , branchial appendages ; Chi , chilarium; Chl , chelicera ; Dbr , doublure rib ; Ept , epistome ; Evs , exuvial suture ; Flb , flabellum ; Flg , flange ; Gol , genital operculm ; Ivl , interados of vault; Mls , mesial spine ; Mrp, marginal process ; Mth , mouth ; Mvs , moveable spine ; Pms , promeso-sternite ; Pts , posterior slope ; Sfa , sub-frontal area ; Spo , spatulate organ ; Tlt , telson trunk ; Trm , terminal membrane ; Tvr , transverse ridge ; Vlf , ventral longitudinal furrow of telson ; Vso , ventral sense organ ; Prp, propodite.

43 PROSOMA

Chl

IOL

DPB VPB

FL

Doc Vso Ovd

Mth Gnb EM

OPISTHOSOMA

Chi

SL

Mts

Ovd Gol Gop Anl Marginal spines

Dorsal serration

A .

TELSON

A . Dorsal view

B .

Anl

B . Ventr al vie w entral view (showing left half of reproductive system)

Dorsal hinge

Movable spine Lcy

BL

CH

TL

VBL

DPB

C . Lateral view

FMH

Doc

CH

FMH

ƒ

DBL

VPB D. Fr ontal vie w view

Fig.6.

Three Dimensions of a Female Tachypleus gigas Body parts measured :

DPB , dorsal prosoma breadth ; DBL , dorsal body length ; IOL , interocular length ; VPB , ventral prosoma breadth ; TL , telson length ; FMH , frontal marginal height ; CH , central height ; FL , sub-frontal length ; Sl , length between dorsal opisthosomatic spine and median mating scar ( Mts ) ; Gnb , gnathobases ; Ovd , oviducts ; Gop , female gonopore ; Gol , genital operculum ; EM , egg-mass ; Anl , anal slit ; Doc , dorsal ocelli; Lcy, lateral comp.eye;Chl, chelicera; Chi, chilarium,VBL, ventral body length.

44 COMMON CURVE for C. rotundicauda

COMMON CURVE for T. gigas

0.45

0.40

0.35

0.30

Log

Weight ( gm. )

0.25

0.20

O +

O +

Œ

0

0Œ

0.15

0.10

0.50

O

0.5

1.0

1.5

2.0

Log

Length ( cm. )

0

0.5

1.0

1.5

2.0

Fig .7. Parabolic growth curves of Tac hypleus gig as (A) and Fig.7 ach gigas

Carcinoscorpius rotundicauda (B)indicating faster growth rate in females than that of males.

160

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

T

80

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 1234

250 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

200

100

1234 1234 1234 1234 1234 1234 1234 1234

123 123 123 123 123 123 123 123 123123 123 123 123123

50

T

20

T

Ä

150

T

T

123 123 123 123 123 123 123 123

350

T

T 40

123 123 123 123 123 123 123 123

400

T

T

60

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

T

123 123 123 123 123 123 123 123 123

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

450

300

T

100

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123123 123 123 123123 123123 123

T

120

123 123 123 123 123 123 123 123 123 123 123 123 123

500

T

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

140

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

T

T

180

1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234

550

T

MEAN

O

♀

T

SD

T

200

1234 1234 1234 1231234 1231234 1234 123 1231234 1234 1231234 1234 123 1231234 1231234 1231234 1234 123 1234 123 1231234 1234 1234 123 1231234 1234 1234 1234 1234 1234

RANGE

T

T

1234 123 1234 123 1234 123 1234 123123 123 1234 123 1234 123123 123 1234 1234 123 123 1234 123 123 1234 123123 1234 123123 1234 123123 123 1234 123 123 1234 123 1234 123123 1234 1234 123 1234 123

T

123

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

T

220

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

123 123 123 123 123 123123 123123 123123 123 123 123123 123123 123 123 123 123123 123123 123 123 123123 123 123 123123 123 123123 123 123 123123 123123 123 123 123 123

Fig.8.

Morphometric

Wt.

IOL

DPB

VPB

DBL

TL

in gm

Different

CH

FL

FMH

RL

123

Variables

Graphical presentation of Nine (9) morphometric variables ( mm ) and hypleus ach body weight (gm) of 23 mated males and females of Tac gigas gigas..

Ä

46

Mating Scars

A

B

Trailing Scars

C

D

47

Fig. 16 . (A-D)

(p.46)

A , A male C. rotundiacauda in ventral view showing its prosomatic appendages , comparatively broad opisthosoma with very small marginal spines . The telson is comparatively larger and stout ; ; B Dorsal view of a gravid female, 40cm. in length including the spine ( T. gigas), showing three vivid corroded marks , the ' mating scars ', C , Dorsal view of a juvenile horseshoe crab of approx. 5 cm. length ,whose sex was not idtifiable; D, Ventral view of a female C. rotundicauda ( 16 cm. excluding the spine) showing its prosomatic as well as opisthosomatic appendages ( genital operculum and gill flaps );irregularities in the trailing edges of the opisthosoma due to damage to the carapace during amplexus i.e. most of the movable spines were dropped off.

A

Fig.17.

A , One male T. gigas is in upturned posture ; B , Adult horseshoe crabs ( T. gigas ) rest on the sand beach of CHANDIPUR, ORISSA for taking individual weight with a pan-balance.

B

48

Male

Female

A.

Ventral view of Carinoscorpius rotundicauda G

I

J

K

L

2nd pair E

C

ale m

rs pe s cla

D

Tachypleus gigas

s ite d po ro p ale fem

s ite d po ro p ale 1st pair m e f

s sper e cla mal

Carinoscorpius rotundicauda

B

* H

A

F

B. Morphological Differences & Sexual Dimorphism in C. rotundicauda and T. gigas.

1st Walking leg

1st Clasper organ 2nd Clasper organ

2nd Walking leg

Female

Male

Genital operculum

B. Ventral Appendages of Tachypleus 1st Clasper organ

1st Walking leg

2nd Clasper organ

Male

D.

gigas

2nd Walking leg

Female

Ventral Appendages of Carcinoscorpius rotundicauda

Fig. 18 (A-D) A, Ventral view of C. rotundicauda showing comparative body size of the male (smaller ,left one) and the female(larger , right one); B & D, Comparison between five walking legs : 5B, with genital operculum in T. gigas and 5D , first walking legs with attached chelicerae in C. rotundicauda; C, Shows the morphological difference and sexual dimorphism in two species of horseshoe crabs . A & B , dorsal carapace ; C & D , structure of genital operculae ; E & F , telson structures ; (*H) in T. gigas only ; and I,J & K,L show the most distinguished characters of sexual dimorphism .

50 DISCUSSION

The publication of vander Hoeven (1838) provided the first comprehensive treatise on the morphology of horseshoe crab " Limulus moluccanus " , presently known as T. gigas and thereafter only few fragmentary informations on morphology , including that of the larvae , were added by Packard , Lankester , Patten and others in the latter half of the 19th century . From 1927 to 1932 , Shoji studied the American and Japanese horseshoe crabs from the view point of comparative morphology and comparison of their larval forms just after hatching , and in 1929 he gave an account of the anatomy of Tachypleus tridentatus in detail . The morphology of Limulus polyphemus in the modern sense was given by Snodgrass in 1952 . Recently a vast collection of information regarding horseshoe crabs morphology consists of data on intraspecific variation , specially in body size and length of marginal spines . The morphological variation was given in L. polyphemus by Shuster (1957) , in T. gigas by Sekiguchi et . al . , (1976) , and C. rotundicauda by Sekiguchi et. al. (1978) . The body of the horseshoe crab in distinctly divided into two parts , an anterior large prosoma or cephalothorax and a posterior , small and hexagonal opisthosoma ; the latter is freely movable up and down with its broad base attached to the prosoma on a strong transverse hinge . The dorsal surface of the body is elevated along the middle . The glossy carapace is hard and is composed of an organic combination of chitin and protein (Lafon , 1941) . The telson , sharply elongated posteriorly , together with marginal spines arranged along the postero-lateral margins of the opisthosoma , gives the animal a unique appearance . In outline , the prosomatic carapace is suggestive of a horseshoe as used in the common name of the animal . The dorsal convexity of the prosomatic carapace enables the animal effective for feeding and respiration when the animal is embedded in muddy or sandy substratum . The swelling of the carapace is moderate in T. gigas and weak in C. rotundicauda . The antero-frontal margin forming an arc of the prosoma is relatively more in female than in the males . But in C. rotundicauda this arc is almost same in both sexes . Only the male of T. tridentatus has a pair of notches on this arc anterolaterally (Sekiguchi , 1988) . The dorsal ocelli or dorsal eyes , sometimes called as median eyes , are a pair of very small sports situated at the starting point of the median ridge . The distance between the two ocelli varies according to the species ; they are far apart in L . polyphemus , moderately distant in T. gigas and C. rotundicauda , and close together in T. tridentatus (Yamasaki et al. , 1988) .Compound , lateral eyes are situated on and near the opthalmic ridges . The average diameter of a compound eye is more than 10 mm. in L. polyphemus , whereas it is below 10 mm. in Asiatic species . Yamasaki , et. al . , (1988) showed the progression in three Asian species as C . rotundicauda , T . gigas and T. tridentatus . It can be said that C. rotundicauda has large eyes relative to its small body size . According to Waterman (1954) , the maximum number of ommatidia was 1,161 in L. polyphemus and 446 in T. tridentatus. In C. rotundicauda the distance between the two compound eyes (IOL) is shorter than in T. gigas . Ratios of male to female morphometric characters (Table -4) indicate that it is 0.7787 + 0.0373 and 0.8460 + 0.0373 in T. gigas and C. rotundicauda, respectively . These results actually tell the comparative body size of male and female that is in the former species the male is nearly 78% of the female , while in the latter species it is nearly 85% . But in respect to body weight the male T. gigas is almost 37% of the female , while it is 52% in C. rotundicauda . In every aspect it is evident that the female specimens of C. rotundicauda are smaller than the females of T. gigas , but the situation is reversed in case of male . Perhaps the comparatively large body of female has potentially effective for lengthening the breeding cycle in the estuarine environment . This has also been further confirmed by the availability of C. rotundicauda in Sundarbans right from Frbruary to August or Spetember , at least solitary females on the muddy shore .

51

Among the four morphometric variables , e.g. interocular length (IOL) dorsal prosoma (DPB) , dorsal body body length (DBL) and ventral prosoma breadth (VPB) , simply or partially correlated (Table - 5) , it was the DBL and DPB , between which the perfect correlation had occurred . In the 2nd r

step , it is only 23.14 where both sex category of either species showed significant P (0.05 , P < 0.01) . It is , therefore , justifies to make a length-weight relationship (Le Cren , 1951) considering only DBL as size parameter with that of body weight . In the present discussion horseshoe crab , T. gigas showed n = 3.08995 and 2.52090 for males and females respectively ; while it was 3.09554 and 2.25312 in C. rotundicauda . They may be vital units of taxonomic consideration of the species . Presently it has been found that T. gigas , the males increased in weight by an exponent of 3.08 and females by 2.52 ; while in C. rotundicauda males and females increased by an exponent of 3.09 and 2.35 ; respectively . Although these 'n' value higher in males than in females but did not differ significantly (Table -12, 't' test) . Such higher values for males were common in fishes too (Sekharan , 1968 ; Chanchal et . al . , 1978 ; Lazarus and Reddy , 1986).

A female of

T. gigas

A female of C. rotundicauda

In every aspect it is evident that the female specimens of C. rotundicauda are smaller than the females of T. gigas , but the situation is reversed in case of male . Perhaps the comparatively large body of female has potentially effective for lengthening the breeding cycle in the estuarine environment . This has also been further confirmed by the availability of C. rotundicauda in Sundarbans right from Frbruary to August or Spetember , at least solitary females on the muddy shore .

52

An excellent review of length-weight relation was presented by Peters (1983) where Pough (1980) showed the ranges of regression coefficient were 2.98 - 3.02 in reptiles , 2.94 - 3.64 in salamanders , 2.69 in turtles , 3.24 in frogs ; while Jerrison (1973) showed 2.53 in world record of fishes and Smock (1980) recorded 2.62 in aquatic insects . McMahon's (1973 , 1980) explanation of the basal metabolic rate (Rb) in terms of body mass (Rb

α

W 3/4) has been deduced from the length-weight relation and he finally

concluded that Rb varied as body mass to the three-fourth power (i.e. W3/4). So in this context the present investigation may open a new line to manage horseshoe crabs in captivity . Deducing the two parabolic growth equations of T. gigas and C. rotundicauda it is found that the former species will stop its growth completely when it attains 248 mm in maximum length and 766 gm in maximum weight , while the latter species ceases growth at 247.28 mm and 756.40 gm of maximum length and weight , respectively . The above assumptions are simply the theoretical values which usually do not corroborate with the natural findings ; as for example , horseshoe crabs in Indian coastal region observed maximum 629 gm in weight and 209.6 mm in DBL . Increase in weight was found to follow the cube law in females of C. rotundicauda and two curves of males and females intersected between the carapace length 130 - 135 mm which indicated the length in which the animal attains sexual maturity (Chatterjee et. al. , 1988) . But in the present study it has been found that the females reach maturity sooner than the males , and growth of the female is even faster in C. rotundicauda ; while combined growth patterns are more or less identical in both species (Fig. 7) . Biometric analysis of Tachypleus (T.gigas) matting indicates the following points : 1) Large males tended to 'prefer' large females and medium-sized males 'prefer' medium females . Small males , however , donot show a "preference" for small females , but rather for mediumsized ones ( Table - 8 A,B,C) Although X2 -test couldn't be done due to the appearance of expected value more frequently less than 5 (Bailey , 1959) . Su\huster , el. al. , (1992) has confirmed this hypothesis in L. polyphemus , where in no case the male size was homogamous with that of the female partner (Pomerat , 1933) ; Cochen & Brockman (1983) and Brockman (1990) also demonstrated assortive matting in L. polyphemus . According to them (opt. cit.) competition between males for females was also independent of size i.e. , attached males were not significantly larger than the unattached males . ii) It has already been established that the female horseshoe crab is larger than the male (shuster , 1982 ; Sekiguchi 1988) , it might , therefore , be deduced that higher correlations represented closer approximations in size for characteristics studied (Shuster et. al. 1992) . If this is true , then , if a charteristic of one member of a mated pair diverges from the overall male to female regression line , then it is specific with sex . Hence , sexual dimorphism occured in the above morphometric features . But less significant levels and low coefficients (Table -7 ) make such conclusions dubious . iii) There are three " mating scars " an axial one with two laterrals on either side (Fig. 6) are caused by the motion of the male prosomal arch abetted by sand , while amplexus has been in a long time (Shuster , 1955/58) . These " mating scars " are mute evidence of the duration of amplexus and the numbers , and positions of scars can be used to idicate the age of the female as well as the size and the number of previously attached suitors (Shuster et. al. , 1992) .

53

In the present study it was also ecident that in terms of weight the male assumed 35% of the total body weight of the female , while in all other morphotic characteristics the males were of 75.8% of the females ( Table -16) . The total egg mass of gravid females varies from 55 to 195 gms with a mean value of 133.56 gm. It was found after serveral trials that 1 gm of eggs counted 28 in number which will help to determine the total egg-number in a female . For instance , a female T. gigas of 505 gm total body weight yielded 5,497 eggs , while another female of same weight gave the figure as 3295 . Interestingly enough , a female severely infested with epifaunal associates (confirming its old age) with only 355 gm of total body weight gave only 9 mature eggs (brownish , rather than usual light yellow-greenish appearence) . The wide variation in egg numbers contained by the females is indicative to the frequent and usual egg laying behaviour of the animal. When regression as well as correlation were made between DPB of male and female egg mass it showed a very weak but negative coefficient ( r = -0.224 ) (Table -17) . From the same table -17 it was found that only CH , FMH and RL show significant correlation and comparatively higher tangent angles of regression lines .

Comment : Since all the four living adult horseshoe crabs are not known to molt, the term " growth " in adults is an area of enquiry needing more study. ......... Prof. C.N. Schuster, Jr. (1993)

An excellent review of length-weight relation was presented by Peters (1983) where Pough (1980) showed the ranges of regression coefficient were 2.98 - 3.02 in reptiles , 2.94 - 3.64 in salamanders , 2.69 in turtles , 3.24 in frogs ; while Jerrison (1973) showed 2.53 in world record of fishes and Smock (1980) recorded 2.62 in aquatic insects . McMahon's ( 1973 ,1980) explanation of the basal metabolic rate (Rb ) in terms of body mass (Rbα W 3/4) has been deduced from the length-weight relation and he finally concluded that Rb

varied as body mass to the three-fourth power (i.e. W3/4). So in this context the present investigation may open a new line to manage horseshoe crabs in captivity .

54

C . Absolute population estimates

55 INTRODUCTION It is usually possible to estimate the size of population and communities of sessile and sedentary organisms by using methods which involve fixed sampling units i.e. , quadrate or line-transect methods . But with mobile individuals i.e. , insects , fishes , snails , horseshoe crabs and other animals different procedures , particularly capture-recapture techniques are required . The application of recapture method for such purpose was first made by Lincoln (1930) following the principles of proportion of capture-recapture of marked animals introduced by Peterson as early as 1889 . Lincoln estimated the total number of ducks in North America , and the formula he used was sometimes called the "Lincoln Index" . The principle is very simple : a number of animals are captured , marked and released . When the marked animals are thought to have mingled thoroughly with the unmarked population , a further sample is captured and the proportion of marked individuals noted . Then an estimate of the total population size is given by duviding the number of marked animals released by the proportion found to be marked in the second group of captures . Jackson (1937 a,b) independently used the same method for estimating the density of tsetse flies . In a subsequent extension of this method Jackson (1937 , 1939) used his "positive" and "negative" methods allowing certain range of complications imposed due to birth-and death-rates . There are also certain other methods of estimating a population that depened on the presence of marked individuals , employing different principles to the Lincoln Index . A comprehensive survey of these marking techniques as well as capture-recapture methods has been reviewed by Seber (1973) .There are at least eight assumptions (Southwood , 1978) which underline all methods of capture-recapture analysis , such as :1. The marked animals are not affected (neither in behaviour nor in life expectancy) by being marked and the marks will not be lost . 2. The marked animals become completely mixed in the population . 3. The probability of capturing a marked animal is the same as that of capturing any member of the population sampled randomly with respect to its marked status , age and sex , termed as 'equal catchability'. This assumption has two aspects : i) that all individuals of the differen age groups and of both sexes are sampled in the proportion in which they occur , ii) that all the individuals areequally available for capture irrespective of their position in the habitat . 4. Sampling must be at discrete time intervals and the actual time involved in taking the samples must be small in relation to the total time . 5. If the population is not a closed one , immigration and emigration can be measured or calculated . 6. There are no births or deaths in the period between sampling or , if there are , allowances must be made for them . A few horseshoe crabs unlike Limulus become stranded .So the number of death is is negligible in this study. 7. Being captured one or more times does not affect an animals subsequent chance of capture. This is a further extension of assumption no.3 i.e. , equal catchability . 8. Every marked animal has the same probability of surviving through the sampling period .

Comment : Unless animals of the same age are marked , survival probability becomes biased.............. Prof. C.N.Shuster, Jr.( 1993 ).

