THE„SEROLOGICAL RELATIONSHIPS OF SOB/IE PACIFIC COAST DECAPOD CRUSTACEA by Terrance Henry Butler A Thesis submitted i n p a r t i a l f u l f i l m e n t of the requirements for the Degree of MASTER OF ARTS i n the Department of ZOOLOGY We accept t h i s thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS. Members of the Department of Zoology THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1953-ABSTRACT The se r o l o g i c a l relationships of P a c i f i c coast decapod Crustacea were investigated by use of blood sera and protein extracts. Blood sera were obtained from eight species representing s i x f a m i l i e s ; the protein extracts were produced from f i f t e e n species as representatives of ten f a m i l i e s . Protein extracts gave negative r e s u l t s . Testing with blood sera demonstrated no relationship between anomuran and brachyuran crustaceans. Within the Anomura a close r e l a t i o n -ship was found between the Lithodidae and Paguridae. In the Brachyura the relationships among the families Cancridae, Atelecyclidae, Grapsidae and Maiidae were found to be generally i n accord with those established by morphological studies. Testing of three species of Cancer demonstrated that they are clos e l y related, yet d i s t i n c t species. TABLE OF CONTENTS Page Acknowledgements i Introduction i i Materials and Methods..... 1 Antigens . 1 Antisera 4 Testing 5 Experimental Results 9 Tests with Lopholithodes foraminatus A n t i -serum 9 Tests with Cancer g r a c i l i s Antiserum 9 Tests with Cancer ma^ister Antiserum 10 Tests with Cancer productus Antiserum 10 Tests with Telmessus cheiragonus Antiserum 13 Discussion 18 The c l a s s i f i c a t i o n of decapod Crustacea 18 Interpretation of Results 21 Summary 2? Literature Cited 28 i ACKNOWLEDGEMENTS The writer wishes to express his appreciation to Dr. W. A. Clemens of the Department of Zoology, University of B r i t i s h Columbia for his assistance concerning the systematics of the Decapod Crustacea. Sincere thanks are extended to Dr. W. S. Hoar for his advice, encouragement and p r a c t i c a l assistance at a l l times during the study. Thanks are extended to Mr. W. R. Hourston for his generous co-operation i n a l l phases of the study. Many of the samples were collected while the writer was conducting research on marine Crustacea for the Fisheries Research Board of Canada. Appreciation i s expressed to Dr. J . L. Hart, Director of the P a c i f i c B i o l o g i c a l Station, Nanaimo, B. C , for t h i s p r i v i l e g e . F i n a l l y , the writer extends thanks to fellow students for assistance i n some technical operations. i i INTRODUCTION The schemes of crustacean c l a s s i f i c a t i o n now i n effect are based l a r g e l y on comparative studies of anatomical, embry-o l o g i c a l and physiological characters. The biochemical characters generally have not been considered, i n spite of increased knowledge i n the f i e l d of biochemistry. Of a l l the biochemical constituents of animals the proteins are probably the most useful to the taxonomist. The methods i n a n a l y t i c a l chemistry generally are not able to show s i m i l a r i t i e s and differences i n proteins that would be of value to the taxon-omist. The procedures developed i n the f i e l d of serology for the comparative study of proteins provide a " t o o l " of d e f i n i t e p o s s i b i l i t i e s . Serology i s based on the quantitative s p e c i f i c i t y of proteins or the possession by each animal species of s p e c i f i c or c h a r a c t e r i s t i c proteins which are less s p e c i f i c to the proteins of other animals i n direct proportion to t h e i r r e l a -tionship. The method by which s p e c i f i c i t y i s demonstrated depends on the a n t i g e n i c i t y of proteins. The a n t i g e n i c i t y of a protein i s i t s a b i l i t y to produce immunizing substances or antibodies when i t i s injected into an animal under appropriate conditions. I f these antibodies are reacted with the protein or antigen which has produced them a f l o c c u l a t i o n w i l l r e s u l t . Further, i f the antibodies react with antigens other than t h e i r i i i associated antigen the f l o c c u l a t i o n w i l l be d i r e c t l y propor-t i o n a l to the degree of relationship of those animals. Reference may be made to the degrees of relationship which have been demonstrated by serology. The American lobster Homarus americanus and the European lobster are considered to be separate species by most a u t h o r i t i e s , yet Leone (1950b, and personal communication) was not able to separate the two forms s e r o l o g i c a l l y . The relationship between the phylum Chordata and invertebrate phyla i s uncertain. Wilhelmi (1942) found that s e r o l o g i c a l l y the chordates were more clos e l y related to the echinoderms than to the annelids and arthropods. The relationships shown by serological