Vitellogenin is the blood protein precursor of lipovitellin, the

main yolk protein. Many crustacean workers have used electrophoresis

to detect the vitellogenin and lipovitellin in the blood

and ovary respectively. However, authenticity of identical

relative mobilities in homologizing vitellogenin with lipovitellin

has been sometimes questioned because fluctuating current,

buffer strength and gel composition may lead to variations in

the relative mobility of the same component

Ezhilarasi, S

Subramoniam, T

CMFRI Digital Repository

„>A»;S!£S " * • \ % 3 • U ^ ' CMFRI SPIXIAL PUBLICATION Number *> MANUAL OF RESEARCH METHODS FOR MARINE INVERTEBRATE REPRODUCTION Issued on the occasion of the Workshop on MARINE INVERTEBRATE REPRODUCTION joint ly organised by the Department of Zoology, Universi ty of Madras and the Centre of Advanced Studies in Mar icu l t i i re , Central Mar ine Fisheries Research Inst i tu te, Cochin held at the Universi ty of Madras f rom 25th October to IOth November 1982 The Centre of Advanced Studies in Mariculture was started in 1979 at the Central Marine Fisheries Research Institute, Cochin. This is one of the Sub-projects of the ICAR/UNDP project on 'Post-graduate agricultural education and research'. The main objective of the CAS in Mariculture is to catalyse research and education in mariculture which forms a definite means and prospective sector to augment fish production of the country. The main functions of the Centre are t o : —provide adequate facilities to carry out research of exce-llence in mariculture/coastal aquaculture ; —improve the quality of post-graduate education in mari. culture; —make available the modern facilities, equipments and the literature; —enhance the competence of professional staff; —develop linkages between the Centre and other Institutions in the country and overseas ; —undertake collaboration programmes ; and —organise seminars and workshops. Under the programmes of the Centre, post-graduate courses leading to M.Sc. (Mariculture) and Ph.D. are offered in colla-boration with the University of Cochin since 1980. Front cover: SEM picture showing surface topography of Strepto-cephalus dichotomus egg. Manual of Research Methods for Marine Invertebrate Reproduction EDITED BY T. SUBRAMONMM Unit of Invertebrate Reproduction, Department of Zoology, University of Madras, Madras-600 005 & CMFRI SPECIAL PUBLICATION Number 9 ISSUED ON THE OCCASION OF THE WORKSHOP ON MARINE INVERTEBRATE REPRODUCTION JOINTLY ORGANISED BY THE DEPARTMENT OF ZOOLOGY, UNIVERSITY OF MADRAS AND THE CENTRE OF ADVANCED STUDIES IN MARICULTURE, CENTRAL MARINE FISHERIES RESEARCH INSTITUTE HELD AT THE UNIVERSITY OF MADRAS FROM 25TH OCTOBER TO 10TH NOVEMBER, 1982 . (LIMITED CIRCULATION) Published by: E. G. SILAS Director Central Marine Fisheries Research Institute Post Box No. 1912 Cochin 682 018 PRINTED IN INDIA AT THE DIOCESAN PRESS, MADRAS'7—1982 C3626 PREFACE The technologies of controlled reproduction, induction of spawning, sex reversal, artificial fertilisation, sterilisation and preservation of gametes are increasingly applied in aquaculture to obtain quality seed, quality fish stock and better yield. In this context, researches on different aspects of reproduction, developmental biology and physiology have assumed consider-able importance besides their values in understanding of the ontogeny of the organisms. Extensive researches carried out in recent years from several laboratories in the world have not only accumulated a body of information, but also broughtforth several new concepts to our understanding of the development and reproductive behaviour of finfishes and shellfishes-In India, directed research on reproductive physiology and biology is taken up only recently and the field is still in an infant stage. In view of its emerging importance, it is identified as an area for priority research and for expertise development in the programmes of the Centre of Advanced Studies in Mariculture at the Central Marine Fisheries Research Institute, and several programmes of research are being taken up in this field with particular reference to the reproductive behaviour of the culti-vable finfishes and shellfishes. Advances made on the frontiers of