56

A

B

C Fig. 19 (A-C) A,Shows the making device with steel needle on the dorsolateral carapace of one horseshoe crab (T. gigas); B, Shows the coded mechanical mark on the right dorsolateral surface of prosoma; C , One marked and segregated female of T. gigas released in the falling tide .

57 OBSERVATIONS A. Application of Jackson's Principle in the present investigation : Horseshoe crabs are mobile in nature and they prefer a wide territorial range (habitat) in the sea particularly in the shelf region , come to the shore only during the breeding season , mostly in pairs in Indian coastal region (Debnath , 1985) . So their population size was measured only by capture - recapture method and it was not possible to estimate the population size of such mobile animals by quadrate method or by using any other fixed sampling units . This author used Jackson's "Negative" and "Positive" method (Jackson , 1939) to estimate the population size of adult horseshoe crabs , Tachypleus gigas that came to breed at Chandipur sea shore in Orissa State from March to July , 1985 . The principle of "Positive" method is , in contrast to the "negative" method , to release a large number of marked animals on a single occasion . Capture is then made on a number of later occasions , the number of marked animals being recorded each time . The majority of animals captured on these later occasions should be released again , otherwise one has to consider special account to the diminution in the number of marked animals available for recapture . On the other hand , the principle of the negative method is to catch , mark and release groups of animals on several successive observation days , making no record of the numbers of recaptures until the last occasion . Such a method is suitable when the preliminary marking of successive batches can be carried out by relatively unskilled workers (Bailey , 1952) . However , the idea of Jackson's captur-recapture method is simple enough , but it is very difficult to attribute a precise variance to the estimates (Bailey , 1952 ; Andrewartha , 1970) . CALCULATIONS AND RESULTS a) Crude - Recaptures : The complete data showing the numbers of captures , releases and recaptures can be demostrated by a triangular array of " trellis" diagram . Three "trellis" diagrams have been constructed to accommodate the crude-recaptures ( Table 13 A-C) in which total of 653 , 1082 and 680 individuals of horseshoe crabs were counted in March , May and July , respectively . Although 692 , 1120 and 703 animals were captured , 39 , 38 and 23 had been rejected because of their moribund condition or physical injury caused by the ravens Corvus splendens , an effective predator bird (Debnath and Choudhury , 1988a). The numbers in each row of the Table 13 A-C (horizontal) indicate the number of horseshoe crabs marked on the date indicated in the left margin . The vertical columns indicate the numbers recaptured on those that were marked on the dates shown in the left margin . In respective three months the total number of animals recaptured were 73,307 and 107 . Thus the ratios of total captured , total marked and released and total recaptured are 1.05:1:0.11 , 1.03:1:0.28 and 1.03:1:0.15 for the successive three months as mentioned above . b) Corrected Recaptures : In Table - 14 A-C each row provides the row material for estimating the size of the population (on the day of release) by Jackson's "negative" method . The values in the body of the Table 14 A-C are corrected recaptures which have been calculated with the help of the equation No. 1.

59 Population size :

The values for 'r' calculated from each row and column by using equation 2 or 3 (equation 2 that covers 3 entries only). Equation No. 4 is then used to calculate 'a' for each row and column and the population size N obtained by using equation No. 5 . Thus the table shows the calculated values for r+ , r , a0 and N. In March 1985 , the number of individuals that arrived on the 7th , 8th and 9th date respectively were calculated by positive method , while for the 10th date it was calculated by negative method . Similar methods were followed to estimate the population of T. gigas in May and July (Table 15) .

59

Equations: Yn

=

r+/-

=

r+/-

=

a

=

0

N+/ -

Where ,

Rn

100 Cm

100 X

......................( 1 )

Cn

Y2 + Y3 + Y4 +......+ Yn

......................( 2 )

Y1 + Y2 + Y3 +......+ Yn-1

Y3 + Y4 + Y5 +........+ Yn

......................( 3 )

Y1 + Y2 + Y3 +........+ Yn-2 ( Y1 + Y2 + Y3 +........+ Yn-1) r+/-

- ( Y1 + Y2 + Y3 +.......+ Yn-2) ........( 4 )

10, 000

=

Yn

X

a

......................( 5 ) 0

=

Corrected recaptures ,

Rn

=

No. of recaptures on date n ,

Cm

=

No. of marked on date n ,

Cn

=

Total captured (including recaptures) on date n ,

Yn

r

=

The weighed ratio of

a0

=

Corrected values for the date of estimation ,

N+/- =

Yn-1

Estimated population on date n.

Animal dispersion and sexual selection : Out of the total 50 amplexed animals released within 50-200 mts. , 9 pairs of T. gigas were recovered from Burhabalanga sand flat , 8 at a distance of 200 mts or within from the point of release and other pair at a distance of 1000 mts opposite to the site of release . These findings (Table 22) , recovery rate , R = 18 % ) helped to document the dispersal behaviour of the species , however , in the restricted sea shore only . One interesting observation was made that out of 15 mating pairs segregated and released , one amplexed pair was recovered after 3 days from the date of release and they showed similar marks on their cararapaces (Debnath and Choudhury , 1988a) .

60 B. Application of Lincoln 's method

:

In the present part of the tagging study the horseshoe crabs were captured , marked and released at a time and then recaptured in the following day only . Between the receding tide of 26th March and the following high tide of 27th March provided almost 24 hrs. to intermingle the released population with the unmarked population that required to be determined . The calculation is very simple enough following the Lincoln - Peterson Index as shown below : <

a.n r

N = <

Thus ,

Where , a = n=

300 X 284 20

N =

=

total marked and released . total second sample including recaptures (r)

4,260

Bailey (1951 , 1952) has suggested that with small samples (if r < 20) a less biased estimate can be obtained if 1 is added to n and r , thus ; <

a (n+1) 300 (284 +1) = = 4071.42 r +1 20 + 1 The 'variance' and the 'standard error' of N are :

N

=

<

Var N

=

a2 (n +1). (n - r ) ( r + 1)2 . (r + 2) <

S .E .

=

Var N

= 697959.18

= + 835.439

( C ) Application of Jolly - Seber stochastic method : Jolly (1965) and Seber (1965) have , independently , developed a common method to cover situations in which there is both loss (death and emigration) and dilution (birth and immigration). Their methods give similar solutions , except that Jolly's makes allowance for any animals killed after capture and hence not released again . At Digha (West Bangal) horseshoe crabs population comprising only adult T. gigas has been severely affected by human interferrence and a predator , C . splendens ( Debnath, 1985 ; Debnath and Choudhury , 1988a) . So the factor , like "loss" due to death and emigration is more inevitable than "dilution" . However , dilution in the population may occur to some extent due only to immigration but not with birth . The Jolly-Seber method efficiently groups the data and is fully stochastic , " an extra advantage over any deterministic method , however its reliability strictly depends , effectively , on the probability of any animal surviving through any period is not affected by its age at the start of the period". The Jolly - Seber 's estimate of population is usually reliable when 9% or more of the population is sampled and the survival rate is not less than 0.5 (Bishop and Sheppard , 1973) . Although this method may seriously overestimate the survival rate , it properly remains the most useful method to date and has been used for a number of different insect population (Parr , 1965 ; Sheppard et. al . , 1969 ; Fletcher , 1973 ; Ito et. al. , 1974)

61 Calculation and Results : The basic equation in Jolly's method (1965) is :

^ .n M i i

^

Ni =

...................................... (I)

ri

^

^

where , Ni = the estimate of population on day i , Mi = the estimate of the total number of marked animals in the population on day i (i.e. , the counterpart of 'a' in the simple Lincoln Index) , ri = the total number of marked animals recaptured on day i and ni = the total number of animals captured on day i . The procedure of calculation may be demonstrated as follows : 1)

The field data are tabulated as in Table 16 A according to the date of initial capture or mark and the date on which the animals were last captured . The columns are then summed to give the total number of animals on the ith occasion ( = Si of Jolly , 1965) , recaptured subsequently (Ri)

2)

Another table is drawn up (Table 16 B ) giving the total number of animals recaptured on day i

bearing marks of day j (bold printed in the Table 16 B) or earlier (Jolly's a i j ); this has been done by adding each row in Table 16 B from left to right and entering the accumulated totals . The number marked before time i which are not caught in the ith sample , but are caught subsequently (Zi) , is found by adding all but the top entry in each column . The top entry in each column represents the number of recaptures (ri) for the day on its right . 3) Then the estimate of the total number of marked animals at risk in the population on the samplind day is obtained thus (Table - 17) ai . Zi i ^ ......................... (II ) Mi = +r Ri 4)

The proportion of marked animals in the population at the moment of capture on day "i" is found and enterd in the final table (Table 17) , thus : ri

∝i

5)

..................................................(III)

=

ni

The total population is then estimated for each day by the following equation as constructed using equation -I and II :

^ N

i

=

^ M i

∝i

.................................................. (IV)

62

6) The probability that an animal is alive at the moment of release of the ith sample will survive till the time of capture of the sample ( i + 1) th sample is obtained :

^ Øi

=

^ γ

=

^ M i+1

...................... (V)

^ -r +a M i i i For technical as well as biological reasons this survival rate may be converted into a "loss rate" ( the effect of the predation , Debnath and Choudhury , 1988a ) and emigration due to human interference . ^ Ø i ..................................( VI )

1 -

i

7) The number of new animals joining the population ( due to fresh emergence of T. gigas

from the shallow shelf region and from adjoining areas) in the interval between the ith and (i+1)th samples and alive at the time (i+1) is given by :

^ B i

^ - ^( ^ = N Øi N i - n i + a i) .......( VII ) i+ 1

^

This equation no. VII may be converted into the dilution rate, â , thus :

^ B

1

^ â

= 1 -

^ N

i

................................( VIII )

i+ 1

8) Now in the final calculation (Table 17) standard errors, i.e. the square root of the variances ^ , and B^ are obtained by using very cplicate equations ( discussed in detail by of N^i, Ø i i Jolly, 1965 ).

^ ) and survival rate (var^ ) 9) According to Jolly(1965) the variances of the population(var N Øi i estimates given above contain an error component due to the real variation in population numbers. These errors of estimation may be obtained with the following formulae:

i)

^ M i- ri + a i var ( ^ Ni / N i ) = ^ Ni ( ^ Ni - ni ) M i

x ( 1 Ri

1 ) ai

+

1 - αi ri

63

^/ ) = ii ) var ( Ø i Øi

^) var ( Ø i

^2( 1 - ^ ) Øi Øi ^ M

i+1

Now , results obtained by mathematical equations following procedures , number 3 to 9 are tabulated in the final Table -17 . The special problems involved in the computation of the summation term of var ( N )

What the author contends is that an organised team with working facility in the feild and assistance for a longer duration taking many more stations along the coast may help to bring out a more comprehensive population picture of the Indian horseshoe crabs .

But

an interesting observation in recovering two segregated opposite sexes further amplexed (coupled) 3 days after release has rendered the author to assume that specific sexual selection might had occurred during the breeding activities. But how they choose themselves and found together and returned to the breeding ground remained a great mystery .

64

Table 13 (A, B & C) . Showing crude recaptures in March and July , 1985 at Chandipur (Orissa)

Trellis Diagram .

13 A

Date Total Total marked captured marked & released March,6th 7th 8th 9th 10th 11th 12th 13th

12 70 193 202 74 63 50 28

11 68 188 194 72 60 36 24

No. captured 692 653 Date captured. March'1985

DATE 7th 8th 1

1

2 4

6

RECAPTURED

9th

10th

11th

12th

18 26

2 12 2

2 1

1 1 -

44

68 188 194 7th 8th 9th

16 72 10th

3

2

60 36 11th 12th

Total recaptured 13th 1 1 24 13th

= 73 = 642 + 11 = 653

Ratio of total captured , total marked & released and total recaptured , respectively = 1.05 : 1 : 0.11

65 Trellis Diagram 13 B Date

Total

marked

DATE

Total

captured

RECAPTURED

Total recaptured

marked & released

1985

May'

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th

2nd 3rd

1 2

4th

1 28

5th 6th

15 32

1 27 32

7th

5 12 83 186 197 223 155 92 57 51 36 23

4 12 80 184 190 218 152 88 54 48 34 18

-

1 12 24 24

0

3

29

47

60

64

No.Captured 1120

1082

12

80

184

190

218

2nd

3rd

4th

5th

6th

8th

9th

8 7 19 17

8 7 8

10th 11th

12th

13 5 2

7 1 2

4 5 -

51 23

20

10

9

152

88

54

48

34

18

7th

8th

9th

10th 11th 12th

= 307 = 1078+4 = 1082

Date Captured

May'1985

Ratio of total captured , total marked and released and total recaptured respectively = 1.03 : 1 : 0.28

Trellis Diagram 13 C Date

Total

marked

captured

DATE

Total

RECAPTURED

Total

marked & released

recaptured

June - July'1985

June - July'1985

30th

29th 30th 1st 2nd 3rd 4th 5th 6th

9 8 59 56 97 94 185 182 167 164 98 94 63 60 25 22

No. captured 703 Date captured June-July '1985

680

1

1st

1 3

2nd

20 18

3rd

4 16 3 15

4th

5th

10 4

6th

2 5 1

1 -

1

4

38

35

17

8

1 2

56

94

182

164

94

60

22

= 672 + 8

2nd

3rd

4th

5th

6th

= 680

30th 1st

= 105

Ratio of total captured, total marked and released and total recaptured respectively = 1.03 : 1 : 0.15

66

Table 14. Corrected recaptures in March and July,1985 at Chandipur,Orissa.

A. Date marked & released(DMR) 6th 7th 8th 9th 10th 11th 12th 13th N (-) Date captured

7th 13.368

DATE 9th 13.644 7.128

8th 9.671 3.128

7th

(7 th - 13 th March'1985)

8th

(DMR) 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th N(-)

2nd -

3rd 31.251 20.832

-

-

Date captured

2nd

3rd

4th 4.524 19.005

29/6 30/6 1st 2nd 3rd 4th 5th 6th N (-) Date captured

Average = 2142 11th

12th

13th

6th 3.816 6.714 7.709

-

-

2583

1946

2284

4076

368

4th

5th

6th

7th

8th

9th

10th

1st

22.312

13.287 5.692

30/6

13th 6.994 -

5th 9.862 9.139

30/6

-

N (+) 785 1,144 5,820 -

(2nd - 12th May'1985)

C. (DMR)

820 10th

9th

B.

RECAPTURED 10th 11th 12th 4.084 8.865 1.477 1.431 1.718 1.431 2.314 -

7th 8th 9th 10th 11th 12th 3.285 4.281 4.934 8.293 4.182 11.687 7.222 9.885 6.782 12.721 12.687 8.512 17.790 16.821 11.831 23.386 7.710 5.443 6.126 206

N(+)

178 225 1,042 3,850 1,857 2,273 1,541 - 1725

11th 12th

(29th June - 6th July)

2nd 19.599 10.504

3rd 4.348 10.357 5.015

4th

5th

3.389 5.835 2.589

2.029 5.070 1.771

6th 2.767 7.571

-

-

935

1490

1873

-

1st

2nd

3rd

4th

5th

6th

N(+) 476 473 980 2042 1181

67

Ta b le 15. The values for r+ , r- , a0 and N in March , May & July 1985 at Chandipur (Orissa) POSITIVE r

+

METHOD

a0

N(+)

1985,March 7th 8th 9th 10th

1.0570 0.6466 1.0000

1985,May

0.3281 0.4552 0.7919 0.8861 0.8163 1.2402 1.2291

56.2573 44.3782 9.6001 2.5972 5.3861 4.3991 6.4891

178 225 1042 3850 1857 2273 1541

0.9462 0.6583 0.7181 1.0233

21.0187 21.1552 10.2043 4.8962

476 473 980 2042

2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th

1985,July 30/6 1st 2nd 3rd 4th 5th

12.7395 8.7387 1.7180

NEGATIVE METHOD

785 1144 5820

r-

a0

N(-)

Average of March= 2142

0.7951

12.2018

Average of May =

1.3692 1.2481 1.4083 1.6562 0.6593 0.4012

820

1725

3.8712 5.1392 4.3783 2.4531 27.1582 48.4533

2583 1946 2284 4076 368 206

Average of July = 1181

0.9773 0.9131 0.9632

10.6972 6.7103 5.3378

935 1490 1873

48 57 83 187 197 225 260 147 92 68 107 56 38 49 57

d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d11 d12 d13 d14 d15

Ri =

ni

i 48 56 80 185 192 224 255 142 90 68 100 56 36 48 56

ai

3

1 2 1 0 0 0 0 0 0 0 0 0 0 0 0

capture released

capture

Total

Total

3 28 15 0 4 0 0 0 0 0 0 0 0 4 32 27 12 8 0 0 0 0 0 0 0 5 32 24 7 0 0 1 2 3 0 0 6 22 19 8 0 6 0 5 0 7 7 17 9 13 2 1 2 5 0 8 8 4 0 3 0 3 1

9 17 2 0 4 0 5

10 9 4 2 0 0

11 13 4 1 4

12 11 13 2 10 14 0 5 8 15

Day when last captured (j)

4 47 79 69 67 49 19 28 15 22 12 15 8 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14

2 2 1 0 1 0 0 0 0 0 0 0 0 0

The tabulation of recaptured data according to the data on which the horse shoe crabs were last caught for analysis ( afterJolly , 1965)

Date of

A.

Table 16.

68

1 2 1 0 0 0 0 0 0 0 0 0 0 0 0 1 Z2

d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d11 d12 d13 d14 d15

Z (i-1) =

3 29 15 1 4 0 0 0 0 0 0 0 0 4 47 28 16 8 0 0 0 0 0 0 0 5 60 40 15 0 0 1 2 3 0 0 6 62 34 8 0 7 2 8 0 7 7 51 17 13 9 3 10 5 7 8 25 17 9 6 10 8 8 9 34 11 6 14 8 13

10 20 10 16 8 13

11 23 20 9 17

ri

12 31 13 11 21 14 17 22 30 15

2 20 52 61 66 64 58 52 57 46 28 22 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 Z11 Z12 Z13 Z14

2 3 1 0 1 0 0 0 0 0 0 0 0 0

on a given day bearing marks of day or earlier (after Jolly , 1965)

B. Calculated table of the total number of marked animals recaptured

69

17 18 19 20 21 22 *FM 23 24 25 26 27 28 29 30 31

0.035 0.036 0.155 0.238 0.266 0.238 0.346 0.271 0.500 0.186 0.410 0.815 0.428 0.526

∝i

0 16.0 6.404 75.835 191.695 263.940 405.469 529.315 211.428 269.733 279.091 237.666 98.2 153.0 -

Mi

risk

No. marked

animals at

Proportion

May , 1986 of recaptures

Days of

457.142 177.888 489.258 805.411 992.255 1703.651 1520.812 780.177 539.466 1500.489 579.673 120.490 357.476 -

Ni

Total population

0.3333 0.0914 0.9092 0.8268 0.7839 0.9474 0.8844 0.3408 0.9757 0.9188 0.6618 0.3628 1.4825 -

0i

Survival rate

0.3213 0.0414 0.0396 0.0411 0.0546 0.0685 0.0878 0.0643 0.1274 0.1857 0.1098 0.0882 0.2562 -

V(0i) 523.7608 85.6532 87.4896 125.9477 149.2452 300.2092 374.4005 181.4503 136.3980 424.6594 174.4444 21.5786 128.5370 -

V(Ni / Ni)

0.3139 0.0231 0.0240 0.0327 0.0497 0.0676 0.0868 0.0614 0.1854 0.1850 0.5750 0.0832 0.2693 -

V(0i / 0i)

Standard errors due toerrors in the estimation of parameter itself

79.6189 93.9395 106.0502 97.9702 118.7688 290.7265 147.5546 154.2766 362.3066 249.4151 39.2232 78.8038 -

V(Bi)

STANDARD ERRORS

524.1970 86.6849 90.2383 129.1030 152.5308 303.0322 376.4252 183.5881 138.3489 426.4219 176.0974 204.1878 129.9201 -

V(Ni)

* FM , Full moon

136.196 330.249 402.546 364.812 764.536 27.525 260.521 -219.801 1004.827 - 408.718 - 89.815 171.814 -

Bi

No. of new animals

Table 17 . Estimated population of T.gigas at Digha sea shore by Jolly - Seber (1965) method

70

1 sq.km.=106 sq.mt.