comparison are not applic-able to the construction of a phylogenetie "tree" as envisaged by N u t t a l l (1904). The formation of such a tree would require " f o s s i l " biochemical evidence which has been found only r a r e l y . The s p e c i f i c i t y of proteins depends on t h e i r chemical constitution and their s t r u c t u r a l c h a r a c t e r i s t i c s . Haurowitz (1952) uses the term "determinent group" to describe the chemical r a d i c a l s and isomers which may determine spec-i f i c i t y . The twenty to t h i r t y amino acids i n theory may combine i n a great many ways to form proteins. This may account i n part for the possession by each animal species of c h a r a c t e r i s t i c proteins. Closely related species probably possess the same amino acids, and here i t i s l i k e l y that the s p a t i a l configuration of the protein molecule may d i f f e r . i v I t i s suggested that protein evolution may proceed f i r s t by an a l t e r a t i o n i n the configuration of the molecule, then second-a r i l y by a change i n amino acid composition. Proteins are probably the only substances which are antigenic (Landsteiner, 194-5). Their antigenic property i s not yet f u l l y understood. Landsteiner (op. c i t . ) believed that the possession of aromatic amino acids might contribute to an t i g e n i c i t y . Gelatin i s a protein which i s non-antigenic; i t lacks the aromatic amino acids tyrosine and cystine. However Haurowitz (1952) describes unsuccessful attempts to produce antigenic a c t i v i t y by coupling tyrosine to the g e l a t i n molecule. The non-antigenicity of g e l a t i n i s a t t r i b u t e d to other factors; i t i s a denatured protein and has molecules fibrous i n shape, and g e l a t i n i s excreted into the urine before i t can be deposited into the sit e s of antibody formation. Haurowitz (op. c i t . ) also states that the determinant groups mentioned e a r l i e r must be strongly polar i n nature and r i g i d i n structure. For example, the long-chain f a t t y acids are non-antigenic, because they are e a s i l y distorted. Antibodies are proteins, or to be more s p e c i f i c , they are modified serum globulins (Haurowitz, 1952). The antibodies d i f f e r from normal globulins i n t h e i r immunological a c t i v i t y and the i r greater resistance to proteolytic enzymes. Antibodies are formed i n the r e t i c u l o e n d o t h e l i a l c e l l s (Haurowitz, op. c i t . ) . The mechanism of antibody formation V i s not d e f i n i t e l y understood. An early theory which i s des-cribed by Landsteiner (1945), proposed that antibodies were the normal constituents of blood serum. However, i t i s incon-ceivable that a mammal such as a rabbit should contain a n t i -bodies for the sera of a l l e x i s t i n g animals. This theory has been discarded. Recently, Haurowitz (op. c i t . ) has proposed that the antigen may i n t e r f e r e with the synthesis of normal globulin molecules. The modified globulin molecule i s complementarily adapted to the determinant groups of the antigen molecule. The a c t i v i t y of the antigen molecule w i t h i n the animal body has been studied by the use of radioactive isotopes (Haurowitz). I f a " l a b e l l e d " antigen i s injected, i t i s found to disappear rapidly from the blood stream and to be deposited i n c e l l s of the r e t i c u l o e n d o t h e l i a l system, e.g., l i v e r and spleen. The antigen then i s deposited i n c e l l s which produce the antibodies. The mechanism of the antigen antibody reaction i s also not c l e a r l y understood. The recent theory described by Haurowitz (1952) i s that the a f f i n i t y between antibodies and antigens i s due to the complementariness of t h e i r combining s i t e s . The determinant groups of antigen molecule are not able to approach the surface of another large molecule unless the surface i s shaped complementarily. In the case of an antigen molecule and the antibody molecule which has been v i modified by the former, mutual complementariness e x i s t s , r e s u l t i n g i n a t t r a c t i o n between the molecules. The water of hydration i s displaced from the surfaces of the molecules, and the determinant group of the antigen w i l l combine fir m l y with the complementarily shaped group of the antibody. Tissue proteins, respiratory pigments, and the blood serum proteins have been used for serological compari-sons. The c i r c u l a t i n g f l u i d or blood of the decapod Crustacea has the property of coagulation. After coagulation, a clear f l u i d or serum remains. Biochemical analysis ( A l l i s o n and Cole, 1940) has shown that t h i s serum contains a single protein, haemocyanin. Due to t h i s fact Boyden (1943) regarded the decapods as p a r t i c u l a r l y favourable material for serological research, as compared with the sera of vertebrates where several proteins are found, and these may