invertebrate reproduction in recent years have been significant enough to organise a national workshop and to prepare a manual on research methodologies for the study of the subject. Several histological, histochemical and biochemical methods and sophisticated instru-ments have been introduced in these studies making it essential that the scholars who desire to work and specialise in the field are given adequate basic information on the research methods so as to enable them to appreciate and advance research to understand the problems confronted in the field. The present manual, the third in the series, is prepared and compiled by Dr. T. Subramoniam, Leader of the ' Unit of i i i Invertebrate Reproduction ' of the Zoology Department of the University of Madras, Tamil Nadu. During the past decade, a team of research scholars are working on different aspects of marine invertebrate reproduction including the cultivable crustaceans such as Scylla serrata, Panulirus homarus and Macrobrachium spp. under his leadership. Contributing to our knowledge on the subject, the research results achieved so far in these aspects by the Unit have unfolded several new concepts in oogenesis, spermatogenesis, sperm transfer strategy, ferti-lization and endocrine control of reproduction and gamete formation. I wish to express my great appreciation to Dr. T. Subramoniam and his team of Scholars, who by their dedication and interest evolved a series of tested research methods and set a theme of investigation through insight and skill on marine invertebrate reproduction. I am sure that this manual will be of immense use to the research scholars and scientists who would like to specialise in the subject and cognate fields. This is the second workshop we are organising in close colla-boration with the University of Madras. I wish to express my gratitude to Dr. M. Santappa, Vice-Chancellor, University of Madras for the keen interest evinced in such collaborative pro-grammes and for the advice. I am also indebted to Dr. K. Ramalingam, Professor and Head of the Department of Zoology, University of Madras for productive discussions, continuous support and suggestions. I wish to thank Shri P. T. Meenakshisundaram and Shri K. Rengarajan, Scientists of the Central Marine Fisheries Research Institute for their help in the preparation of this manual. E. G. SILAS, Director, C.M.F.R.I. iv 4 SEROLOGICAL IDENTIFICATION OF VITELLOGENIN AND LIPOVITELLIN IN SCYILA SERRATA AND EMERITA ASIATICAVSSNG BMMUNO ELECTROPHORESIS* 4.1. INTRODUCTION Vitellogenin is the blood protein precursor of lipovitellin, the main yolk protein. Many crustacean workers have used electro-phoresis to detect the vitellogenin and lipovitellin in the blood and ovary respectively. However, authenticity of identical relative mobilities in homologizing vitellogenin with lipovitellin has been sometimes questioned because fluctuating current, buffer strength and gel composition may lead to variations in the relative mobility of the same component. It is therefore necessary to confirm the results of electrophoresis by serological investigations. Immunoelectrophoresis has been tried for study-ing the relationship between lipovitellin and vitellogenin in a few crustacean forms (Kerr, 1969 ; Croisille et al„ 1970 ; Ezhi-larasi, 1982). Many of the crustacean Wood proteins are immunogenic and produce antibodies in the mammalian blood. Therefore, cross reaction of these antibodies with the suspected identical proteins will produce specific precipitation arcs thus enabling real comparison. In addition, this method is advanta-geous in tracing the origin of vitellogenin into the extraovarian sources, even if the concentration of such precursors is very low. •4.2. PRINCIPLE Antibodies to vitellogenin and lipovitellin are raised by active immunization by injecting the antigen directly to albino rabbits. Antigen-antibody reaction is carried out in the gelified medium •either in agarose/agarose or polyacrylamide/agarose and bands of precipitation form wherever an antibody and its corresponding * Prepared and verified by S. Ezhilarasi and t . Subramoniam, •unit of Invertebrate Reproduction, Department of Zoology, Univer-sity of Madras-600 005. 