5000

* toa , terms of area , and tod terms of days

Population in terms of area (toa* )

7500 2500

3333 3333 4167 4167 3333 2500 3333 3333 Total..............

0 1 0 1 0 0 0 0 6

0 0 1 2 1 2 0 0 4

4 4 5 5 4 3 4 4 2

0 0 1 0 0 1 0 0 2

0 0 0 1 0 0 0 1 1

0 0 0 0 0 0 1 0 2

1250

1 1 2 0 0 0 0 0 3

2 1 0 0 0 0 0 0 3

0 0 1 0 0 0 2 0 3

1 0 0 1 1 0 0 0 2

0 1 0 0 1 0 0 0 3

0 0 0 0 0 0 1 2 2

29/3

4432

3437

Population in tod* ( 12 days )

30/3

2500

28/3

31/3

2500 ---------

27/3

26/3

3750 3750

Number of T. gigas in Eight Transects ( 102 mt) counted from 20th to 31st March , 1986 at Chandipur in area of 1 sq. Km. 25/3

24/3

23/3

22/3

1 0 2 0 3 0 4 0 5 1 6 0 7 0 8 1 800 sq.mt.

21/3

2500

20/3

3750

sq. mt.

(10 X 10)

Transect

2500 3750

Table 18 .

71

Table 19. Correlation and Regression between estimated populations (N) with

captured , marked and released (C) and recaptures (R) at Chandipur and Digha , India

JACKSON'S METHOD

JOLLY - SEBER'S METHOD

Chandipur sea shore ( March - July , 1985)

Correlations: N = 20 , r = 0.647 , df 19 , P< 0.01 Se = + 0.130, t = 3.600 , df 18 . P < 0.01

Regression (I):

y = 0.8822 X + 1.2854

Digha sea shore ( May , 1986)

N = 13 , r = 0.687 , df 12 , P < 0.01 Se = + 0.146, t = 3.134 , df 11 , P = 0.01

y = 0.8578 X + 1.0709

tan 0 = 41.420

tan 0 = 40.620

Relationships between N and R

Correlations:

N = 17 , r = 0.655 , df 16 , P< 0.01 Se = + 0.138 t = 3.632 , df 15 , P < 0.01

Regression (II): y = 0.6608 X + 2.2259 0

tan 0 = 33.45

Estimated population (N) :

y = 4.85 (Log) 70,795

N = 13 , r = 0.481 , df 12 , P > 0.05 Se = + 0.213 t = 1.819 , df 11 , P > 0.05

y = 0.3621 X + 2.2871 tan 0 = 19.900

y = 3.26 (Log) 1,820

..........."unauthorised sacrifice"............... "unauthorised sacrifice" because in India the "donor crabs" are not returned to its natural habitat ; rather they are sacrificed in whole for its total blood . Like U. S - F. D. A. regulation , neither such exists in India nor it's taken up in action ...........this is why the author has stressed upon the term "unauthorised sacrifice.

73 Table 20. Showing

the relation between total marked and total recaptured horseshoe crabs , T. gigas in respect to its sex with recovery rate .

MONTH Total captuof 1985 red , marked

Sexual isolation

and released

Total re-captured and re-

Sexual isolation and recovery rate (%)

covery rate (%)

MARCH

653

333

320

73(11.17)

MAY

1120

568

552

307(27.41)

JULY

680

350

330

105(15.44)

Correlation between captured and recaptured animals , r*

38(11.41)

33(10.93 )

r = 0.699 , p < 0.05

165(29.04) 142(25.72)

r = 0.739 , p < 0.01

60(17.14)

45(13.63)

* "r" represents the correlation coeficient of the relationship of day to day captured and recaptured animals

Amplexus occurs in the off-shore shallow water but not in the mud or sand-beach . This inference can be made from the recovery of males and females in isolation bearing similar marks / rubber bands days after capture , tagging and segregation .

r = 0.937 , p < 0.01

74 FULL MOON DAY

123456 123456 123456 123456 123456 123456 123456 123456 123456 123456

123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456

123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456

1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567

6

7

8

9

110

100

Individual No. Tachypleus gigas

90

80

70

60

50

40

30

20

10

Date of High Tide

Number of adult T.gigas

FULL MOON NIGHT

123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 10

123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 11

123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 12

1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 1234567 13

MARCH ' 1 9 8 5 on or near the breeding beach of Chandipur

Fig.9. Presents the number of spawning horseshoe crabs T. gigas in two respective high tides of day and night .

O ♀ SR 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

550

500

123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

350 O

♀ 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

300

250

FULL MOON TIDE

200

150

100

No of

Tachypleus gigas

50

6 7

8

9

10 11 12 13 Total 1 2

M A R C H

Fig .10 Fig.10 .10.

SR = 1.04 : 1

D a t

e

FULL MOON TIDE

3

4 5

6 7 8

M A in

= 1.05 : 1

O SR = 1.07 : 1

FULL MOON TIDE

9 10 11 12 Total 31 1

Y

2

3

4

5

♀ 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

6 Total

J U L Y

1

9

8

5

Shows the number of T. gigas counted in successive days of March, May and July, 1985, with respective total of males and females displaying sex-ratio at the tops(this imbalace idicates increasing death rate of the males in comparism to that of females).

5820

4000

3000

Estimated Population of Tachypleus gigas

2000

Full moon

1000 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234

7 8 9 10 MARCH

12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 Full moon 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 123412345 12345 12345 123412345 12345 1234 12345 123412345 12345 12345 1234 12345 12345 1234 12345 123412345 12345 12345 1234 12345 12345 1234 12345 123412345 12345 12345 123412345 12345 1234 12345 123412345 12345 12345 123412345 12345 1234 12345 123412345 12345 12345 1234 12345 12345 1234 12345 123412345 12345 12345 123412345 12345 1234 12345 12345 1234 12345 123412345 12345 12345 1234 12345 123412345 12345 12345 12345 1234 12345 12345 123451234 12345 12345 123412345 12345 12345 12345 1234 1234512345123412345 12345 12345 1234 12345 12345 123451234 123412345 12345 12345 1234 12345123412345 12345 12345123412345 12345123412345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 1234 12345 12345 1234 12345 12345 1234 12345 12345 1234 12345 12345 12345 123451234 1234 12345 12345 1234 12345 12345 1234 12345 12345 12345 123412345 12345 12345 12345 123451234 12345 123412345 12345 12345 12345 1234 12345 12345 1234 12345 12345 12345 1234 12345 12345 12345 1234 12345 12345 12345 1234 12345 12345 12345 1234 12345 12345 12345 1234 12345 12345 12345 1234 12345123412345 12345 12345

2 3 4 5 6 7 8 9 10 11 MAY 1 9 8

76

Full moon

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30

1 2 3 4 5 JULY 5

Fig.13. Showing the number of T. gigas estimated at Chandipur sea shore with the help of Jackson's "positive" (black bar) and "negative" (dotted bar) methods.

77

40

Mated

12345 12345 12345 Solitary 12345 12345 12345

O + ♀ O

O

Solitary ♀

18P 35 16P 15P 30 25

No of Mating Pairs

20 8P 15

7P

10 5

1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234

March

12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345

12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345

1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234

April

May

June

1

9

8

1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234

12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345

July

Aug.

5P 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234

Sept.

12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345

♀

12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345 12345

160

120

80

40

TOTAL

7

FIG.11. Represents the occurrence of mating pairs (P) , solitary males and females of Carcinoscorpius rotundicauda , and showing the sex- ratio (SR) at the extreme right hand column .

No of Solitary Animals

21P

Sex Ratio = 1.41 : 1.00

45

Mated males and females are very hard to separate from the amplexus and when the two are separated forcibly and placed on sand or hard mud-bed the larger ( female ) one moves down toward the lower littoral zone which the smaller (male) appears to be helpless and does not usually follow its mate . But placed close enough , however , the male follows the female's track (Fig. 25) to get its normal position . This field observation was also endorsed by Drs. B.N. c.f. Roonwall , 1944). Chopra and H.S. Rao (c

From the capture-recapture data and from the collection of dry carapaces male to female sex-ratio was found to be 6.89:1 at Digha and 1.04:1 at Chandipur . This disbalance in normal sex-ratio (1:1) ................................................. at Digha fishermen enhanced the predation by throwing them to crows . In Thailand disbalance in sex-ratio of T. gigas and C. rotundicauda is also noticed which is due to the fact that the people collect the eggs to serve on the table as food (Sekiguchi et. al. , 1977 : Sekiguchi and Nakamura , 1979 and Dr. A . Choudhury's personal observation on 1986) . Another possible factor ...............................may be the "unauthorised sacrifice" of the female crabs ( because of their large size) as very important laboratory material for bio-medical research by draining its ' blue blood' to get the precious lectin "carcinoscorpin " (Dorai et. al. , 1981 ; Srimal et. al. , 1985 ; Vijaya Rao and Bhagirathi ,1989; Rudloe , 1983)

ø

W hy the w or ds lik e "unauthorised sacrifice" ? wor ords like

..............asked the foreign expert.............. for explanation please see p.72

79 DISCUSSION :

Population of the American horseshoe crab Limulus polyphemus was estimated following the capture - recapture methods of Baptist et. al. (1957) , Rudloe (1980) , Shuster (1950) and Sokoloff (1978) . For the first time in the Indian sub-continent Debnath and Choudhury (1988a) estimated the population of one of the two Indian horseshoe crabs, Tachypleus gigas by using the above principle at Chandipur sea shore . The breeding horseshoe crabs ( T. gigas ) at Chandipur showed strong lunar and tidal rhythms , with animals appearing at and within few days of fullmoon and within 2 hrs. of the time of flood tide(Fig. 9).Among several variables only lunar cycle , day of the year , and wave- height correlated significantly with number of carbs on the beach (Rudloe , 1980) . Table 20 shows the significant correlation between total marked (after initial capture) animals and recaptured animals in March , May and July . Total recovery rates of recaptures in respect to the total captures of each month are 11.7% , 27.41% and 15.44% respectively . These results indicate that the maxima of population occurred in May i.e. , the mid-breeding season and which started in February - March and declined in July-August (Fig. 10) . Considering the recovery rates in term of sex this table also showed that the males always dominated over the females . This kind of male sex dominance over the females was common in L. polyphemus , but expected to be a normal sex-balance in Indian species . From the capture-recapture data and from the collection of dry carapace , male to female sex-ratio was found to be 6.89 : 1 at Digha and 1.04 : 1 at Chandipur (Debnath , 1985) . This abnormality in normal sex-ratio (1:1) seemed to be a result of severe predatory pressure at Digha than at Chandipur which was caused by C. splendens ( Debnath and Choudhury , 1988a) . Furthermore , the capture-recapture data indicates a clear variation of the total number of arriving animals on or near the breeding habitat . The maximum numbers occurred on 9th March (5,820 animals) . The author , however , failed to capture horseshoe crabs on the following new moon tides (15th to 25th March) . This was also evident in the new moon tides of May and July . Population peak was also recorded between the dates of 4th and 9th May , associated with the full moon tides of 6th , while in July , maximum number of crabs were estimated on or before 5th , the full moon night (Fig. 13) . For animals , dispersion is a common phenomenon in case of mobile species and it has also been proved from the result of the capture-recapture experiment performed at Chandipur sea shore within an area of 2 X 3 sq. Km. But an interesting observation in recovering two segregated opposite sexes further amplexed (coupled) 3 days after release has rendered the author to assume that specific sexual selection might had occurred during the breeding activities. But how they choose themselves and found together and returned to the breeding ground remained a great mystery . Direct application of Lincoln - Peterson Index on 16th -17th March 1986 at Chandipur revealed 4,260 animals that came to breed which in turn has been corrected as 4,071 with variance and standard error SE ( + 835.439) following Bailey (1951 , 1952) . This observation corrobarates with the findings of the same month of the previous year (1985) following Jackson's principle . While estimating the population size at Digha sea shore , the errors that evolved in Jolly-Seber's stochastic method may be the result of two factors ; a) "small sample biases" inherent in the method and b) biases due to the violation of the assumptions. The former will be minimal when the number of recapture is large . Manly (1971) and Roff (1973) have shown that these errors often express themselves as estimates and their variances . Some improvements could be obtained if N and O are transformed to logarithms before making the calculations ( Manly , 1971 ) . If one looks at Table -17 on the row of 18th May , it becomes clearly evident that error due to "small sample biases" crept

80 i)

^N (457.142) 18

ii)

^N

18

< (457.142) <

^

VN18 (524.197)

^ / ^ VN N18 (523.760) 18

on that very day (18th May) a total 56 animals were marked and released in which the number of recapture ( marked on 17th) was only 2(two) and the survival rate ,^ θ18 is so low (0.0914) that all results gave considerable errors . However , Jolly-Seber's method (1965) applied for the estimation of T. gigas population at Digha in the year of 1986 showed variation in the estimates (120.5 to 1703.6 ). Maximum number of animals occurred at the full moon tides . Numbers of emigrated or immigrated animals were also determined with their variances (Table - 17) .

Discredit should be given to the discrete local population of Indian horseshoe crab, T. gigas at the sea shore of Digha as because the animals were being repeatedly threatened by human interference , continued fishing with long tow nets and over-predation by Corvus splendens (Debnath , 1985 ; Debnath and Choudhury , 1988a) .

81

D. Absolute population estimates of Tachypleus gigas with regression lines: In the foregoing chapters the author has discussed various marking techniques to estimate the population size of T. gigas in which there were distinctively three sets of data i.e. i) estimate population (N) , ii) capture , marked and released (C) and iii) recaptures (R) . Here the author has deployed both correlation and regression between N vs. C and N vs R . Now , theoretically , if in an imaginary area of estimate , the number of total captured , marked and released animals become equal to recaptured animals the area , obviously , will show the absolute estimate (N) . This hypothesis can be obtained from the " Lincoln Peterson Index " as below : nxa N = , i.e. N = n , when a = r r Keeping this in the mind the author has constructed two regression lines and placed them on graph paper . The co-ordinate on which two regression lines intercept each other will indicate the X-value for C = R and thus the Y value for absolute estimate (N) of the population . RESULTS Correlation coefficients were determined between N & C and N & R for both Jackson's and Jolly-Seber's methods , with its standard errors (se) and t-values (Table 19) . Then two regression lines were constructed for both the methods and fitted them on graph papers (Fig. 14) with intercepting co-ordinates and tangants to X-axis . The intercepting co-ordinates ( X and Y-values) were then antilogged to find out the theoretical estimates . E. Population estimation of standared T. gigas at Chandipur beach by Line Transect method In the soft sandy shores of Chandipur , T. gigas , mostly in pairs , remain scattered partly or fully embedded under silty-sand to avoid desiccation and rising temperature , and its anticipated predators Corvus splendens . The alternative explanation for these stranded "crabs" may be placed like this that to avoid to and fro repeated journey between the tide marks during the ebb , they prefer to stay at the upper shore level embedded under the substratum only to wait for the arival of the next tide . An attempt was taken by the author to estimate the population of these standard and sparsely distributed carbs in an area of 1 Km2 of Chandipur from 29th March to 31st March , 1986 following Line Transect method (Vide Chapter : Materials and Methods) . Every day eight transects of 10 m2 each were randomly selected on the stretch of 1 Km to get the count of the crabs . CALCULATION AND RESULTS Horseshoe crabs counted in each transsect (Table 18) are calculated in two ways :1. Population of T. gigas in 1 sq. Km. in terms of area (ITOA) . Mean value of N = 3437.5 + 1541.52 2. Population of T. gigas in 1 sq. Km. in terms of days (ITOD) . Mean value of N = 3437.5 + 499.56 Now if this mean value of N is campared with the estimate (followed by Lincoln's method) of 16th - 17th March the t-test did not show a significant deviation as found the comparison of two means described below :

82

Comparison between two means : Capture-recapture method ( 26th - 27th March , 1985)

Line Transect method ( 2oth -31st March , 1986)

n = 4 X = 2142.25 SD = 2127.958

12 3437.5 1541.52

X 1 - X2 t =

S2 1 n1

+

S2 2

= 1.123, df. = (12+4) - 2 = 14 , p

0.05

n2

Total 5000 and 7500 horseshoe crabs were estimated for 26th and 27th March , respectively by using the line transect method (Table 18) and the average value thus obtained is 6250 . Then following Gaskell and George's (1972) formula an + 2N , where N is the estimate followed after the line transect method . n = r+2 300 X 284 + 2 x 6250 = 20 + 2 = 4,441 . Therefore , the population estimate of T. gigas following the three different principles comes to : 1)Lincoln - Peterson Index (1930) 2)Bailey (1951 , 52) 3)Gaskiell and George (1972)

: N = 4,260 + 918.41 : N = 4,071 + 835.43 : N= 4,441

The above figures of N indicate that the population of T. gigas at Chandipur sea shore was nearly 5,000 that came to breed in the month of March , 1986 and this figure corroborated with the maximum estimate (N= 5,820) of the same month i.e. , March of previous year (determined after Jackson's formula) .

83 5.0

4.85 ( N = 70,794 ) Field Station : CHANDIPUR ( ORISSA )

4.0

Y=

∆ = 33.45

o

∆ = 41.42

o

Y=

2 82 8 0.

X

+

r 4, 5 28 1.

=

5 47 6 0.

Fig . 14 Absolute population of T. gigas estimasted with regression method at two different field stations Chandipur and Digha

2.0

1.0

0 1.0

2.0

3.0

4.0

CAPTURE / RECAPTURE 3.5

3.26 ( N = 1820 )

Field Station :

DIGHA (West Bengal)

3.0

Y

21 X 6 3 . =0

2, r 287 . 2 +

o

∆ = 19.90

(Ñ) ESTIMATED POPULATION

ESTIMATED POPULATION

(Ñ)

3.0

X 08 6 0.6

+

r 9, 5 2 2.2

6 55 6 . =0

Y

2.5

=

78 85 . 0

X

+

1 481 . 0 =

r 9, 0 07 1.