cause inconsistent • r e s u l t s i n serological t e s t i n g . Thus haemocyanin i n the sera of decapods i s the protein or antigen which i s used i n ser o l o g i c a l comparisons. The c l a s s i f i c a t i o n of the order decapod Crustacea as described l a t e r and i l l u s t r a t e d i n Figure 8 i s generally accepted. However, there are groups of species which have an uncertain position within the order. Serological testing may supply information that w i l l c l a r i f y the position of these groups. Such comparisons may also supplement the knowledge of anatomy and embryology upon which the c l a s s i f i c a t i o n of t h i s v i i group has been based. The purpose of the present study was to determine whether or not the proteins of the marine decapod crustacea of B r i t i s h Columbia could be compared s e r o l o g i c a l l y ; and to determine the value of these comparisons i n taxonomic study. The method of serology has been applied to the decapod Crustacea by workers i n other parts of the world. The f i r s t record appears to be that of Von Dungern (1903). This paper was not seen by the author, but according to Boyden (1942) Von Dungern studied reactions "between antisera of mollusk and Crustacea plasma". N u t t a l l (1904) and Graham-Smith (1904) tested the antisera of the European lobster Homarus vulgaris and the crab Carcinus maenas with many animal sera, but only found positive reactions within the decapod Crustacea. Graham-Smith (op. c i t . ) noted weak reactions of Limulus anti-serum with sera of Crustacea. Erhardt (1929) tested the antiserum of the cra y f i s h Potamobius stacus with-the sera from other Crustacea, and found the relationships agreed with the accepted system of c l a s s i f i c a t i o n . In 1937 Boyd tested the antiserum of Cancer i r r o r a t u s with three other species of arthropods. He found that the Cancer antiserum reacted to a considerable degree with serum of the lobster Homarus americanus. gave a weak reaction with the antigen of the Black Widow spider, and did not react d e f i n i t e l y with the serum of Limulus. Boyden (1943) v i i i reported on the serological testing of five families of the Brachyura, and also of Homarus vulgaris and H. americanus. In 1942 Clark and Burnet carried out serological tests covering the four tribes of the suborder Reptantia. They found no relationship among the Palinura, Astacura, Anomura and Brachyura. However, within these tribes they found relationships in accord with the accepted system of classifica-tion. Leone (1949) compared the representatives of seven families of the Brachyura, as an extension of the earlier work of Boyden (1943) and reported on the effect of chemical and physical treatment on decapod sera. Seven families of European Brachyura were studied by Leone (1950a)• His results in general confirmed the existing system of classification. Leone also showed that there was no difference in activity of sera of the same species collected from different localities. The two tribes, Palinura and Astacura, were next considered by Leone (1950b). He found a low degree of corres-pondence between the two tribes, but within each tribe the relationships agreed with the system based on anatomical characters. In a recent paper Leone (1951) reported further on the comparative serology of the Brachyura, with special emphasis on the systematic position of the species Gervon quinquedens. Finally, Leone and Pryor (1952) compared three species of penaeid shrimps, and were able to distinguish these species. MATERIALS AND METHODS Antigens Table I l i s t s the species with l o c a l i t i e s and dates from which sera were collected. The bleeding of the decapods was accomplished by tearing a cheliped from the cepholothorax and allowing the blood to flow into clean test tubes. After a period of twelve hours complete c l o t t i n g had occurred, and the samples were then f i l t e r e d through f i l t e r paper and stored i n s t e r i l e b o t t l e s . A preservative, merthiolate was added. F i n a l l y , i n the labora-tory at the University, the samples were f i l t e r e d through a s t e r i l e s e i t z f i l t e r , bottled and stored i n a r e f r i g e r a t o r . The bloods of some species (Hemigrapsus, and species of the t r i b e Anomura) were found to coagulate almost completely. Centrifugation removed s u f f i c i e n t serum from one anomuran species (lopholithodes foraminatus). However, expression of other sera was not successful u n t i l i t was found that s u f f i c i e n t serum could be extracted by pressing the c l o t through cheese-cl o t h . Many species of decapod Crustacea are too small, even when f u l l y grown to permit the c o l l e c t i n g of s u f f i c i e n t sera by the method outlined above. In several cases, attempts to remove blood from a sinus'using a hypodermic syringe gave unsatisfactory r e s u l t s . Therefore, protein extracts were -2-TABLE 