51 antigen meet at the optimal proportion. Both vitellogenin and lipovitellin of S. serrata are immunogenic in that, they give-rise to antibodies that will specifically react with them. When these soluble antigen and antibody diffuse towards each other in a gel, a precipitin line is formed at their place of meeting and form an impermeable barrier to the antigen and antibody that has formed it. This is permeable to all other substances that are not concerned with that precipitate in question. However^ this barrier persists only as long as some of the forming ingre-dients are present in the gel on either side of it. 4.3. PROCEDURE 4.3.1. Preparation of antiserum 4.3.1.1. Rabbit anti-lipovitellin antiserum 1. Homogenize 500 mg of freshly laid eggs of S. serrata and E. asiatica (0.0167 gm of protein) in Carcinus maenas-Ringer solution (Smith and Ratclifife, 1978). To prepare C. maenas Ringer solution dissolve 33.7 gm sodium chloride, 0.94 gm pottassium chloride, 2.83 gm calcium chloride, 5.38 gm magne^ sium chloride, 0.193 gm disodium hydrogen phosphate and 6.060 gm Tris (hydroxymethyl) methylamine in 42.5 ml of 1 M hydrochloric acid and make it upto 1 litre. The pH is adjusted to 7.4. 2. Centrifuge at 5,000 g and decant the supernatant without the lipid cap. This clear supernatant will serve as the source of antigen. 3. Mix 1 ml of antigen and an equal volume of Freund's complete adjuvant (Difco). 4. Inject this mixture subcutaneously on 1st, 14th, and 21st days. 5. Third injection is a booster injection having double the quantity of antigen. 4.3.1.2. Rabbit anti-haemolymph containing female specific protein (FSP) antiserum 1. Collect 2 ml of haemolymph containing FSP (0.9074 gm protein) from S. serrata in late IVth stage or early Vth stage of ovarian maturation. Add this to a mixture containing 1 ml 52 -of 12.5% sodium citrate and 1ml of C. maenas Ringer solution <To prepare sodium citrate solution dissolve 12.5 gms of it in 100 ml of double distilled water). : 2. Mix 2 ml of haemolymph with 2 ml of Freund's complete adjuvant and inject into the rabbits subcutaneously as described -above. -4.3.1.3. Rabbit anti-control antiserum Prepare the carrier medium by mixing 0.67 ml of C. maenas Ringer with 0.33 ml of 12.5% sodium citrate. Mix 1 ml of this with 1 ml of Freund's complete adjuvant and inject subcuta-neously. -4.3.2. Collection of antiserum . 1. Collect rabbit blood by puncturing: ear vein on 27th •day. Collect blood in the same way on 34th day also* into a •sterilized boiling test tube. 2. Plug the tubes with cotton and leave it overnight in the refrigerator slantingly. 3. Collect the separated serum into sterilized screw cap vials and store in refrigerator. -4.3.3. Purification of antiserum 1. Mix 1 ml of antiserum with 1 ml of freshly collected male haemolymph in C. maenas Ringer. 2. Centrifuge at 6,780 g for 10 minutes to precipitate the reaction products formed by the interaction between the common -antigens of male S. serrata and E. asiatica haemolymph and -antibodies of female haemolymph containing FSP antiserum. 3. Mix the supernatant with 1 ml of male' haemolymph repeatedly after each centrifugation, till no visible precipitate was formed (Fyffe and O'Connor, 1974X 4.3.4. Preparation of agarose gels 1. Prepare 1 litre of 0.015 M Tris-barbital buffer at pH 8.8 4ising a glass electrode pH meter. 53 2. Boil 1 gm of agarose in 100 ml of 0.015 M Tris-barbital buffer at pH 8.8 with 0.1 mg of methiolate. Methiolate avoids bacterial growth. 3. Evenly spread 2 ml of this mixture on a clean microscopic slide and allow to solidify. 4. Make a central trough and two side wells prior to the application of antigen. 4.3.5. Preparation of Polyacrylamide gels (vide Expt. No. 3.) 4.3.6. Separation of antigen proteins by agarose gel electro-phoresis (Fig. 1) 1. Remove the slice of agarose on the well and deposit haemolymph collectd in 40% sucrose or ovary homogeni-zed in 40% sucrose (haemolymph and the ovary of S. serrata and E. asiatica are collected from different stages of ovarian maturation). 