=

72 8 6 0.

o

∆ = 40.62 O

1.5

CAPTURE / RECAPTURE

2.0

2.5

84 DISCUSSION At Chandipur for one breeding cycle during the period of March to July , 1985 , both correlation and regression showed significant results (Table 19) where the maximum likelyhood or theoretical number of capture , marked and released animals (C) became equal to recaptures (R) , i.e. , 8,912 . In that case the author can predict that maximum 70,795 animals (Fg. 14) came to breed at Chandipur in 1985 during the spawning season (spring and summer months) . While at Digha , the correlation between N and R showed non-significant relationship ( r = 0.481 + 0.213 , P> 0.05) , but significant correlation between N and C (r = 0.687 + 0.146 , P< 0.01) . Thus if the non-significant former regression line intercepts with the significant latter line the X value showed 283.79 and Y = 1820.00 . That means almost 1500 T. gigas came to breed at Digha in May 1986 . But this may be an underestimate because of insufficient data regarding recaptures for which" r" showed non-significance in the relation between N and R . The author , in this context , surmises that a complete set of date of one fullmoon breeding season could have yield better result as in the case of Jackson's method applied for Chandipur population . It has already been established in the field of population estimation that Jolly-Seber's stochastic model is the only deterministic approach if it becomes fitted with a complete and unbiased data (Southwood , 1978) . Discredit should be given to the discrete local population of T. gigas at the sea shore of Digha as because the animals were being repeatedly threatened by human interference , continued fishing with long tow nets and over-predation by Corvus splendens (Debnath , 1985 ; Debnath and Choudhury , 1988a) . During the feild investigation of the project programme there were severe constraints regarding field infrastructure facilities in the remote coast line areas of the two states (Orissa and West Bangal) . The author was quite aware of these short coming and tried his level best to bring down the apprehended statistical errors during the generation of data related 'crab' population dynamics . What the author contends is that an organised team with working facility in the feild and assistance for a longer duration taking many more stations amost the coast may help to bring out a more comprehensive population picture of the Indian horseshoe crabs .

85

D . Breeding activity ; Sex-ratio and predation

86 INTRODUCTION The mating or spawning behaviour of all extant horseshoe crabs , comprising three genera and four species , is one of the easiest facets of the Limulidae life cycle to study because the animals come ashore to spawn . Although their breeding behaviour is remarkably similar (Shuster , 1982 ; Sekiguchi , 1988) . Detailed study on the breeding behaviour and spawning activity of American horseshoe crab L . polyphemus have been conducted by various workers as because of its greater abundance and wide range of spawning areas (Barlow et. al . , 1982 , 1986 ; Botton et. al. 1988 ; Brockmann , 1990 ; Cavanough , 1975 ; Cohen and Brockman , 1983 ; Dunton , 1953 ; Finn et. al. 1990 ; Rudloe , 1980 ; Rudloe and Herrnkind , 1976 ; Shuster , 1953 , 1958 ; Shuster , 1979 ; Shuster and Botton , 1985) . Despite the occurrence of three species of horseshoe crabs , widely distributed in the coastal waters of south-east Asia , very little information is available on their breeding behaviour , spawning migrations and other activities in comparison with that of L. polyphemus .While, Sekiguchi and Nakamura(1979), for the first time, provided a comprehensive and comparative account of Asian horseshoe crabs with reference to their breeding biology . In case of the Indo-pacific horseshoe crabs , the breeding season also varies geographically , from species to species (Debnath , 1985 ; Debnath and Choudhury , 1985 ; Sekiguchi , 1988 ; Sekiguchi and Nakamura , 1979 , Waterman , 1953) .

87 OBSEVATIONS AND RESULTS A. Breeding season : In the present study the breeding season of Tachypleus gigas has been found to vary from late February or March to July . On the other hand , Carconoscorpius rotundicauda arrives in the estuaies , especially in Sundarbans (Prentice island and Sagar Island) from March to September . While , in the Gulf of Siam (Thailand) this species lays eggs all round the year (Sekiguchi & Nakamura , 1979) . It is also generally recorded that the egg laying season begins from April both in T. gigas and C. rotundicauda . B. Spawning activity : T. gigas and C. rotundicauda are found in the coastal waters of India , inhabiting the Bay of Bangal , from the sandy shores of Orissa to the muddy-sands of estuarine Sundarbans , West Bengal . Both T. gigas and C. rotundicauda usually make pair in the open water in the infra- littoral zone prior to reaching the spawning area (Debnath , 1985 ; Debnath and Choudhury , 1988a) . During the breeding season horseshoe crabs emerge from the sea and come to the shore line upto the highest mark of high tide associated with fullmoon tides in mating amplexus (Fig. 22 A,B) . The females carries the male on her back , the latter is being attached to the female's opisthosoma by holding her marginal spines with his modified clasper legs (Fig. 22 B , 16A) . In sexually mature males , the 2nd and 3rd prosomatic appendages (1st and 2nd legs) are modified into clasper organs . The male clasper organ is chelate hook-like in C. rotundicauda , while it is non-chelate , bent and stout hook in T. gigas(see Fig.18 C) . The holding mechanism is so strong enough that to separate the male from the female's back is very difficult . At Chandipur (Burabalanga river mouth) the breeding activity of T. gigas has been studied in great detail . It has been observed that the animals come near the sea shore in pairs (amplexed) when the tide level reaches at the upper littoral zone . The pairs are characterized by their larger female partner carrying a male of small size on her back . Mated pairs are usually found to prefer the shore where wave action is minimum . The high tide peak stayed for 20-30 mins . during which time , the amplexed crabs showed a kind of sexual courtship just along the margin of the tide water . All of sudden one pair may emerge from the water's edge , move freely here and there , then disappear in the water and reappear nearby or at a distant place , possibly in search or selection of a suitable site for spawning . On the exposed intertidal beach , when the water receeds back , some characteristic horseshoe shaped swollen impressions on the sand helped the investigator to detect the mating couples . Such markings were indistinct on the mud flats of Sundarbans (West Bengal) and Dhamra (Orissa) due to the presence of heavy mud enriched with silty clays . In that case somewhat depressed area with broken mud cake revealed the existence of C. rotundicauda ( Fig. 21B , 22c) . Studies with mechanical marking as well as tagging with rubber bands on the seggregated males and femals of both species revealed no further formation of amplexus or coupling between opposite sex on or near the beaches of sand- or mud-shores except an exceptional occurrence at Chandipur (Table -20) . This is an indication that the crabs , having spawned on one beach , did not spawn on another.

88 C. Sex -ratio : During high tide pairs of T. gigas were captured from the Burabalanga river mouth . Animals of both species of Indian horseshoe crabs showed 1 : 1 sex- ratio during pairing . But at the low tide when the intertidal sand or mud-flats get exposed showed some sort of disbalance in the above sex-ratio. Because it was observed that in comparison to that of "solitary females" the solitary males are "very common". Since the total capture-recapture data included both the samples of high tide and low tide , the observed results obviously revealed the presence of higher number of males over the females in the spawning area ( Fig. 10) . Similarly, the total net collection of T. gigas at Digha sea shore usually showed more males than females . Solitary males were very common at the mud-flats of Prentice island , Sagar Island (Chemaguri ) and Dhamra (Koithkola, Orissa) . In all the above study sites C. rotundicauda occurred except at Dhamra where both T. gigas and C. rotundicauda orrurred in the same creek (Fig. 12). D. Predation by crows , Corvus splendens L. :

(Debnath & Choudhury , 1988a)

At Chandipur and Digha several mating pairs of T. gigas were found on exposed beach (fig. 27 A) during low tide when they were unable to make burrow quickly and hide themselves in the sand( Fig. 27 B) . It was the appropriate time for the predator birds to locate their prey . The crow sat beside the pair and upturned them with their beaks and claws . The crab always tried to protect itself by moving the swordlike telson around . But once they were uoverturned , they were susceptible to predation easily , especially the female crabs . The predator pierced the ventro-lateral soft chitinous layer of females prosoma and took eggs one by one , while the male still remained clinged to the marginal (opisthosomatic) spines of the female with his clasper legs . In most cases it was observed that the male remained unhurt by the crow and the predator hardly took the flesh of the crab , although injured by mistake ( Fig. 27 C) . It was also observed that more than one crow shared the paired crabs , but normally one crow was found to be engaged in this operation ( Fig. 27 D) . A gravid female usually carried 100 to 180 gm of eggs in an average of 600 gm of total body weight . The whole prosoma was being filled with mature eggs , (in an average 6,000 in number ) . Approximately , each egg measuring about 3.7 - 4.0 mm. in diameter and looked like the seeds of the ladies finger (Fig. 28 A) .

E. Tandem amplexus in C. rotundicauda : During June 1988 , the investigator found 5 to 25 mating pairs of C. rotundicauda in each of the seven narrow channels (sub-creeks) of Chemaguri Creek situated at the south-eastern part of Sagar Island , West Bengal . While stdying the breeding behaviour of these pairs ( Fig. 23 A) it has also been observed that an unusual coupling of two males , one behind the other (in tandem) . When the mud was cleared away , in front of the forward male , a female was exposed (Fig. 23 C) . All these three animals were in a linear row (Fig. 23 B) . When pulled out of the mud and the manner of attachment examined , the first male was found to be attached to the female , hence a "clasping" male , with the second male attached to the first male , forming a "satellite" * . These two males were about equal in size , shape and weight . Table 21 presents the total counts of male and female horseshoe crabs of both species ( T. gigas and C. rotundicauda) and their sex-ratio (SR) obtained from five stations , ranging from estuarine Sundarbans of West Bengal to Dhamra of coastal Orissa 6.89 :1 was the highest SR determined at

89

Digha , while the lowest one was 1.04 : 1 obtained from Chandipur ; in both the cases the studied specimen was only T. gigas . Both T. gigas and C. rotundicauda have been found to occue in creeks and mud-bottoms of estuarine Dhamra as sympatric population , where they showed almost similar sex- ratio. The X2 - test of male versus female horseshoe crabs reveals that it is being significantly disalanced in the three coastal stations of West Bengal , amongst which Digha sea shore showed the most vulnerable area for severely damaged sex-ratio (X2 = 83.62 , P< 0.001) . On the other hand , the coastal range of Orissa was not as that much under predator pressure to reverse the desired sex-ratio .

A combined histogram (Fig. 12) has been presented for showing the occurrence of horseshoe crabs in two mud - flat mangrove ecosystems of Chemagari and Dhamra , respectively in W.B. and Orissa . Total male and female counts revealed a high deviation in sex balance of C. rotundicauda in the former station (SR = 1.44 : 1 , P= 0.01) , while the SR (1.09:1) for the same species in Dhamra showing no significant deviation i.e. P> 0.05 . such is the case for T. gigas (SR = 1.21:1, P> 0.05) at Dhamra .

SOLITARY CRAB

PAIRED / MPLEXED CRABS

90

Table 21 .

Study area , period , total males and females of C. rotundicauda are T. gigas are shown to determine the male to female sex-ratio and Chi-square (X) tests* with significant (P) levels

Study Area

Period

Species type

Total

Total

Male Female Prentice Island West Bengal

Feb'86 to Nov. '87

Sex Ratio

2

X - test

P

(Male/Female)

C. rotundicauda

218 177 (197.5) (197.5)

1.23 :1

4.255

0.05

Chemaguri Sagar March -June C. rotundicauda Island , W.B. 1988

296 205 (250.5) (250.5)

1.44:1

16.528

0.001

Digha Sea Shore West Bengal

May-June 1986

131 (75)

6.89:1

83.626

0.001

Chandipur Orissa

Feb - March T. gigas 1985

333 320 (326.5) (326.5)

1.04:1

0.258

------

Dhamra Orissa

April-May 1987

75 (68.5) 69 (66)

1.20:1

1.233

------

1.09:1

0.272

------

T. gigas

T. gigas C. rotundicauda

19 (75)

62 (68.5) 63 (66)

*Theoretical values in parenthes is below the observed values

A bunch of Eggs seems to be the bunch of Pearls

C. rotundicauda (May -June) 1988

25

C. rotundicauda (April-May) 1987

Chemaguri , Sagar Island , W.B. (Low tide)

25

T. gigas

Koith-Kola , Dharma Orissa (Low tide)

50

-

T. gigas (May-June) 1986

Digha sea shore West Bengal (High tide)

50

15

10

10

10

15

Marking device

Do

50-200 mts

Do

Nil

Total = 9,R = 18%

5-20 mts 2 at 5 mts apart from 2 at 15 mts one another 2 at 50 mts 1 at 100 mts 1 at 200 mts 1 at 1000 mts

Release of Release of Recovery of intact pairs seggregated pairs : number at distance animals at and distance from the site distance from of capture one another

Nil

R = 6.66 %

1 pair , both sexes with similar marks recovered at 200 mts away from the orginal site of capture

Recovery of seggregated animals ; if any

Do

Manipulated Random release Total 17 pairs released in I in III - IV sub- recovered from to V sub creeks creeks sub-creeks & (ave. distance mud flats with 100 mts.) random distri bution R=34%

Sluggish

Active

Active

Remarks

Nil , but with 6 Sluggish similar marked males & females in same creek (IV) distributed with in 50 mts .

Colored rubber bands Manipulated Random release Total =8( R=32% ) Nil tied on telson trunk released in I-II in I & IIsub =10 (R=40%) sub creeks creeks Recovered from sub(ave. distance creeks and mud flats 200 mts.) with random distribution.

Do

Mechanical markings (with steel needle) on carapaces

Number of Number of intact pairs seggregated pairs

T. gigas (March-July) 1985

Species type and period

Chandipur sea shore Orissa (High tide)

Study Area

Bengal , India .

Table 22 . Movement , dispersion and recovery rate (R) or the breeding populations of two Indian horseshoe crabs at four coastal stations of Bay of

91

92

A quack practitioner sacrificed the crabs, cut off the limbs, boiled in mustard oil and sold it to the people !

possible factor for the depletion of the female horseshoe crabs in coastal West Bengal of Orissa may be the unauthorised sacrifice of the female crabs ( because of their large size)

FM

+

T. gigas

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1

2

3

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Population Occurrence: 12345 12345 12345 Sympatrism , ie. both 12345 12345 12345 12345 C.rotundicauda & T.gigas 12345 12345 12345 12345 12345 12345 12345 12345 12345 1234 12345 10 12345 12345 1234 12345 12345 1234 12345 12345 1234 12345 123456 12345 12345 1234 12345 123456 12345 12345 1234 12345 123456 12345 12345 1234 12345 123456 12345 12345 123456 1234 12345 12345 123456 12345 12345 1234 12345 123456 12345 12345 1234 12345 123456 12345 12345 1234 12345 123456 12345 12345 1234 12345 123456 12345 12345 1234 12345 123456 12345 12345 1234 12345 123456 12345 123456 12345 123456 1234 12345 12345 123456 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 123456 1234 12345 12345 123456 123456 12345 123456 12345 1234 12345 5 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 123456 12345 123456 12345 1234 12345 12345 123456 12345 123456 12345 1234 12345 12345 123456 12345 123456 12345 12345 123456 1234 12345 12345 123456 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345 123456 12345 123456 12345 12345 123456 1234 12345 12345 123456 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345 123456 12345 123456 12345 12345 1234 12345 12345

17

18

O Õ

+

C. rotundicauda

NM 12345 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456

4

15

19

APR I L' 1 9 8 7 MAY ' 1 9 8 Fig.12 A. Histogram showing the total number of C. rotundicauda at

with respective sex-ratio (SR)

20

21

7

DHAMRA, Orissa

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SR= 1.21 :1

O Õ

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SR= 1.09 : 1

Counting of horseshoe crab population at field station,DHAMRA,Orissa during April & May ' 1987 1234

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Õ +

∆

20

NO OF ANIMALS

10

O

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2

3

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FM

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4 MARCH

1

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FM

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18

19

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1

2

3

SR = 1 . 44

O Õ +

: 1 . 00

FM : Full Moon NM : New Moon

Counting of horseshoe crab population at field station,Chemaguri,West Bengal during March 12345 & June ' 1988 12345 1234 12345

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16 17 APRIL

9

NM

18

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31 1

2

FM

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3

16

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FM

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17

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31

1

2

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3

MAY

8

Fig 12 B. Histogram showing the total number of C. rotundicauda at West Bengal with respective sex-ratio (SR)

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15 JUNE

8

Chemaguri ,

NM

16

94 DISCUSSION The differential sex-ratios in the population of the Indian horseshoe crabs in the coastal West Bengal and in coastal Orissa (Table 20) may underline the following ecological consequences : i) In comparison to the coastal West Bengal , people of Orissa were found to be more conscious about these marine creatures and attaching some religious superstition on the pairing of the 'crabs' as "Rama-Laxman" the mythological two brothers of the holy Epic the Ramayana and hence unknowingly helped in their partial conservation , ii) More and more Anthropogenic interference at the sea resorts of Digha , Junput , Bakkhali , Frazerganj , Sagar Island and other places of coastal West Bengal inflicting random habitat destruction , alarming sea resource exploitation , and others may be the major causative factors for disbalancing the sex-ratio, iii) Natural predation by Corvus splendens is common everywhere , but this predation pressure is enhanced manifold by the fishermen at Digha as they throw the 'crabs' to the exposed sandy beach believing them as enemies to their fishing nets , iv) Furthermore , the largest freshwater draining rivers like Ganga , Matla and several others carry a vast amount of chemical pollutants , industrial effluents , oil spills and a variety of toxic substances ( Botton & Loveland , 1989) into the estuarine Sundarbans which may severely affect the normal sex balance and fecundity of Indian horseshoe crabs in coastal Bengal than in coastal Orissa , and finally, v) Although the local people do not eat the eggs of horseshoe crabs like the people of Thailand (Sekiguchi and Nakamura , 1979) , the female specimens are much preferred by the animal traders (quacks , Debnath, 1987) due to its large size . The present chapter communicates the first report on the predation of horseshoe crabs ( T. gigas) by the shore birds ( Corvus splendens) in Indian sub-continent (Debnath and Choudhury , 1988a) . A unique prey-predator relationship was observed in coastal Orissa (Chandipur) and West Bengal (Digha) which revealed that population of crows increased at the time of breeding time of the 'crabs' . Predator birds have reported to arrive at the Cape Shore within a week or two with the onset of Limulus polyphemus spawning in May and generally depart by July ; by this time Limulus abundance has been found to decline and most eggs of the horseshoe crabs were hatched into trilobite larvae (Botton , 1984c) . From the capture-recapture data and from the collection of dry carapaces (Fig. 28 B) male to female sex-ratio was found to be 6.89:1 at Digha and 1.04:1 at Chandipur (Table-21) . This disbalance in normal sex-ratio (1:1) seemed to be a result of severe predatory action at Digha than at Chandipur as in the later case crows get access to the 'carbs' in natural way , but at Digha fishermen enhanced the predation by throwing them to crows . In Thailand disbalance in sex-ratio of T. gigas and C. rotundicauda is also noticed which is due to the fact that the people collect the eggs to serve on the table as food (Sekiguchi et. al. , 1977 : Sekiguchi and Nakamura , 1979 and Dr. A . Choudhury's personal observation on 1986) . Another possible factor for the depletion of the female horseshoe crabs in coastal West Bengal of Orissa may be the "unauthorised sacrifice" of the female crabs ( because of their large size) as very important laboratory material for bio-medical research by draining its ' blue blood' to get the precious lectin "carcinoscorpin " (Dorai et. al. , 1981 ; Srimal et. al. , 1985 ; Vijaya Roa and Bhagirathi ,1989; Rudloe , 1983) . The breeding activity of Indian horseshoe crabs , particularly T. gigas has been studied readily at high tide time at Chandipur . In and around full-moon day-night observations (March , 1985) it was revealed that the number of mating animals reached its peak during the day tide than night tide (Fig. 9) . No solitary animal for mating pairs could be recovered during the approaching new moon tides .