1 L i s t of species, with code letters, sources and dates, whose sera are compared. Species Code Source Date Cancer magister Dana CM1 Hecate Strait • Aug. 8, 1950 Cancer gracilis Dana CGI Hecate Strait Aug. 8, 1950 Cancer productus Randall CP1 Hecate Strait Aug. 13, 1950 Telmessus cheiragomis TCI Masset Inlet Aug. 13, 1950 (Tilesius) Lopholithodes foraminatus LF1 Burrard Inlet Dec. 29, 1950 (Brandt) Chionoecetes bai r d i i CB2 . Strait of Georgia Feb. 2, 1952 (Rathbun) Hemigrapsus nudus' HN2 Departure Bay Feb. 2, 1952 (Dana) Pagurus alaskensis PA1 Departure Bay Jan. 18, 1952 (Benedict) -3-TAELE II Li s t of species, with, code letters, sources and dates, from which protein, extracts were prepared. Species Code Source Date Crago nigricauda Stimpson CN1 Stanley Park, Vancouver Jan. 8, 1951 Spirontocaris brevirostris SB1 Stanley Park, Jan. 8, 1951 (Dana) Yancouver Pa gurus hirsutiusculus PEL Stanley Park, Jan. 8, 1951 (Dana) Yancouver Pandalus platyceros Brandt PP1 Howe Sound Feb. 1, 1951 Brandt Hemigrapsus nudus (Dana) HN1 Stanley Park, Yancouver Feb. 2, 1951 Cancer oregonensis (Dana) C02 Stanley P ark, Vancouver Feb. 5, 1951 Cancer magister Dana CM4 Stanley Park, Vancouver Feb. 5, 1951 Pinnixa l i t t o r a l i s Holmes ELI Stanley Park, Vancouver Feb. 5, 1951 Hemigrapsus oregonensis H02 Spanish Banks Feb. 12, 1951 (Dana) Callianassa californiensis CC1 Spanish Banks Feb. .27, 1951 Dana Pasiphaea pacifica Rathbun PPA1 Eraser River mouth Mar. 19, 1951 Pandalopsis dispar Rathbun PD1 Eraser River mouth Mar. 19, 1951 Pandalus borealis Krover PB1 Fraser River mouth Mar. 19, 1951 Chionoecetes bairdi Rathbun CB1 Eraser River mouth Mar. 19, 1951 Spirontocaris suckleyi SSI Fraser River Mar. 19, 1951 (Stimpson) mouth prepared from the whole animals. The procedure1, which Leone (1947) used for insects was followed with modifications. The viscera of a l l animals were removed before extraction. These extracts gave a precipitate with trichloriacetlc acid and were considered to contain sufficient protein. Table II lis t s the species with localities and dates from which the protein extracts were obtained. Antisera The antisera containing the antibodies were produced in rabbits. The injection technique was the same as that recom-mended by Leone (1949). Each rabbit was injected in the lateral ear vein with 0 . 2 5 ml. of serum and allowed to rest for thirty days. Then four subcutaneous injections, each of 0 . 2 5 ml., were given on alternate days. After a period of a week from the last subcutaneous injection, a small sample was removed from the lateral ear vein. The potency of the serum (antiserum) was determined by testing with the decapod antigen. A positive ring test indicated that the antiserum was sufficiently potent. Each rabbit was then bled completely by cannulation of the carotid artery. The blood was collected in test tubes and allowed to coagulate for twelve hours. The serum contain-ing the antibodies which had separated from the clot was decanted and centrifuged. Finally, the antiserum was filtered through a sterile Seitz filter and stored in sterile bottles in the refrigerator. - 5 -Testing The f l o c c u l a t i o n procedure was used exclusively i n preference to the r i n g te s t . In the f l o c c u l a t i o n procedure the antigen and antiserum are mixed i n certain proportions and the precipitate may be described q u a l i t a t i v e l y , or expressed as weight, volume, or nitrogen content of the p r e c i p i t a t e ; or the t u r b i d i t y of the mixture may be measured photometrically. The t u r b i d i t i e s of the antigen-antiserum mixtures were measured photometrically by the photronreflectometer (photron'er) developed by Libby (1938). The photron'er i n the Department of Zoology was b u i l t , i n 1949 by a technician i n the Physics Department according to the diagram i n Figure 1 (taken from Libby 1s paper). The method of testing was performed by t i t r a t i n g a constant amount of antiserum with each of a series of solutions containing a varying amount of antigen arranged i n a series of doubling d i l u t i o n s i n test c e l l s . In the f i r s t c e l l was placed a d i l u t i o n of protein of Is250, and i n the second tube a d i l u t i o n of protein of 1:500, and so on, u n t i l the l a s t c e l l i n the series contained a d i l u t i o n of one part protein i n 1,024,000 parts of d i l u t i n g medium. A l l the sera used were analysed for protein by the micro-Kjeldahl technique and the protein values thus obtained were used as a basis for the pro-t e i n d i l u t i o n . The medium used i n preparing the antigen d i l u -t i o n series was a buffered physiological saline solution, made - 6 -v a n a b l e r e s i s t o r p a r a l l e l b e a m o f l i f ch t ac + ive Surface P.P ' ' i / ' ' K TL \ \ » \ I » X I » \ t^ i i,,,,. tj} G ligKf bulb l e n s . d iaphragms . g lass ee l • black c i r c l e C in a c t i v e ) - p h o t o e l e c t r i c c