2. Before application mix the samples' with the marking dye, bromophenol blue. 3. Fill the electrophoretic tank with 0.06 M Tris barbital buffer at pH 8.8. Place agarose smeared glass slides, in the tank. mmimml**tJm*m+tiMm Fig. 1. Immunoelectrophoresis: Both fractionation and diffusion of antigen-is carried out in agarose : Antigen : Female matured S. Serrata haemolymph containing FSP. Antibody: Rabbit anti FSP antiserum. Note the thick precipitate arc formed against FSP. Thin precipitin arc-corresponds to haemocyanin fractions. (Slow moving fractions are-not represented). ; 54 4. Prepare two paper wicks with Whatman No. 1 filter paper and place it on the two edges of the glass plate dipping into the buffer solution, so that the gel is connected to the buffer by the paper wicks. 5. Supply 1.6 mA/slide till the marker dye reaches the anodic end of the slide. 6. After the completion of electrophoresis switch off the current, remove the paper wick and slide. 7. Remove the agarose gel in the centre to have a longi-tudinal groove and procure appropriate immuno serum-into it. 8. Incubate the slides in a humid chamber to allow immuno diffusion at 34°C for 24 hours. 4.3.7. Separation of antigen proteins by polyacrylamide gel electrophoresis (vide Expt. No. 3 ; Fig. 2). 4.3.8. Transfer method (Fyffe and O'Connor, 1974) 1. Transfer the polyacrylamide gels containing the resolved: antigenic proteins onto a slide. 2. Mix O.S ml of antiserum with two ml of agarose-buffer mixture and pour it around polyacrylamide gel. 3. Incubate the slides in a humid chamber to allow immuno-diffusion at 34°C for 24 hours. 4. Note the predpitin arcs developed as white curved line*. after the incubation period (Fig. 3). 5. Wash the gels with 1 % saline for about 16 to 20 hours with frequent changes of saline and wash finally in double-distilled water for an hour to remove saline. 6. Place the gel slides horizontally and filter paper strips over the gel to absorb excess water in the gel. After drying, filter paper comes out of the gel of its own. 7. Stain the gels with 0.1 % amido black (W/V) for 10 minutes and destain them in 2% acetic acid till the gel becomes, transparent and the arcs are clearly visible. 55-Fig. 3. PolyacrylaHiide discfractionation of S, serrata haemolymph and ovarian •proteins. 1. Male S. serrata haemolymph. 2. Female immature 5. serrata •haemolymph. 3. Female maturing S. serrata haemolymph. 4. Female mature S. serrata haemolymph. Arrow indicates, the appearance of female "specific protein (FSP) in late stage IV and early stage V. 5. Mature ovarian proteins of S. serrata. Note the homologous proteins in the ovary and 'haemolymph. :56 » I . 1 . " " I 1 "• • I " < ' . ' • i Bs-£ Fig. 3. Immunoelectrophoresis: Transfer method. Antigens separated on polyacrylamide is diffused through agarose containing antiserum. 1. Female S. serrata containing FSP x Rabbit anti haemolymph contai-ning FSP antiserum. 2. Female 5 , serrata containing FSP x Rabbit antiiipovitellin antiserum. 3. Freshly laid egg proteins of 5. serrata X Rabbit anti haemolymph containing FSP antiserum* 4. Freshly laid egg proteins of S. serrata x Rabbit anti-lipovitellln antiserum* (Slow moving fractions are not represented as they are not immunogenic). 5 m REFERENCES CROISIIXE, Y., J. J. MEUSY AND H. CHARNIAUX-COTTON, 1970 Etude immunochimique chez differents crustaces superieurs de la specifi-city de la' fraction |proteique femelle' de 1* hemolymph. Acad. Sci. Paris, 271 : 527-529. EZHILARASI, S. 1982. Biochemical investigations on the vitellogenesis of an edible crab Scylla serrata. Ph. D. Thesis, University of Madras, 170 p; FYFFE, W. E.AND J. D. O'CONNOR, 1974. Characterization and quantification of a crustacean lipovitellin. Comp. Biochem. Physiol, 47 : 851-867, KERR, M. SJ 1969. The haemolymph proteins of the blue crab,, Callinectus sapid us. II. A lipoprotein serologically identical to oocyte lipovitellin. ,Develop. Biol., 20: 1-17, SMITH, U; J. AND N. A. RATCLIFFE, 1980. Host defence reactions of the shore crabsi Carcinus maenasiJL.) in vitro. / . Mar. Biol. Ass.. U.K., 58 : 367-379. ; 

Serological identification of vitellogenin

and lipovitellin in Scylla serrata and

Emerita asiatica using immuno

electrophoresis

http://eprints.cmfri.org.in/3858/1/Spl_9-7.pdf

Serological identification of vitellogenin and lipovitellin in Scylla serrata and Emerita asiatica using immuno electrophoresis

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