95

Similar observations were made in May and July in the same year . From the whole result (Fig. 10 ) it becomes apparent that the mid-breeding time approximated at May when maximum population came to brred at Chandipur . The histogram results (Fig. 10) also indicate that the sex-ratio (SR) increases very slowly from the start to the end of spawning like 1.04:1 in March to 1.05:1 in May and finally 1.07 :1 in July . Recovery of mating pairs as well as solitary individuals of either sex from the exposed sandy beach or muddy substratum have ecological bearings . like i) the animals have tried to shorten their to and fro-journeys at the coast of each and every high tide and ebbe tide , ii) hide themselves from the predators partly or fully submerging (Fig. 22 C) in the sand or mud , and iii) protect themselves from desiccation , rising temperature , sun-burn and oxygen defieiency in the gill-flaps due to loss of moisture . Onece these temporarily buried animals were thought to be as egg-laying " crabs " , but no eggs were obtained from such burrows in the intertidal exposed beach (Fig. 20 A , 21 A) . However , a soft pressure at the base of the genital apertures situated undersurface to the genital operculum ejaculated eggs one by one (Fig. 21 C) and milting sperms . Similarly , a sizeable number of L. polyphemus buried in the intertidal beach throught the subsequent low tide which return to the beach to breed again on the following high tide (Rudloe , 1980) . The mating activity of horseshoe crabs is related to the environmental changes produced by periodic motions of the earth and moon (Barlow et. al. 1986) . Annual orbital movement of the earth around the sun produces seasonal changes in day lengh and in ocean temperature . While the daily rotation of the earth about its own axis also creates diurnal changes . On the other hand the monthly rotation of the moon around the earth modulates the tidal amplitudes of the ocean and produces periodic inequalities in the daily tides in the northern and southern hemispheres . All these astronomical events affect the physical environment of intertidal zones , and for the above reasons horseshoe crabs , T. gigas and C. rotundicauda come to shore during spring and summer months associated with full moon tides only . Though a sizable number of solitary males and few pairs of C. rotundicauda have been obtained from the exposed mud-flats of Prentice island , Chemaguri and Dhamra . Such shoreward migration was also reported for L. polyphemus during full-moon tides (Teale , 1957 ; Rudloe , 1980). She (Rudloe , 1980 ) also reported maximal mating activities during full-moon high tides , but without any breeding activity during neap tides . However substantial mating also occurred during new moon , µ specially at the begining of the season when overall numbers were large . Cohen and Brockmann (1983) instead , reported the arrival of more animals at the seashore Bay (Florida) in the same Atlantic coast during the full-moon only for breeding purposes . Again , Cavanaugh (1975) , and Barlow et. al. , (1986) observed the arrival of horseshoe crabs during both the full and new moon tides . On the contrary , no mating activity has been reported from the Indian coast during new moon (Fig. 10 & 13). A study of neuroplysiological light intensity test on Limulus median ocellus (Lall and Chapman , 1973) revealed the sensitivity to illumination as low as 1.4 X 10-10 w/(Cm2. nm) at 360 NM , whereas the moon-light intensity measured by Kampa (1970) is well within the range of adult horseshoe crab's vision . Beside those factors , tide height and sunlight ( as it varies according to the days of year) were also correlated significantly with the migration of Limulus towards the shore (Howard et. al. , 1984 ; Barlow et. al. , 1986) . Seasonal variation in Limulus visual sensitivity was determined with both physiological as well as behavioral studies which indicated the sensitivity was highest in the summer new moon , while lowest in the winter full-moon (Ross Farhang et. al. , 1990).

96

A comparable lunar breeding patterns for the two Indo-pacific horseshoe crabs ( T. gigas and C. rotundicauda ) have been reported ( Sekiguchi et. al. , 1977 ; Debnath , 1985 , Debnath and Choudhury , 1985 ; Choudhury and Hafizuddin , 1980) . Tagging study with rubber bands as well as mechanical marking (Table -22) has revealed the following facts :a) T. gigas prefers the sand shore having potentially low wave action and comes in the same spawning ground day after day during full-moon only . But C. rotundicauda prefers the muddy shore associated with mangrove vegetations (Fig. 20 A-C , 26 A-C) . b) Dispersion is a common rule in T. gigas , while C. rotundicauda tends to remain in the same breeding ground . c) Amplexus occurs in the off-shore shallow water but not in the mud or sand-beach . This inference can be made from the recovery of males and females in isolation bearing similar marks/rubber bands days after capture , tagging and segregation . d) Mated males and females are very hard to separate from the amplexus and when the two are separated forcibly and placed on sand or hard mud-bed the larger (female) one moves down toward the lower littoral zone which the smaller (male) appears to be helpless and does not usually follow its mate . But placed close enough , however , the male follows the female's track (Fig. 25) to get its normal position . This field observation was also endorsed by Drs. B.N. Chopra and H.S. Rao (c.f. Roonwall , 1944) . Cohen and Brockmann (1983) have classified the breeding activity of the males and three catergories : (1) Clasping - the male grasping the posterior opisthosomal angles of a female ; (2) Satellite - a male not clasping a female but within 10 cm of her or of the males eigher clasping her or also satellite to her , and (3) Solitary - a male neither clasping nor satellite . While this and other studies (eg. Rudloe , 1980 ; Brockmann , 1990) have been conducted along the shores of the Gulf of Mexico , the behaviour is essentially the same in other populations. Thus , the main focal point of this chapter , tandem amplexus was reported as early as 1870 by the Rev. S. Lockwood . The first record of tandem amplexus in C. rotundicauda (Debnath and Choudhury , 1992b appears to be the equivalent of the behaviour reported by Lockwood (1870) and others like Cavanaugh (1975) for L. polyphemus . The difference , if any , would be when and where amplexus took place , relative to the breeding area . In the American species there are generally more males than females on the breeding beaches . Also , those males that have not found a mate tend to proceed to the foot of a beach and await the arrival of an unattended female . Attachment occurs when a female moves up the beach slope to the water's edge (Shuster , 1953) . Extra males may accompany the pair onto the beach and participate in the spawning process . Usually most of the males , attached or satellite , are milting profusely . The relative contributions (of sperms ) of all of the males in a spawning group have been analysed by Brockmann (1990) . In the case of C. rotundicauda , where extra males are not common and where amplexus occurs prior to the adults reaching the breeding area , it is surmised that the tandem attachment reported here probably occurred off-shore (Debnath and Choudhury , 1992b) .

97

Fig . 20 ( A-C) A , Mating pair of C. rotundicauda embededv party on the intertidal soft mud bottom of Prentice island ; B , Upturn posture of the same showing typical amplexus , C, Shows a morphologically injured female of C. rotundicauda being clasped with the normal male .

C

98

A

B

Emergence of eggs through genital pores

C

Eggs of C.rotundicauda (enlarged)

Eggs of T.gigas (enlarged)

Fig . 21 ( A-C) A , The female "crab" carrying a male on her back fails to makes burrow in the hard mud-bottom ; B , Shows a clear depression in the mud with mud-cakes , below which one mating pair of C. rotundicauda resides ; C , Mature egg emerges out of the gonopore situated at the base of the genital operculum with a gentle pressure . 22C

99

A

B

An injured female has been selected by a healthy male as the sexual partner !

C Fig . 22 ( A-C) A , Two mating pairs of T. gigas in the soft sands of Chandipur sea beach during low tide ; B , The large female carries (undersurface) a comparatively smaller male posteriorly on her back ; C , Shows an amplexed pair of C. rotundicauda at Chemaguri mud flat . Numberous juvenile Cerithedia and a small mud-skipper are found to occur with the amplexed pair .

Embeded F emale Female

B

A Satellite Male

EXPOSED FEMALE

C

Clasping Male

Fig . 23 ( A-C) A , Shows an unusual amplexed pair of C. rotundicauda on the muddy sub-creek at Chemaguri , Sagar Island ; B , Three horseshoe crabs , C. rotundicauda occur in a linear row - the first one is almost embedded in the mud ; C , all the three specimens are clearly exposed just after the removal of mud , the first one is the female while the later two are males . The last male is being the "satellite" .

101

Third-rail track

A

B Foot prints

C

Fig . 24 ( A-C) A , Lateral view of a male T. gigas ; B , Dorsal view of the same showing its "third-rail tracks" produced by its shell edges and tail spine , punctuated by foot prints ; C , A moving pair of T. gigas ; the frontal one is female that carries the smaller male on her back .

102

A A

B B

C C

D D

Fig . 25 ( A-D) A , A readily segregated female of T. gigas having dorsally situated barnacles , moves towards the film of standing water to make a burrow quickly ; B , Trailing activity of segregated male partner along the path of female T. gigas ; C , Closer approximation of the male near the posterior end of the female partner and burrowing instead of making further amplexus ; D , A normal mating pair of T. gigas , engaged in making burrow quickly into the sand (lateral view).

103

A

B Male claspers

C Feast on eggs by Corvus splendense

Fig . 26 ( A-C) A , Shows the mechanism of clasping by the male claspers with the lateral opisthosomatic margin of the female in C . rotundicauda ; B, Figure shows a mating pair of C. rotundicauda partly embedded in the hard mud bottom decorated by the faecal pellets and holes of Uca sp. Proteresia and Psueda Mangrove herbs are also seen to be associated ; C , Shows of solitary male of C. rotundicauda on the muddy substratum covered with Psueda sp.

104

Fig . 27 ( A-D) A & B , Shows the mating pair of T. gigas on exposed sandy shore with crab holes and faecal pellets and a pair of C . rotundicauda on the exposed mud bottom in association with juvenlie Cerithedia and smaller Proteresia grasses . The upper furface of both pairs seems to be dried up which indicates a long time of receding tide water ; C & D , Corvus splendens , the predator bird sitting beside the pair and enjoying the flesh and eggs . Sand deposition over the vacated carapace the day after the predation is seen .

105

E . Epifaunal associates

106

INTRODUCTION

The carapaces , Particularly , of the adult horseshoe crabs are suitable habitats for a number of epifaunal associates ; so much so that the American species , Limulus polyphemus has been described as "walking museum" (allee , 1923). Most of the organisms attached to Limuli may remain attach to several other hand surface . Thus these are considered as ectocommensals , some of which showed symbiosis and other relationships (shuster , 1982) . Presence of epifaunal organisms of Indian horseshoe crabs has been reported by Roonwal (1944) . Rama Rao and Surya Rao (1972) presented a comprehensive study on the ectocommensals infesting the two species of Indian horseshoe crabs , T. gigas and C. rotundicauda are termed them as "mobile museums of natural history " . The present investigation elucidates a further description of the fouling organisms in detail to relate the occurrence of epifauna with the milting phenomenon (Debnath and Choudhury , 1991) .

Bryozoans,Anemones,Barnacles, and many others constitute the epifaunal community on the shell of Horseshoe crab's dorsal surface.

ONE MALE Tachypleus gigas WITH HEAVY INFESTATIONS ON THE DORSAL SURFACE ( on both PROSOMA and OPISTHOSOMA ) OF THE BODY

107 OBSERVATION A brief description of the epifaunal organisms with their taxonomic categories and size is presented here . 1. Sea anemones : Metridium schillerianum (Stoliczka) was observed infesting heavily the dordal surface of the body of T. gigas and C. rotundicauda . It ranged from 5 to 21 mm in its column length (Fig. 20 A) . 2. Sessile barnacles : balanus amphitrite Darwin , measuring 2.5 to 19.8 mm in length were found to occur profusely on the dorsal surface (rarely ventral surface) of the body of eigher species . Very frequently goose-barnacle , Chthamalus stellatus (Poli) measuring 1.8 to 9.7 mm in length were found to infest the carapace (Fig. 28 C) . 3. Bivalves : Several specimens of Ostrea sp. , measuring 10.5 to 28.5 mm. length were collected from the dorsal surface of C. rotundicauda and T. gigas . 4. Gastropods : Few specimens of Cerithidea sp. measuring 8.9 to 15.7 mm. in length were collected from both the species . 5. Amphipods : Cheiriphotis megacheles ( Giles) ranging from 4.2 to 9.2 mm. in length were collected from the abdominal appendages , and very rarely from the empty shells of the barnacles of T. gigas . 6. Isopods :Few specimens of Cleantis megacheles Barnard ranging from 13.0 to 15.0 mm. were found along with amphipods from T. gigas . 7. Polychaetes : Specimens of Gattayana deludens Fauvel ranging from 14.5 to 25.0 mm . were obtained from empty barnacle shells or from crevices of the ventral appendages . These were collected from both T. gigas and C. rotundicauda . 8. Brachyura : A few juvenile specimens of Thalamita sp. were collected from the empty barnacle shells present on the dorsal surface of T. gigas . 9. Bryozoa : The gymnolaemate , encrusting , amt-like bryozoan , Membranipora sp. was found on the dorsal surface of both T. gigas and C. rotundicauda (Fig. 28C). Beside those epifaunal associates (Rama Rao & Surya Rao , 1972) as described above , there were also other organisms like protozoa , diatomes , algal mat particularly the colonies of Obelia sp. But following are the two categories of ectocommensals found to occur in both T. gigas and C. rotundicauda . I. Turbellaria : A marine triclad , possibly belonging to the genus Ectoplana sp. were collected from the bases of the ventral appendages and genital opercula of T. gigas and C. rotundicauda.In most cases these were found in aggregate , mostly , on either side of cephalic swellings , i.e. below the subfrontal area (above the ventral organ). Its tiny size ranges from 1.0 to 2.5 mm. in length . The whitish-creamy body showed a prominent ventral sucker , a pair of cephalic eyes and distint intestinal cecae (Fig. 15 A) (reviewed by Kawakatsu and Sekiguchi , 1989). II. Scale - worms : Polychaete scale-worms (Fig. 15 B) , Polynoe sp. measuring 7.8 to 12.5 mm. in length , were collected from the ventral appendages , particularly the gills and ventral grooves of both the species of Indian horseshoe crabs . These are ectocommensals , because the similar animals were also obtained from hermit-crabs of associate community . In aquarium they were found to swim from horseshoe crabs to hermit crabs or hide underneath the rocks or other surfaces or vise-versa .

Epifauna with systematic position

Class : Anthozoa , Order : Actinaria Metridium schillerianum

Class : turbellaria , Order : Seriata Sub-order : Tricladia . Ectoplana sp.

Class : Gymnolaemata (Bryozoan mat). Membranipora sp.

Class : Polychaeta i) Gattyana deludens ii) Polynoe sp. (Scale - Worms)

Class : Crustacea , Order : Thoracia i) Balanus amphitrite ii) Chthamalus stellatus Order : Amphipoda Cheriophotis megacheles Order : Isopoda Cleantis natalensis

Class Gastropoda Cerithedia sp.

Class : Bivalvia Order : Pseudolamellibranchiata Ostrea sp.

Class : Brachyura Thalamita sp.

Sl. No.

1.

2.

3.

4.

5.

6.

7.

8.

9

19

45

670 87 25 4

5 147

+++

130

235

No.

T. gigas

0.65

1.38

3.28

48.69 6.32 1.82 0.29

0.36 10.68

-

9.44

17.07%

%

2

35

25

590 108 -

10 221

+++

265

149

No.

C. rotundicauda

Table 23 . The % of infestations of major epifauna of Indian horseshoe crabs (based on numberical abundance)

0.14

2.49

1.78

41.99 7.68 -

0.71 15.73

-

18.86

10.60%

%

108

109

Table 24 . The number of infested horseshoe crabs collected from 5 different stations ; figures in parentheses indicate actual no. of animals observed . Carcinoscorpius rotundicauda Male Female Prentice Island (West Bengal)

15(28)

Sagar Island (West Bengal) Digha (West Bengal)

51(77)

Tachypleus gigas Male Female

8(21)

0

0

16(63)

0

0

82(131)

4(19)

0

187(333)

69(320)

38(66)

7(50)

43(71)

11(44)

60.8%

23.13%

58.32%

21.93%

0

0

Chandipur (Orissa)

0

Dhamra (Orissa)

% of infested animals

Head

Eye-pot

Gut

Scale-like segment

Ventral sucker (Pharynx) Parapodia Intestinal divertculae

Triclad (Turbellarian) flat worm, Ectoplana sp.

Polychaete scale-worm, Polynoe sp.

110

Table 25. A&B presents the 2 X 2 contingency Chi-square tests of infested and non-infested horseshoe crabs , C. rotundicauda and T. gigas respectively

C. rotundicauda 0

T. gigas

+0

Total

0

0 +

Total

Infested Non-Infested

104 67

31 103

135 170

312 223

84 299

396 522

Total

171

134

305

535

383

918

A. E 75.688 95.3111 59.311 74.688

B.

0 104 67 31 103

E__0 -28.311 +28.311 +28.311 -28.311

C. rotundicauda

X2

230.784 304.215 165.215 217.784

-81.215 +81.215 +81.215 -81.215

T. gigas

312 223 84 299

X2

(E__0)2 801.512 801.512 801.512 801.512

= 43.243

= 120.472

(E__0)2 / E 10.589 8.409 13.514 10.731

df = 1

P< 0.001 6595.987 6595.987 6595.987 6595.987

28.581 21.682 39.923 30.286

P< 0.001

df = 1

111

Fig .A15 The ventral view of a female C. rotundicauda showing the positions of triclad turbellarion (A) & polychaete scale-worm (B), two prominent ectocommensals .

B

A

B

112

A

Egg-mass of Tachypleus gigas

B Massive Epifaunal Infestations on T. gigas

c

Morphometric measurement of Hoseshoe crabs on sandy shore of CHANDIPUR, Orissa.

Fig . 28 (A-C) A, Enlarged view of a mass of eggs ( T. gigas ) ; B, Shows the collection of dry carapaces for morphometric measurement at Chandipur sea shore ; C , Shows an aged male T. gigas at Chandipur sea shore with heavy infestations over the carapace .

113

RESULTS AND DISCUSSION Table 23 shows the % of epifaunal infestations based on numberical abundance of organisms obtained from 32 T. gigas and 18 C. rotundicauda . The data clearly indicate that major infesting animals are Balanus amphitrite , Metridium schillerianum , triclad turbellaria (Ectoplana sp) and polychacte scale worms (Polynoe sp.) . Allee (1923) , Andrewa (1942) , Gorham (1904) , Humm (1942) , Pearse (1947 , 1949) , Shuster (1955 , 1958) , Verrill (1893 , 1895) , Wheeler (1894) have reported a variety of epifaunal organisms from L. polphemus . But Rama Rao & Surya Rao (1972) are the first persons to report these from Indian horseshoe crabs . In their paper (Opt. Cit.) they tried to find out an inter-relationship between the infesting animals and the horseshoe crabs . The present investigation does not agree with the statement - "the amphipods , isopods and polychaetes are probably attracted for eating the egg mass clustered around the abdominal appendages of King crabs" (Rama Rao & Surya Rao , 1972) , since the evidence of possessing clustered eggs in the ventral appendages of the female 'crabs' , has already been discarded (Debnath , 1985 ; Iwanof , 1933 ; Roonwal , 1944 ; Sekiguchi et. al. , 1977) . The number of horseshoe crabs infested with epifaunal organisms in given in the table 24 which clearly indicates that male horseshoe crabs are being infested with ectocommensals (58.92%) more than that of the female crabs (22.24%) . Out of 305 ( 171 M + 134 F ) C. rotundicauda observed only 31 female (F) showed epifauna with less or sometimes moderate infestations . Heavy infestations in females were very rare . On the other hand , amongst 918 ( 535 M + 383 F ) T. gigas only 84 females (F) showed epifaunal infestations . But for male (M) C. rotundicauda and T. gigas it accounts 60.81% and 58.31% infestations respectively . The Chi-square test of the infested to non-infested horseshoe crabs reveals that in both cases males are highly vulnerable to sustained growth of epifaunal organisms on their carapaces than the females (for C. rotundicauda , X2 = 43.243 , P < 0.001 and for T. gigas X2 = 120.471 , P< 0.001) (Fig. 28C) The high frequency of epifaunal infestations in males than in the females may be explained in two ways : i) Since mating amplexus takse place in the shallow seas before the commencement of spring and summer months (debnath , 1985 ; Debnath & Choudhury , 1988a) the males remaining attached to the dorsal carapace of the female get exposed for easy access to the epifaunal organisms . This hypothesis can be approved by the findings of fouling organisms mostly on the solitary females , very rarely on the mated females . ii) The more appreciable second assumption is that males do not molt further after attaining sexual maturity (Shuster , 1955;58) and thereby this shells become suitable habitat for epifaunal growth ; while as the females molt more frequently , at least onece in a year (shuster , 1950 , 1954) ; its shells are generally remain free of fouling organisms and it seemed likely that the larger size attained by females is due to successive moltings (Rudloe , 1980) . The frequency of molting is probably a deterent to sustained attachment of ectocommensals . Thus the first attachment is noticeable on Limuli larvae and is most prevalent on adult Limuli, because they do not molt as frequently if at all , paricularly the males , permitting continued individual growth as well as population development of the attached animals (Shuster , 1955/58) . This is further confirmed by the fact that all immature and most young (adult) Limuli are found free of "encrusting" organisms (Shuster , 1957) .

114

Although distinction between ectocommensalism and parasitism has not made , the marine triclad Bdelloura has been categorised as ectoparasite of Limulus (Ryder , 1982 ; Verrill , 1893) and Ectoplana for T. gigas and C. rotundicauda (Sluys , 1984). Carapaces of adult horseshoe crabs are found frequently with encrusted epifaunal invertebrates , particularly barnacles , blue mussels and slipper limpets which could potentially be useful in approximating ages of adult horseshoe crabs (with Crepidula fornicata , a gastropod ectocommensal cf. Botton & Ropes , 1988) . In addition to providing insights into the age-structure of an adult horseshoe crab population , studies of epifauna may be useful in determining whether discrete spawning populations throughout a coastal range exist or not . In fine , horseshoe crabs provide a better shelter to a variety of epifaunal associates i) for their transport from one locality to the other (since the host 'crab' has effective migrating habit) as in the case of transportation of thousands of oyster-drills on the carapace of single Limulus (Mackenzie , 1962) , ii) for facilitating feeding of barnacles and other sessile forms , and iii) for chemouflaging itself from the predators . According to Shuster (1957) infesting organisms provide a clue to the identification of horseshoe crabs of different geographical localities and "encrustations" , if identified , on fossil Limulidae may give information on the ecology and growth stage of the fossil .

115

PART - II STUDIES ON FEEDING BEHAVIOUR ; FOOD ; FEEDING AND DIGESTION

................ it is to be noted that the functional activities of the neuroendocrine system of horseshoe crabs are , probably attributed with the moulting phenomena , visual system and mating behaviour.

116

INTRODUCTION From several recent reviews of the natural history of horseshoe crabs (Shuster ,1979 ,1982 ; Rudloe , 1979a , 1981 ; Sekiguchi and Nakamura , 1979 ; Sekiguchi , 1988) , it is evident that relatively very little is known of the merostomate's feeding biology , inspite of its familiarity to a large number of zoologists and palaeontologists , and its growing importance in biomedical research (Cohen et. al. , 1979 ; Pearson and Weary , 1080 ; Nobitsky , 1982 , 1984) . Information on the feeding biology of horseshoe crabs is limited (lockwood , 1870 ; Fowler , 1908 ; Shuster , 1950 ; Smith and Chin , 1951 ; Smith , 1953 ; Smith et. al. , 1955 ; Botton , 1981) . The first comprehensive study of feeding behaviour of Limulus polyphemus is of Botton (1984 a, b) which revealed a resonable methodology to conduct the gut-content analysis in Indian horseshoe crab , Tachypleus gigas (Debnath , 1985 ; Debnath and Choudhury 1987) . Botton and Haskin (1984) provided a more intensive study of the food habits of L. polyphemus recovered from the continenta l shelf of Delaware Bay and the subsequent analysis of their stomach-contents . Except Schlottke (1934a, 1934b, 1935) ; Lockhead (1950) and Tanimoto and Kondo (1982) , so far no attempt has been made to study the digestive physiology of Merostomates . It is for the first time that the gut contents of T. gigas have been examined in context of the digestive enzymes secreted by the organism ( Debnath et. al., 1989). This part of the thesis discusses in brief the digestive system , feeding and food-items in context to digestive physiology and finally deals with the trophic level to ascertain the possible position of the Indian horseshoe crabs in coastal or estuarine trophic level .

Corvus predator Deliceous dish

Carnivore predator

? Small goboid fish

?

OE

Crab's eggs

FIG S LE IMP .3 L V 2B TH ELS IFIE D ES & CR F T AB UDIE FOOD ORM S So f IN WEB OF DIA as TRO N H BA PHI OR SED C SE SH on

Large predator fish

Tachypleus

Gastropods Copepods Bivalves

Carcinoscorpius

Ophiuroids

A

Polychaetes

Shrimp

Plants & Detritus (organic decompose)

117

OBSERVATION DIGESTIVE SYSTEM The early works of merostomate digestive system were on L. polyphemus ( Patten and Redenbough , 1899 ; Schlottke , 1935 ; Shuster , 1948) , but not on that of the Asian horseshoe crabs , except for a short description in T. tridentatus ( Shoji , 1929) . The digestive system of horseshoe crabs consists of two main parts ; the digestive tract and the mid-gut gland . The digestive tract is a J-shaped simple duct sub-divided into 3 parts : i) the fore-gut , comprising mouth , oesophagus , and proventriculus ; ii) the mid-gut or stomach-intestine ; and iii) the hind-gut , comprising the rectum and anus . The mid-gut gland , filling most of the prosoma , consists of two different tissues ; the hepatic diverticulae i.e. the blind branches of the hepatic ducts arising from the intestine , and the yellow connective tissue surrounding the mid-gut diverticulae . In adult horseshoe crabs , the mid-gut diverticulae are located only in the prosoma , but the yellow connective tissue is seen in the opisthosoma , on either side of the intestine . 1. Digestive tract : The mouth is a longitudinal slit that opens on the ventro-median surface of the prosoma surrounded with three kinds of mouth parts : a pair of chelicerae , coxae or gnathobases of five pairs of walking legs , and a pair of chilaria . The elastic peristomal cuticle enables the mouth parts to move freely The oesophagus , the wall of which is provided with developed muscles , passes upwards from the mouth , through the vascular ring within which the circum-oesophageal nerve ring is situated , in fornt of the endosternite it then runs forwards to the more swollen proventriculus . It is often regarded as the "stomach" or "gizzard" ; however , it is not a true stomach of endodermal orgin , rather it belongs to the ectodermal foregut and called as the crop . At the half-way point , it is strongly bent backwards , divided into front and hind parts . The wall of the proventriculus , i.e. the crop (front) or gizzard (hind) , is provided with well-developed muscles , is the thickest one in the whole digestive tract . Externally , a dorsal notch is often seen that marks the distinction between crop and gizzard ( Fig. 32 C) . The posterior end of the proventriculus (hind-part = gizzard) narrows sharply into apyloric valve , protruding into the intestine . The first part of the intestine thus has a swollen appearence that covers the pyloric valve and regarded as the true stomach . A marked constriction indicates the boundary between the gizzard and the stomach-intestine . The intestine is a soft and wide tube stretched between the prosoma and opisthosoma , lying longitudinally under the heart and running to near the posterior end of the opisthosoma . A short rectum leads to the anus , opening ventrally at the base of the telson which is guarded with a pair of anal valves . There is no boundary such as valve or constriction between the intestine and the rectum . The wall of the rectum , with its well developed muscles , is thicker than that of the intestine . One on each side of the anterior part of intestine lies the hepatopancreas ; paired ducts from each of them enter into the intestine. Openings of the first pair lie slightly antero-ventral and the second one slightly postero-dorsal (Fig. 32C) . The length and diameter of five major parts of the alimentary tracts (10 mated pairs of T. gigas ) are given in the following table as mean values expressed in mm. and it was also found that the total mean length of male tract as 110.38 mm (range : 105.0 - 119.4 mm) ; while it was 153.80 mm (range : 147.5 - 163.5 mm) in female .

118

Length and diameter of different parts of the digestive system MALE (mm)

FEMALE (mm)

Length

Diameter

Length

Diameter

28.75 +5.97

3.52 + 0.56

34.50 ±4.08

8.12 + 2.03

2.Proventriculus (Crop) 14.53 + 2.19

8.75 + 1.87

21.76 ±1.25

12.45 + 3.16

3.Gizzard

16.98 + 1.83

7.42 + 1.34

20.44 ±1.40

7.75 + 1.13

4.Intestine

39.45 + 2.41

4.52 + 0.81

50.25 ± 2.89

8.87 + 2.29

5.Rectum

25.25 +2.25

7.76 + .047

37.12 ± 3.42

11.05 + 5.24

1.Oesophagus

The inner surface of the oesophagus is deeply folded into 6-7 longitudinal ridges and furrows , which are covered with a transparent moltable cuticle . In fore part of the proventriculus (crop) the inner surface has many longitudinal ridges as extensions of those present in the oesophagus . But in contrast , the hind part i.e. , gizzard has 13-15 , usually 14 , deep longitudinal ridges and furrows , the ridges are remarkably deeply notched into several chitinous denticles; the hardened cuticle of which often coloured blackish brown . These features show that this part is undoubtedly a place for mechanical manipulation or a masticatory stomach (Yamasaki et. al. , 1988) . In the pyloric valve , the toothed lines are reduced into 6 fine serrated ridges . This valve opens into the stomach or the first swollen part of the intestine with 6 papillae (Fig. 32 C) . The oesophagus , the proventriculus , the gizzard including the protruding pyloric valve into the stomach-intestine , all are characteristics in having the muscular wall on the outer part and transparent or thick moltable cuticle in the inner surface . These common features show that the parts have the same ecotodermal or the stomodeal orgin (Yamasaki , et. al. 1988) . The inner surface of the intestine is brownish in colour , has a thin mucous layer , instead of the cuticle . The excreted faeces of the horseshoe crab are surronded by a mucous envelope . One or more such faecal blocks or threads enveloped with fine mucous layer were observed . These features of the intestine , which are quite different from those in the oesophagus and to the proventriculus , show that the intestine is of endodermal orgin (Yamasaki et. al. , 1988) . In the final phase the inner surface of the rectum is again folded into 6 or 7 longitudinal furrows and ridges and covered with a transparent moltable cuticle , which further confirms the rectum originating

119

2. The mid-gut gland : According to Yamasaki et.al. ( 1988 ), the mid-gut diverticulum is a blind branch of the hepatic duct , 100 - 200 µm in diameter . These are distributed throughout the prosoma surrounded with yellow connective tissue in an anastomosis with the midgut gland and the gonad . The wall of the midgut diverticulum consists of two layers - the inner hepatic epithelium and the outer muscle layer . The former is columnar cells and a small number of conical cells . The former cell types include several large secretory granules , the distal part of which is often cut off from the cells and floating in the lumen of the diverticulum. The conical epithelial cells lie sporadically among the columnar epithelial cells . In form and stainibility the granules in the conical cells are similar to the granules stored in the yellow connective tissue . A remarkable level of non-specific esterase enzyme activity has been noted in these granules of the conical cells (Tanimoto and Kondo , 1982) . Fage (1949) described the midgut diverticula as partly located in the opisthosoma in L. polyphemus . In young L. polyphemus of the 1st and 2nd instars , some branches of the hind pair of hepatic ducts enter into the opisthosoma (Shuster , 1948) , but the mid-gut deverticulae are located only in the prosoma in adults of any species . 3. Yellow connective tissue : Yamasaki et. al. (1988) described this specialized tissue in great detail . It is a soft mesenchyme-like tissue , yellow or light brown in colour ; consists of a single type of large yellowish cells , which are often filled with many eosinophilic granules . Nutritive material (oesinophilic granules) may be stored in this tissue and released into the blood. This tissue on the other hand , may be homologous with the chloragogen tissue of certain annelids , because of its colour , its celluar constituents , and its location around the intestine . Most of the yellow connective tissue composes the midgut gland together with the midgut diverticula . For this , several authors described the opisthosomatic midgut gland as the hepatopancreas or liver in adult crabs (e.g. , Fage , 1949 ; Shoji , 1929) . The digestive system of the four species of horseshoe crabs have a good many morphological similarities (except a few) in minute details , such as longitudinal ridges in the inner surface of the oesophagus , the proventriculus , and the rectum . These similarities may be due to evolutionary conservation , rather than convergence , and the basic feeding behaviour remains same and unchanged in all extinct and extant horseshoe crabs , which leads to the final conclusion that the horseshoe crab's digestive system retains some primitive features like the well-defined yellow connective tissue (Manton,1977; Yamasaki et. al. , 1988) . GUT - CONTENT ANALYSIS Major food-items of T. gigas occurring at Digha sea shore , were comprised of vascular plant parts , foraminiferan shells , cnidarians , polychaetes , tabanid larvae , crustaceans , specially amphipods and junvenile shripms , soft-shelled molluscs (both bivalves and gastropods) , ophiuroids , and goboid fishes . The major food-items recovered from the gut-contents may grossly be grouped as below :

120

1. 2. 3. 4. 5. 6. 7.

Polychaetes Gastropods Bivalves Crustaceans Tabanid larvae Ophiuroids Miscellaneous

: : : : : : :

Male

Female

11.46% 11.64% 19.04% 6.35% 35.62% 5.46% 10.40%

16.28% 16.94% 16.28% 8.47% 18.89% 16.94% 6.18%

The miscellaneous category includes unidentified prey items of various taxa and few zooplanktons besides few goboid fishes and synthetic fibres . Several varieties of foraminiferans , Casuarina needles , vascular plant debris and fruits mixed with sand-particles and pebbles were also found in the guts (Fig. 29 , 30 & 31) . Now , a detailed description of food items recovered and identified upto possible lowest taxa can be presented : Bivalve molluscs : Several kinds of bivalve shells (intact very rarely) were found . Usually fragments of the bivalve shells made it difficult to identify the prey-item. In most cases shells were found in complete corrossive form , although remained intact . But juvenile bivalves less than 0.5 mm in length were frequent in the gut contents as indicated by unbroken shells . The possible taxonomically identified catgories are : Anadara sp. , Dosinia sp. , Placenta sp. , Macoma sp. , Solen sp. , Neosolen sp. and Teredo sp. etc. (Fig. 30B). Gastropod molluscs : These were also very difficult to identify , because most of the shells became corroded , crashed or fragmented . However few of them were identified as Cerithedia sp. , Littorina sp. , Assimenea sp. , Nerita sp. , Cassidula and Cymia sp.etc. (Fig. 30C) . Annelids

: Polyehaetes were the only annelid worms recovered with intact head or tail and sometimes complete bodies of juvenile forms . The major polychaete worms are Gattyana sp. , Phyllodoce sp. , Nereis sp. , Perinereis sp. , Glycera sp. , Sabellaria sp. , Polydora sp. etc. (Fig. 31A) . Arthropods : Amongst this category the major food items consist of crustaceans , mostly unidentified . These were mysid shrimps , gammarid amphipods , copepods (e.g. Microstella sp. ) , decapods (e.g. Crangon sp.) and Cirripeds (Fig. 31B) . Under Insecta , the most dominating food item is a tabanid larva , identified as Atylotus agrestis . The other rare insect larva was Chironomus sp. Frequently body parts , usually , appendages of unidentified insect were also recovered from the gut (Fig. 29C) . Miscellaneous food matters : These include numerous foram shells like Ammonia beccarii , A. tepida , Asterorotalia dentata , A . multispinosa , A . trispinosa , Bolivina sp. , Elphidium sp. , Ouinquloculina sp. These benthic foraminiferans are found along the south-east coast of Bay of Bengal (Mazumder , 1992, pers.

121 communication ) . Other miscellaneous food matters include unidentified bryozoans , nemarteans , turbellarian trematodes , few nematodes , eggs , synthetic fibres and calcareous spicules of Echinoidea , siliceous crystals etc. mixed with sand particles . (Fig. 29A , 30A) . Vascular plant matters : Vascular plant parts include Casuarina needles , plant fruits , algal bodies etc. These plant matters constitute a major part of the gut-content (Fig. 29B) Teleost fishes : Three goboid fishes with distinct ventral suckers (?) , fins with fin rays and scales were found from the gut-content of T. gigas collected from Chandipur sea shore (Fig. 29 C(A) . Remark - I : Razor clams (Solen sp.) brachyuran crabs ; and polychaetes etc. were the major food constituents of C. rotundicauda collected from Prentice Island (West Bengal , Sundarbans) and estuarine mangroves of Dhamra (Orissa mangroves) . Remark - II : Beside the above food items , T. gigas of Digha sea shore possessed several juvenile anthozoan actinarians (unidentified) . Remark - III : Fragmented shells , spicules , sands and other solids were found in compact ball-forms within the proventriculus , just below the dorsal-notch which marks the junction of crop and gizzard , ready to be ejected out through the mouth . Such "shell-balls" were found in 15 specimen-guts out of 72 dissected for content-analysis (Fig. 32C) . Table 26 shows the measurement of food-items recovered from the gut-contents of T. gigas . This table clearly shows the length and breadth (both expressed in mm) of food matters . The maximun body fragment of polychaete worm measured 25.8 mm and it was 30.0 to 40.0 for goboid fishes ; while the minimum length ranges from 0.3 to 0.5 mm for juvenile molluscs . Most of the junenile molluscs were observed in the semidigested body of polychaete worms and tabanid larvae ; few from both gizzard as well as rectal content of horseshoe crabs . Similarly foram shells were obtained from both gizzard and rectal content which also measured less than 1 mm. in diameter . In Table 27 the frequency ( f ) and number of food items along with their ratio ( r = n/f ) have been presented . The data are arranged in three major columns , that is , for 2 solitary males , for 15 mated males and 15 mated females . The 'r' values clearly indicate that the solitary males consumed more food than the mated males except in the cases of miscellaneous category . At the same time , it is found that mated females had consumed food much more than either the solitary or the mated males . In terms of percent if 'r' values compared the following assumptions can be drawn : i) Adult males (both solitary and mated combained) consumed only 65.78% food than those of females . ii) Mated males alone can consume as much as 92.65% food if compared with the 100% of solitary males , i.e. 7.35% less . iii) 61.65% consumpsion by mated males and 66.65% consumpsion by solitary males , if compared separately with that of the mated females . Table 28 presents the weighed values of gut-contents of 15 mated males and females , respectively .This observation apparently reveals the high gut-content value in females (0.952 + 0.47) than the males (0.862 + 0.80) irrespective of their body weight .

122

DIGESTIVE PHYSIOLOGY The results of the examination of the gut content of 20 specimens, 10 of each sex , are recorded in Table 29 in relation to their body weights and inter - ocular length (IOL in mm.) . Abundant food matters were found in seven females out of ten , whereas only one male contained abundant food . Vascular plant debris were found in all females and in all males but one . Numerrical abundance showed that major food items are molluscs , polychaetes and crustaceans (mostly gammarid amphipods) . Numerous foraminiferan shells mixed with sand particles were found in the gut-contents (Table 29) . The results of digestive enzyme assay have been presented in table 30 . The activity of acid protease is highest in gizzard and lowest in oesophagus , whereas alkaline protease is highest in intestine and lowest in gizzard . Esterase shows maximun activity in oesophagus , whereas in the other three regions studied , the activity is reduced by 50% . Cellulase activity is highest in gizzard and oesophagus , but for invertase and amylase , highest activities are found in digestive gland and lowest in the oesophagus . TROPHIC LEVEL : A simplified food web based on Shuster (1982) , comprising various trophic levels , has been constructed (Fig. 32B) after analysing the gut contents and predatory phenomena of horseshoe crabs . In this study it has been observed that horseshoe crabs preyed on various prey forms from micro-to macro-organisms . For instance , an important dietary components of horseshoe crabs was polychaetes which were dependent on juvenile gastropods and bivalves . Similarly larger soft shelled molluscs are detritus feeders . On the other hand , no ophiuroids are used as food by man , and none are venomous ; but they have a considerable indirect economic importance in view of their immense number and consequent significance in natural food chains involving commercially sought species like horseshoe crabs , T. gigas . Most of the pelagic forms of polychaetes and zooplanktons like calanoids ( a copepode) , mysid shrimps , and Microsetella sp. etc. are common food items of the mated males of horseshoe crabs , as they did not come access to the benthic communities , were dependant on the pelagie microorganisma , while in motion along the back of the carrier female in amplexus . The goboid teleosts , on the other hand , are either micro-carnivores or detritivores . In the above category horseshoe crabs can be regarded as a predator . While , the crow , Corvus splendens was found to predate on horseshoe crab's egg. Thus a very complex food chain in the form of a net-work (food web) is being obtained . In the present investigation , it is evident that horseshoe crabs might be regarded as primary , secondary tertiary consumer as and when the circumstance demands certain trophic level for any individual nutritional flow system . Higher trophic level like crows , jackles or even human-being ( in Thailand, coastal China including Hong Kong as referred by Sekiguchi and Nakamura , 1979; & also a pers.comm. with Huang Qin, Helen Chiu and Brian Morton, 2003....."the fact that they are, locally and elsewhere in China, fished, sold in markets and eaten as a specialist sea food." ) seem to be under the tertiary consumer category as observed in

the present investigation . FEEDING The mechanism of feeding in horseshoe crabs have been described by a number of authors , begining with Lockwood (1870) . Usually small macrofauna and juvenile forms are initially captured by the animal using the chelate walking legs . These ventral appendages along with chelicerae , chilaria and genital operculum , manipulate the food-items to the gnathobases of the legs . The mouth lies between the 1st pair of legs . Posteriorly from it , and between the dentate process of the coxae which form the gnathobases , runs a groove called the food basin ( Fig. 15; * it may be comparable with the pro-mesosternite as suggested by the expert, Prof.C.N.Shuster,Jr.1993) . The chitinous gnathobases are fortified

123

Table 26 Measurements of various food items (all expressed in mm) of T. gigas

Taxonomic category Items Length (mm) Annelida 0.9 - 1.2 Polychaete jaw 2.0 - 25.8 Polychaete body(fragmented) 1.3 - 1.6 Polychaete setae 3.7 - 5.4 Polychaete head (complete) Juvenile Polychaete (complete) 1.8 - 2.0 Mollusca Bivalve shell (complete) Bivalve shell (fragmented) Juvenile bivalve Gastropod shell (complete) Gastropod shell (crashed) Gastropod shell (fragmented) Crustacea Amphipod body (complete) Crustacean head(semidigested) Crustacean appendages Zooplankton (complete)

5.0 3.5 0.3 4.0 3.2 2.3

-

Breadth (mm) 0.2 - 0.3 1.8 - 2.2 1.6 - 2.1 0.3 - 0.4

8.0 6.2 0.5 7.2 5.8 2.5

2.5 - 4.3 0.2 - 0.3 3.1 - 4.3 2.8 - 5.0 -

2.7 - 4.0 3.5 - 4.0 1.0 - 4.2 0.4 - 0.7

0.8 - 1.3 0.7 - 1.5 -

4.5 - 5.0

0.2 - 0.3

3.0 - 6.8 3.8 - 4.2

0.5 - 0.7 -

Insecta Tabanid larvae(Atylotus agrestis) Cnidaria Juvenile anemones (?) Hydroid colony

Foraminifera ( shells of various kinds)

Pisces Goboid teleosts Eggs

Unidentified eggs

-

0.3 - 0.7

30.0 - 40.0

5.7 - 10.5

-

0.1 - 0.7

with sharp spines to grind molluscan shells and other prey (Shuster , 1982) . Anteriorly along the food basin , food is passed to the mouth while being triturated , and then enters a muscular , chitinous gizzard or proventriculus , where additional grinding occurs . Food is grounded in the gizzard and admitted to the stomach-intestine through the pyloric valve . Coarse fragments are ejected through the mouth ( Lockhead , 1950) as bolus forms , found in the present investigation , at least in 15 guts dissected out .

124

Table 27 . Occurrence of food items in digestive tracts of 72 Tachypleus gigas collected from Digha sea shore , West Bengal , India ; during the summer of 1985 ( f = frequency of food items occurred at least one in the gut-content analysed ; n = total number of food items counted ) Taxonomic category of food items

42 solitary males 36 with food

15 mated females 15 with food

f

n

f

n

r

f

n

Arthropoda Crustacea Insecta

18 29

29 63

1.611 2.172

5 6

7 10

1.400 1.666

10 15

26 35

2.600 2.333

Annelida Polychaete

28

35

1.250

4

4

1.000

13

42

3.230

Echinodermata Ophuroidea

11

17

1.545

2

3

1.500

10

15

1.500

Mollusca Bivalvia Gastropoda

20 16

75 36

3.750 2.250

4 3

12 7

3.000 2.333

12 11

40 33

4.166 3.000

28

31

1.107

7

12

1.714

15

28

1.866

28

22 + 5 ++ __ 1 +++

3

2+ 1 ++ __ 0 +++

15

2+ 7 ++ __ 6 +++

Miscellaneous Unidentified and others Vascular plant matters

r

15 mated males 7 with food r

*r = ratio of n / f ; + , present ; ++ , moderate ; +++ , high ; and Foraminiferan shells were excluded from the count .

125

Table 28 . Weight of gut-contents of 15 segregated males and females (expressed in gm.) UG , EG & GC , weight of undissected gut , emptied gut and gutcontents ; FP , SP & DP , food , sand and debrices present .

UG

EG

GC

FP

SP

DP

Body weight

3.75 4.28 5.03 3.43 2.95 2.57 4.87 5.25 5.04 4.85 5.86 6.23 4.28 3.05 2.57

3.24 3.87 4.25 3.03 2.85 2.55 3.75 3.96 3.87 3.25 3.28 3.95 3.57 3.05 2.56

0.51 0.41 0.78 0.40 0.10 0.02 1.12 1.29 1.17 1.60 2.58 2.28 0.71 0.01

+ ++ ++ + ++ ++ + ++ + + -

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

+ + ++ + + + ++ + ++ ++ +++ -

130.0 155.0 120.0 175.0 200.0 135.0 180.0 140.0 210.0 125.0 120.0 170.0 155.0 140.0 135.0

4.87 5.95 6.35 6.54 5.85 FEMALE 6.83 N=15 6.25 5.56 4.95 4.87 5.08 6.05 5.34 4.87 6.35

4.53 5.45 5.03 5.15 5.54 5.59 4.83 4.71 4.50 3.92 3.56 4.47 4.09 4.55 4.50

0.34 0.50 1.32 1.39 0.31 1.24 1.42 0.85 0.45 0.95 1.52 1.58 1.25 0.32 0.85

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

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

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

405.0 490.0 510.0 425.0 485.0 515.0 355.0 505.0 480.0 545.0 490.0 505.0 410.0 405.0 540.0

MALE N=15

130.0 140.0 120.0 155.0 101.0 120.0 150.9 120.0 195.0 125.0

Weight (g)

73.0 76.0 71.0 74.0 75.0 69.0 73.0 71.0 78.0 71.0

+ + ++ ++ + + ++ +++ ++ -

IOL(mm) Gizzard content

++ + ++ + + + + + + -

Plant debris

8 150 7 4 -

1 1 -

Foraminiferan Ophiuroids shells (Echinodermata)

22 4 3 2 5 -

Mollusc Shells

2 1 4 3 3 1 -

Polychetes

1 1 1 1 1 -

Crustaceans

Female N=10

405.0 409.0 510.0 425.0 485.0 515.0 355.0 505.0 480.0 585.0

92.0 101.0 103.0 92.0 101.0 105.0 89.0 102.0 103.0 107.0

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

5 150 100 75 15 8 2 12 3 5

1 15 1 1 3 19 2

2 8 6 4 11 7 15 12

IOL , Interocular length . + , present ; ++ moderate ; +++ , high

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

1 3 2 4 3 1 4 2

1 1 2 2 1 1 1

------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Male N=10

Sex

Table 29 . Data on gut contents of 20 mated Males and Females of Tachypleus gigas .

126

127

Table 30 Dig esti ve enzyme assa y of T. gig as Digesti estiv assay gigas (Mean + SD values are presented ) 1

2

Enzymes Assayed

Oesophagus n=20

Acid Protease (mg. tyrosine)

0.192 +

Alkali Protease (mg. tyrosine) Esterase (mg. B-napthol)

Gizzard n=20

0.015

3

4

Intestine n=20

Hepatopancreas n=20

0.679 + 0.061

0.464 + 0.037

0.259 + 0.023

0.406 + 0.053

0.372 + 0.059

0.756 + 0.113

0.642 + 0.098

0.500 + 0.055

0.260 + 0.031

0.273 + 0.035

0.296 + 0.034

Cellulase (mg. glucose)

10.944 + 1.061

11.529 + 1.037

7.736 + 0.618

9.351 + 0.912

Invertase (mg. glucose)

3.462 + 1.290

4.683 + 0.601

5.899 + 0.612

6.059 + 0.557

Amylase (mg. glucose)

13.587 + 1.290

15.640 + 1.801

15.658 + 1.733

16.608 + 2.011

MO SO

PH

UT

H

HEPATOPANCREAS

CR OP

OE

AG

US

A

M

ID

-G

U

T

RECTUM HIND-GUT ANUS

INTESTINE STOMACH

LO NG RID ITUDI NA GE L S

GIZ ZAR D

FOOD BOLLUS

CU TE TICU ET H LAR

LVE LVA ICA CON

D

B

C Fig. 32 C , Shows different parts of the gastro-intestinal tract of horseshoe crab .

128

A

B

C

Fig . 29 (A-C) A, Shows various kinds of foram shells obtained from the stomach content ; B , Plant parts comprising fruits , vascular structures , Casuarina needle and others ; C , spicules , polychaete jaw , body of tabanid larva , segments of ophiuroid arm and full goboid fish body have been recovered from the stomach content of T. gigas .

A

B

C

Fig . 30 (A-C) A, Various parts of food matters , the most important being the razor clams (RS) occur in the gut contents of C. rotundicauda ; B , Different bivalve prey shells of both fragments and intact juveniles occurring in the gut ; C , Shows the broken as well as intact gastropod shells in the gut of T. gigas .

130

Fig . 31 (A&B) A, Shows pelagic forms of polycheates and intact body parts , particularly the head and tail regions ; B , Plate showing the intact as well as broken or partly corroded crustacean body parts . The most conspicuous is being the gammarid amphipod (C) .

131

A

B

C Fig . 32 (A-C) A, Dissection of the alimentary tract for its primary preservation in 3.7% formalin . Numerous mature eggs are also seen in the tray recovered from the female's prosoma ; B , A simplified trophic structure showing its various levels of organization for both T. gigas (Tg) and C. rotundicauda (Cr) . Polychaetes (P) , gastropods (G) , mysid shrimps (M) , bivaves (B) and ophiuroids (O) that depend primary on the benthic detritus and plant life ; C , Shows various parts of the gastro-intestinal tract of horseshoe crab .

132 DISCUSSION Molluscs are the most importent food item in terms of numerical abundance to adult T. gigas and L. polyphemus of Delaware Bay (Botton 1984a,b) during the season .Next to its stand the numerical abundance of arthropods e.g. gammarid amphipods and tabanid larvae . On the continental shelf of New Jersey , crabs preyed on a wide variety of benthic organisms , primarily molluscs , arthropods and annelids (Botton and Haskin , 1984) . The size inequality between adult male and female horseshoe crabs generated the hypothesis that diets of males and females might differ . Females are larger than males , which might enable females to feed on large prey . Futhermore females can dig deeper than males and might therefore have access to such type of fauna which are not available to males . However , based on gut contents , males and females in the present study show similar diets and food preferences . In the aquarium experiment , Botton (1984b) observed similar response by the males and females , furthermore , males were to feed more . The above results of L. polyphemus do not corroborate with the results obtained in the present investigation . Adult mated females of T. gigas were found to consume more food than either the solitary or mated partner males . On the other hand , solitary males are found to consume more food than the mated males . These findings lead to the dorsal opisthosoma of the female partner for a long time they do not have access to the bottom benthic community ; while this was more easy for the carrier females . At the same time solitary males come in direct contract to the benthic community for feeding . This hypothesis is further confirmed by the presence of all zooplanktons or pelagic prey found in the gut-content of mated males . The tenable alternative hypothesis (Botton , 1984b) explaining the food selection in horseshoe crabs involves chemically mediated preferences . Both gnathobase spines and chelae of the walking legs possess chemorecaptors (Barber , 1956 ; Wyse , 1971) which are supposed to be involved in food selection . As regards the preferential consumption of prey materials of L. polyphemus , Botton's (1984 a,b) analysis shows that the 'crab' lays its preferential importance on the large over small molluscan shells and soft shells over the thicker one in both the sexes . Of course this type of observation in the gut contents of T. gigas and C. rotundicauda could not be established , at least in this geographical locale. Effects of predator's differential prey digestibility may induce bias results in the identification and enumeration of ingested prey materials in favour of taxa with shells and exo-skeletons (Botton 1981) . Keeping this in mind , the gut contents were meticulously examined , and molluscs were found to be the most abundant animal prey followed by the arthropods and polychaetes (Botton , 1984b) . Consumption of plant detritus of T. gigas is supported by the report of ( Botton , 1984a,b) i.e. 90% L. polyphemus consumed heavy amount of vascular plant matters , although the nutritional value of plant to this predator is not known . In the present investigation , gut content was seen to reflect similar food preferences between the two sexes of the species studied , through the females were found to have more ingested matter in gut than the males . This may be due to the fact that in the coupled T. gigas , the smaller males clings to the back of the larger female , which has its chelae free for foraging . These observations led the author to infer that T. gigas which grazed along the sea shore , was a dietary generalist (Debnath and Choudhury , 1987) . This observation has also been endorsed by Botton and Haskin (1984) . The presence of proteases , esterases and carbohydrases in the digestive tract and associated gland lends further evidence to the above assumptions (Debnath et. al. , 1989) . Lockhead (1950) reported in Limulus ( = Xiphosura) polyphemus , that digesion occurred in the stomach-intestine by means of enzymes from hepatopancreas (midgut gland) , except for splitting dipeptides , which occurred (Schlottke , 1934a,b) . A second set of enzymes active in acid medium in

133

the foregut has been reported which may be the reason why acid protease activity is highest here . But the stomach - intestine which received secretions from hepatopancreas has high alkaline protease activity (Lockhead , 1950) . Among proteases , the alkaline and among carbohydrases , amylase are the most abundant . A remarkable amount of non-specific esterase has been reported in T. tridentatus by Tanimoto and Kondo (1982) in the conical cells of the hepatopancreas , but how this esterase has occurred abundantly in the oesophagus of T. gigas is yet to be ascertained . Presence of celluase even after through cleansing of gut lumen by flushing indicates that the cellulase in question is perhaps not bacterial in origin (Debnath et.al. , 1989) . Because , so far the amount of concentration is concerned , cellulase stands next to amylase (Table 30) . That Whether this should be regarded as a preadaptation for its omnivorous habit or as retention of its ancestral enzymic complement is not clear . Fage (1949) reported the hepatopancreas to be the site of enzyme production and digestion , the digestion being not entiraly extracellular , but also occurring intracellularly in hepatopancreas (Schlottke , 1934a,b) . This is further coroborated by the present observation that most of the hydrolysing enzymes are present in large amount in the hepatopancreas A synopsis of horseshoe crab's food-items with special reference to L. polyphemus is given below ( based on Shuster 1982 ) : 1. Trilobite stage : Experimental feeding with a variety of organic matter and organisms e.g. bits of Mytilus , Nereis , frozen brine shrimps , marine nematodes , small whole polychaetes , and alga , Enteromorpha sp. (French , 1979) . 2. Immature crabs : Worms - Cerebratulus , Nereis and Cistenids (Shuster , 1950) ; Bivalves Mya , consumption of 99 items in 72 hours (Turner et. al. , 1948) ; Macoma , Gemma , Ensis , Spisula and Mytilus (Shuster , 1950) . 3. Mature (adult crabs) : Comparatively soft-shelled molluscs , both gastropods and bivalves ; annelids (mainly polychaetes) , arthropods , plant materials and miscellaneous category including bryozoa , nematodes , teleosts , turbellarians and foram shells . (Shuster , 1982 ; Botton , 1981 ; 1984a,b etc.) A considerable amount of ophiuroids reported for the first time T. gigas as food item (Debnath and Choudhury , 1987; Debnath et.al. , 1989) .

NEXT CHAPTER ON

CENTRAL NERVOUS SYSTEM & NEURO-ENDOCRINE SYSTEM

134

PART - III STUDIES ON ANATOMY AND CYTOMORPHOLOGY OF THE CENTRAL NERVOUS SYSTEM WITH SPECIAL REFERENCE TO THE NEURO - SECRETORY ELEMENTS

Olfactory nerve Optic nerve Cheliceral nerve PROTOCEREBRUM (Supra-pharyngeal ganglion)

TRITOCEREBRUM (Circum-oesophgeal Ring) ORAL COMMUSSURE Nerves to walking legs Nerve to 6th walking legs

VIII

SUB-PHARYNGEAL GANGLION VENTRAL NERVE CORD Stomodeal nerve Nerve to genital operculum

IX X Nerves to book-gills

XI XII XIII XIV Caudal nerves

Central Nervous System of Horseshoe Crab, Carcinoscorpius rotundicauda

135 INTRODUCTION

Research literature on arthropod nervous and neuroendocrine system is immense (Bullock and Horridge , 1965 ; Gabe , 1966 ; Highnam , 1965 ; Novak , 1975) . The organization of the arthropod nervous system has a striking correlation with their increased cephalization . This cephalization in arthropods is seen not only in greater abundance of cephalic sensory organs and their specialisation but is also reflected in a more highly developed brain . The arthopod brain , in general , consists of three major regions - an anterior protocerebrum , a median deutocerebrum and a posterior tritocerebrum . Of these three , the deutocerebrum receives the antennal nerves ( 1st antennae of crustaceans) and contain their associated organs. Antennae are lacking in the chelicerates like merostomates (horseshoe crabs) and arachnids (spiders and scorpions). As a result the chelicerate brain is devoid of the deutocerebrum . The brain in arthropods is joined to a ventral cord via circum-oesophageal connective . Arthropod nervous system , like those of annelids contains many sensory cells and interneurones . The flexibility of arthopod behaviour and their impressive power of association learning , may thus seem surprising . Part at least of the explanation , however , is doubtless , to be searched in the extensive branching of their nerve processes , and the variety of connections thereby made possible , so that the nervous system is very much more flexible in action . The nervous system in all invertebrates contains in addition to "ordinary" or " typical" neurones (as concerned with the propagation of nerve impluses and the secretion of chemical transmitter substances) a second type of neurone containing secretory product that is readily stainable and that can be seen with the light microscope . It is referred to as neurosecretion . This material , like the neurohumours , arises in the cell body , passes down the axon , and is released from the nerve endings . After being discharged from the cells it passes into the blood stream and circulates round the body , to produce specific physiological effects at targets that may be remote from the region of its release . The cells producing this substance are customarily referred to as neurosecretory cells ; their function is , thus , the secretion of neurohormones , which form part of the endocrine system of the body and thereby termed as the "neuro-endocrine ststem" (Barrington , 1979) . Among the arthropods most of the work on neurosecretion are confined to the mandibulates like Insecta and Crustacea (Bliss and Welsh 1952 ; Matsumoto , 1954 ; Kobayashi , 1957 ; Highnam , 1965 ; Madrell , 1967 ; Novak , 1975 ; Ramadon and Matta , 1976 ; Nanda et. al. , 1984 ; Ghosh and Nanda , 1985) , but information is scanty and unsatisfactory regarding the neurosecretion in Chelicerate arthropods (Scharrer , 1941 ; Gabe , 1955 ; Enami , 1957 ; Habibulla , 1961 , 1970 ; Herman and Preus , 1973) . The earliest report of neurosecretion in Merostomata is that of B . Scharrer (1941) who reported the occurrence , localozation and quantitative distribution of "putative neurosecretory cells" throughout the central nervous ststem of Limulus polyphemus and compared the findings with Tachypleus gigas(collected from Penang , Malayasia by the author as "Limulus moluccanus"in her paper). Later , Enami , 1957 ; and Herman and Preus (1973) reported two types of neurosecretory cells in rudimentary eyes of both L. polypheus and T. tridentatus and the abdominal ganglia of L. polyphemus respectively , on the basis of cell dimension and cytoarchitecture . The present investigation deals with anatomy and cytomorphology of the central nervous system (CNS) and neurosecretory cells (NSCs) of Carcinoscorpius rotundicauda which has hitherto not been recorded so far . Such studies are necessary to correlate the event of histophysiological changes brought about by environmental stimuli. Since all the functional activities of an arthropod , like breeding and feeding behaviours , development , growth , molting , circadian rhythmicities , dispersal and migration etc. , are directly or indirectly controlled by its neuroendocrine system , the present investigation , obviously , is expected to highlight a new area of study on Indian horseshoe crabs .

136 OBSERVATIONS

(A) Anatomy of the CNS

The nervous system displays (Fig. 33A) a large degree of fusion plus the very unique feature of being almost entirely enclosed within arterial vessels (hence the term " vascular-ring") . The brain forms a collar around the oesophagus . The anterior part (suprapharyngeal ganglion) of the collar forms the protocerebrum , while the lateral portions (subpharyngeal portions) represent a fusion of the tritocerebrum plus the ganglia for all the remaining first seven segments . The collar circumscribes and unites behind the oesophagus , giving rise to both a ventral nerve cord with five ganglia and lateral nerves extending through the abdomen . The brain receives nerves from two pairs of lateral eyes as well as from the frontal organ (i.e. the olfactory nerve). The tritocerebrum fuses with the posterior part of the brain and receives the cheliceral nerves . Ganglia II to VIII have moved forward and fused to a large sub-pharyngeal mass , serving not only the prosoma but also abdominal segments VII and VIII . Abdominal ganglia IX to XIV stay in their respective segments and form a nerve chain , the anterior ones close to the appendages , the last three in one mass , possibly a vestige of the 15th segment . So , in general , the nerve ring consists of 3 or 4 post oral commissures on the dorsal part and 1 pre-oral commissure on the ventral side at the posterior margin , an anterior cerebral swelling , the nerves to the appendages , the stomodeal nerves , the olfactory nerves , and the nerves to the median and lateral eyes . (B) Cytomorphology of CNS The CNS is covered by an outer thick connective tissue sheath and the neurosecretory cells (CNCs) are ordinarily distributed throughout the CNS . The NSCs are mainly concentrated in groups extending from ventral to dorsal surface of each ganglia and the arranged symmetrically all around the central fibrous neuropile with respect to the midsagittal plane . It is to be noted that the NSCs stain faintly with CHP and AF , moderately with Masson's trichrome and intensely with Azan method . On the basis of their size , cytoarchitecture and pattern of distribution in the CNS , the NSCs may be classified into two major types : namely, A and B cells . A-cells : These cells are considerably large (diameter ranging from 90 to 100 µm ) , spherical or pear-shaped in appearence ( Fig. 33 B&D) and possess moderately stained cytoplasm . Axonal process may or may not be present . Such type of cells are often distributed beneath the perineurium and usually form groups . A-cells is characterised by the presence of a centrally placed conspicuous round nucleus measuring about 20-22 µm in diameter besides possessiing well defined nucleous (Fig. 33C) . The cytoplasm exhibits clumpy appearence and indications for having cyclical secretory activity ranging from a homogeneous condition to a state of fine and occasionally coarse granulation . Strikingly , majority of the cells possess a ring of cytoplasmic vacuoles in between the cytoplasmic clump and the cell membrane (Fig. 33E) . Axonal bundle ( Figs. 33 E and F) . Seldom transport of neurosecretory material through the axons could be visualised .

137

B-cells : Cells of such types are smaller with wide range of fluctuation in their diameter (30 to 50 µm ) . They are irregular in outline and generally exhibit deep stained cytoplasm having coarse consistency (Figs. 33 B and D) and possess centrally placed prominent nucleus (12 -15 µm ) . These cells are more numerous than A-types and ordinarily distributed , very close to the neuropile forming compact groups of cells . Axonal processes are generally long , filamentous , deeply stained and frequently forming large axon bundles (Fig. E & G) . Discripancies in the pattern of distribution of NSCs in different parts of CNS A and B cells are deterctable in all the ganglionic parts of the CNS . But their distribution varies with respect to different ganglia . The posterior third of the circumoesopageal ring (containing fused 2-3 thoracic ganglia) shows dramatic increase in number of NSCs when being compared with the anterior two third of the ring . The abdominal ganglia also retains increased population of NSCs . Brain : B-cells (mean diameter 35 µm ) are much more in number than the A-cells (mean diameter 90 µm ) and form compact cell groups (Fig. 33 B) . Axon bundles emerging out from these Bcell groups make a configuration of decussation within the neuropile (Fig. 34 B) . Well defined capillary networks that supply the brain are found to encricle the neurosecretory cell groups and even the solitary cell also ( Fig. 33C , 34A) . Besides these NSCs , there are large clusters of conspicuous small cells with delicate axonal processes that are found all around the neuropile in the antero-median portion of the brain . These cells , however , do not show any sign of secretory activity and probably these cells are the ordinary neurones (Fig. 34C) . Thoracic ganglia : A drastic enhancement in the number of both types of NSCs is noticeable . Three important features are on record when compared with the brain NSCs : 1.

Both categories of cells maintain larger dimension (A-cell : 100 µm , B-cell : 40µm ) .

2.

Number of A-cells is relatively enhanced (Fig. 33D and F) .

3.

Distinct of specific groupings of A- and B-cell types are demonstrable despite existence of mixed groups comprising both A - and B - types .

Axnal bundles are , however , common in both categories of cells ( Fig. 33 E & G) . Besides these , involvement of blood capillaries in respect of NSCs remain similar to that of brain . Abdominal ganglia : The abdominal ganglionic NSCs in respect of diameter , number , groupings , supply of blood capillaries etc. have close resemblance with the NSCs displayed in the thoracic ganglia (Fig. 33 F and G) . However , a few A-cells show giant size (100-120 µm ) with coarse cytoplasm , large peripheral vacuoles and long tubular axonal processes (Fig. 34A) . Axon bundles are more abundant in the abdominal ganglia in comparison with other regions of CNS . A comparative account of size range and staining affinities of the NSC types in the CNS of Carcinoscorpius rotundicauda are given in Table 31 .

138

Table 31 Comparative size ranges and staining affinities of the two types of BSCs in the central nervous system (CNS) of Carcinoscorpius rotundicauda .

Cell type

Mean cell diameter(µ m)

Mean nuclear diameter (µ m)

Staining affinity of NSM CHP

AF

Masson's Trichrome

ø

Azan

Brain A

90

20

+

+

B

35

12

+

+/-

++ +++

+++ ++++/+++

Thoracic ganglion A

100

22

+

+

++

+++

B

40

15

+

+

++

+++

+++

Abdominal ganglion A

98

22

+

+

++

B

38

12

+/-

+

+++

ø NOTE : + ++++/+++

++++/+++

= weak/ faintly positive , ++ = moderately positive , = strong (intensely positive) , +/- = doubtful reaction

139

Central nervous system of C. rotundicauda

A

B

C

D

E

F

G

Fig . 33 (A-G) A, Central nervous system (CNS) of C. rotundicauda showing the circumoesophageal ring (brain) and the ventral nerve cord with emerging nerves from them ; B. Section of brain of C. rotundicauda showing distribution of two types of NSCs ( A & B) , Azan x 125 ; C , Section of brain showing individual A cells encircled by blood capillaries , Azan x 532 ; D , Section of thoracic ganglion , showing an increase in the number of A cells . Azan , x 125 ; E , Section showing groups of A cells and B cells forming a large axon bundle , Masson's trichome , x 125 ; G , Section of abdominal ganglion showing a large group of A cells , Azan , x 125 .

B

Blood Capillry B-cells of the Brain

Neuro-secretory granules

Peripheral Vacuole

A

B

Decussation of axon bundles

C

Central Nucleus Nucleolus

Atypical Neurones

Axon bundle

Fig . 34 (A-C) A, Shows " decussation" of axonal bundles emerging from the B cells in the brain region ; B , Shows an enlarged cell-body of A-type displaying peripheral vacuoles containing NSM , central rounded nucleus with a distinct nucleolus . The cell-body is encircled by a capilary system (dark) , Azan , x 1100 ; C , Aggregation of numerous minute ordinary ( "atypical") neurones associated with cellular bodies of NSCs .

141 DISCUSSION

Histological probe of the central nervous system of Carcinoscorpius rotundicauda shows the existence of major two types of cells which maintain characteristic pattern of distribution within the circumoesophageal ring , as well as , the ventral nerve cord . In her pioneering work B. Scharrer (1941) reported a single type of neurosecretory cell throughout the CNS of Limulus polyphemus . She characterised these cells on the basis of the possession of large colloidal mass , marginal vacuolations and eccentric nuclei . In contrast with this observation , Herman and Preus (1973) observed 2-types-Type I and Type II of NSCs in the same species . These observations , however , based on both light and electron microscopic studies and chiefly restricted to the morphological characteristics of the granules despite other cytomorphic features . In the present investigation on C. rotundicauda the author did not observe coloid laden NSCs throughout the CNS as reported by B. Scharrer (1941) . On the other hand , two types of NSCs ( A- and B-types) are encountered and classified on the basis of their distribution , shape , size and other morphological features and is in agreement with the observation of Enami (1957) , Herman and Preus (1973) , as and when , morphological features of cell types as such is taken into account . It is interesting to record that like the ventral ganglionic 'C' cells of mandibulates neurosecretory elements of horseshoe crabs are readily identifiable with Azan , as well as Masson's trichome stains but react poorly with either CHP or AF . This may have some bearing with the histochemical nature of the neurosecretory product in the Xiphosura which possibly retains its strong acidopholic nature that could not be scuttled after oxidation of tissue section with potassium permenganete (Gabe , 1966) . In Carcinoscorpius differential pattern in the distribution of NSCs has been recorded in the CNS . The most "active" neurosecretory region in CNS is posterior part of the circumoesophageal ring . Here the neuroglandular elements are found in clusters that occupy a considerable portion of the ganglia . B. Scharrer (1941) who reported maximum concentration of chromatophototropic principle in posterior third of the Limulus circumoesophageal ring . Increased population and compact arrangement of NSCs and occurrence of more axonal bundles in the abdomical ganglion are all indicative of its functional importance when correlation between physiological and anatomical findings are taken into account (B. Scharrer , 1941) . Although occurrence of well defined axon bundles is a regular feature in the neurosecretory system of Carcinoscorpius rotundicauda , transport of discrete stainable material (NSM) through them , is seldom .It is striking that despite the presence of open type of circulation in Xiphosura , capillaries are found to surround neurosecretory cells throughout the CNS of Carcinoscorpius . Similar arrangement of capillaries around the NSCs had also been reported in the central nervous system of crabs like Paratelphusa hydrodromus (Parameswaran , 1956) and Eriocheir japonicus (Matsumoto , 1954) . These observations indicate that the neurosecretory materials in Carcinoscorpius sp. are possibly discharged directly from the perikarya into the blood and thus reach the general circulation of the body readily without undergoing axoplasmic flow or terminating to the conventional neurohaemal organs . Enami's (Enami , 1957) and our's (Choudhury et. al. , 1991) similar findings suggest that the ventral arteries surrounding the central and peripheral nervous systems play a role in transportation of the NSM , instead of the axons as in many other animals . Such an arrangement of the circulatory system in the CNS also suggests special functions in the event of attributing the species under the category of "Living fossil" (Choudhury et. al. , 1991) . Nevertheless , neurosecretory cells must be expected to have a high metabolic rate and , therefore , require a rich blood supply . This is also the case in vertebrates where nuclei composed of secreting nerve cells are among the most richly vascularised structures of the central nervous system (Scharrer and Scharrer 1954) . In fine , it is to be noted that the functional activities of the neuroendocrine system of horseshoe crabs are , probably , attributed with the moulting phenomena (jegla , 1972 ; Jegla and Costlow , 1979a,b ; Krishnakumaran and Sneiderman , 1970) , visual system and mating behaviour.

142

ret

Cytoplasm

Blood Capillry

Nucleolus

Sec ory Gra

Spherical Nucleus

nul

Cytoplsmic vacuoles

Nucleolus

(Barlow , 1982 , 1983 ; Barlow et. al. , 1977 , 1982 , 1984 , 1985 , 1986 , 1987 ; Lall and Chapman , 1973 ; Rudloe , 1979b , 1980) and other behavioral patters (Botton and Loveland , 1987) . It is also quite possible to control the feeding behaviour as was observed in the red cotton bug , Dysdercus cingulatus that the median neurosecretory cells of the brain are responsible for the stimulation of food consumption and respective enzyme activity through "Secretogogue mechanism" (Muraleedharan and Prabhu , 1979) . Similarly Thomsen and Moller (1959) reported the role of median neurosecretory cells in digestion of Calliphora erythrocephala . The presence of two putative neurohormones (dores and Herman , 1980) in the CNS of L. polyphemus might have some correlation with the present observations.

Central Nucleus

Axoplasm

es

on

Ax

A - type NSC of Thoracic Ganglion

A - type NSC of Brain

Since all the functional activities of an arthropod , ...................... are directly or indirectly controlled by its neuroendocrine system , the present investigation , obviously , is expected to highlight a new area of study on Indian horseshoe crabs

143

SUMMARY of THE THESIS The present study on geographic distribution confirms that the western limit of distribution of the two species of Indian horseshoe crabs ,Tachypleus gigas(Muller)and Cacinoscorpius(=Tachypleus n.comb.) rotundicauda (Latreille) is up to the coastal zone of Orissa (lake Chilka) and West Bengal (Sundarbans) through Bangladesh . Discrete local population of T. gigas occurs in the sandy sea shores . C. rotundicauda dominates the muddy shores of Sundarbans and Dhamra estuaries , while T. gigas also occurs at the later area showing typical sympatrism with C. rotundicauda . The present work also confirms that T. tridentatus does not exist in the Indian coast . Comparative morphology as well as morphometry reveal that the male is nearly 78% and 85% of the female of T. gigas and C. rotundicauda , respectively . But in respect to body weight the male T. gigas is almost 37% of the female , while it is 52% in C. rotundicauda .Considering all the morphometric parameters it is found that the female specimens of C. rotundicauda are smaller than the females of T. gigas , but the situation is just reversed in case of male specimens . It is also evident that a perfect correlation occurs between the dorsal body length (DBL) and the dorsal prosoma breadth (DPB) . So it seems to be justified to make a length - weight relationship taking count the DBL as the only size parameter . In this study , the male increases in weight by an exponent of 3.08 and the female by 2.52 in T. gigas , while in C. rotundicauda males and females increase by an exponent of 3.09 and 2.35 , respectively . The general growth patterns are found to be more or less identical in both the species . The marking study reveals that nearly 5,000 T. gigas come ashore at Chandipur (Orissa) during the fullmoon tides of March , 1985 . The total absolute estimate is obtained after regression method to be nearly 70,000 during the entire breeding season that ranges from March to July (Chandipur) .This figure is rather small i.e. almost 2,000 in the sea shore area of Digha (West Bengal) . During fullmoon high tides of spring and summer months horseshoe crabs are found to emerge from the shallow sea to spawn . The usual 1:1 sex-ratio is obtained for the occurrence of normal amplexus i.e. the larger female carrying a smaller male on her back . Very frequently male biased sex-ratio is found due to stranded male crabs occurring at the exposed sandy beach or mud-flat . It is evident from the present study that deviation in the sex-ratio is significant in all coastal stations of West Bengal , while it is not statistically significant at the coast of Orissa . This deviation in sex-ratio (6.89 :1) at Digha than of Chandipur (1.04:1) might be attributed to the severe predatory pressure exerted by the ravens , Corvus splendens L. and human interference . Preliminary studies on the feeding habit and digestive physiology of T. gigas has indicated its omnivorous food habit . Gut content analysis also enumerates a varieties of food matters e.g. vascular plant parts , bivalves , gastropods , polychaetes , ophiuroids , amphipods and several others to infer the horseshoe crabs also as a " dietary generalist ." Study on central nervous system of C. rotundicauda has revealed two types of neurosecretory cells ( A & B ) differing as regards to their size , cytoarchitecture and distribution . The A cells are large , with fine cytoplasmic granules (NSM) , and are distributed below the perineurium , while the B cells are much smaller , contain coarse granules and are distributed in the vicinity of the neuropile . The posterior third of the circum-oesophageal ring and the abdominal ganglia contain a far larger number of NSCs as part of the CNS . In fine , it is to be noted that the functional activities of the neuroendocrine system of horseshoe crabs are , probably , attributed with the moulting phenomena , visual system and mating behaviour.

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