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The serological relationships of some Pacific coast decapod crustacea Butler, Terrance Henry 1953

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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<UI Figure 1. Diagram of the Libby photron-reflectometer (photron'er) -7-up according to Evans (1922). In making a t e s t , a constant amount of antiserum was added to the solutions of antigen i n each c e l l , and mixed thoroughly. The test c e l l s were then incubated for twenty minutes i n a dry-oven incubator at a temperature of 3 8 0 C. After the incubation period the t u r b i d i t y i n each tube was measured by the photron'er. A graph was constructed of each ser o l o g i c a l reaction. The values for t u r b i d i t y expressed i n galvanometer units were plotted on the ordinates, and the antigen d i l u t i o n s as tube number on the abscissa. The plotted t u r b i d i t y units form a closed curve. The curve representing each s e r o l o g i c a l reaction i s expressed numerically as the summation of the t u r b i d i t i e s over the whole reaction range. In a s e r o l o g i c a l comparison the t u r b i d i t i e s of the homologous reaction, the reaction between an antiserum and the antigen which has caused i t s production, are graphed and summated ar i t h m e t i c a l l y . Then the t u r b i d i t i e s of a heterologous reaction, the reaction between the antiserum and an antigen other than that one producing the antiserum, are summated and the numerical value i s expressed as a percentage of the summa-ti o n of the homologous reaction. This procedure outlined above was f i r s t used by Boyden (1942) and has been adopted by subsequent workers. The v a l i d -i t y of t h i s procedure i s discussed i n a l a t e r section. -Bj-TABLE III Serological relationships among six families of anomuran and brachyuran Crustacea Antisera Antigens i © cd -o •H xs cd O U & s cd At 0) .— <a is id u <D •H o O O o an COan B o o 8 m CO O 3 o o ' 5 U cd cd | | 3 cd o u co Q) <D o CD O Pi o o I cd •H Lopholithodes foraminatus (Lithodidae-Anomura) Cancer gra c i l i s (Cancridae-Brachyura) Cancer magister (Cancridae) Cancer productus (Cancridae) Telmessus cheiragonus (Atelecyclidae-Brachyura) 1 0 0 0 4 6 0 0 1 0 0 7 0 6 2 6 4 1 0 0 7 0 0 6 3 6 8 1 0 0 - 3 7 - 9 -EXPERIMENTAL RESULTS Three reactions were carr i e d out between protein extracts and t h e i r antisera. These were completely negative, i . e . , no t u r b i d i t y was shown on the photron'er. Several reas-ons for these negative r e s u l t s are suggested l a t e r . Since time was l i m i t e d , the writer decided to discontinue the testing of the protein extracts. The r e s u l t s of the testing of the sera are presented as percentage relationships i n Table I I I . Tests with Lopholithodes foraminatus antiserum From Table I I I i t i s seen that there i s no reaction between the L. foraminatus antiserum and the antigens of four species of brachyuran crabs. In Figure 2 the homologous reaction between the L. foraminatus antiserum and i t s antigen i s graphed. The heter-ologous reaction with the serum of the hermit crab Pagurus  alaskensis shows a relationship of 4 6 $ . Tests with Cancer g r a c i l i s antiserum Table I I I shows no reaction between the C. g r a c i l i s antiserum and the antigens of two species of the Anomura, L. foraminatus and P. alaskensis. In Figure 3 , 63$ and 64$ relationships between the £• g r a c i l i s antiserum and the C. productus and C. magister antigens are shown. - 1 0 -The relationships between three families of Brachyura are shown i n Figure 4. Heterologous reactions with the C. g r a c i l i s antiserum (Cancridae) demonstrate a correspondence with the antigens of Telmessus cheiragonus (Atelecyclidae) and Hemigrapsus nudus (Grapsidae) of.41% and 26% respectively. The curve of the heterologous reaction of T. cheiragonus i s d i s -t i n c t l y bimodal; probably i n d i c a t i v e of the presence of more than one antigen and/or antibody. Tests with Cancer magister antiserum Most of the Cancer magister antiserum was used for standardizing the photron'er. A s u f f i c i e n t amount was a v a i l -able to determine the relationship of C. magister to two other species of the genus Cancer. In Figure 5 are shown the curves of the homologous reaction of C. magister, and the heterologous reactions of C. productus and C. g r a c i l i s . The relationships of the C. productus and C. g r a c i l i s antigens are 68% and 70%, respectively. Tests with Cancer productus antiserum The supply of C. productus antiserum was exhausted before the heterologous reactions with Pagurus alaskensis and Hemigrapsus nudus could be performed. Table I I I shows that there was no reaction between the C. productus antiserum and the serum of Lopholithodes  foraminatus. -11-Figure 2. The re l a t i o n s h i p between Lohholithodes foraminatus (LFI) and Pagurus alaskensis (PA1) -12-Flgure 3» The r e l a t i o n s h i p between Cancer g r a c i l i s (CGI), Cancer pro-ductus (CP1), and Cancer magister (CM1) - 1 3 -Figure 6 shows that the relationship of the antiserum of C. produetus to the serum of C. magister was 70$ and to the serum of C. g r a c i l i s , 62$. In Figure 7 the heterologous reactions with the antigens of Chionoecetes b a i r d i (Maiidae) and Telmessus cheiragonus (Atelecyclidae) show relationships of 37$ and 52$, respectively. Tests with Telmessus cheiragonus antiserum Two homologous reactions and one heterologous reac-t i o n were performed with the serum of Cancer productus. A l l these reactions were completely negative, even though a posi-t i v e test was obtained before the antiserum was removed from the rabbit. a -14-Figure 4. The re l a t i o n s h i p between Cancer g r a c i l i s (CGI), Hemigr'apsns  hudus (HN2), and Telmessus cheira-gonus (TCI) Figure 5» The rel a t i o n s h i p between Cancer magister (CM1), Cancer pro-due tus (CP1), and Cancer g r a c i l i s (CGI) -16-Dilution tu be5 Figure 6. The relationship between Cancer productus (CP1), Cancer magister (CM1), and Cancer g r a c i l i s (CGll -17-Dilotiott tubes Figure 7. The relationship between Cancer productus (CP1), Chionoecetes b a l r d i , and Telmessus cheiragonus (TCI) - 1 8 -DISCUSSION The C l a s s i f i c a t i o n of decapod Crustacea The system of c l a s s i f i c a t i o n proposed by Borradaille (1907) i s now generally accepted. The order Decapoda i s divided into two suborders, the Natantia and the Reptantia. The suborder Natantia includes those decapods which are shrimp-like, and are capable of swimming. The Reptantia are characterized by t h e i r crab-like or hermit crab-like (rar e l y shrimp-like) appearance. The abdominal appendages are not developed for swimming. Each of the two suborders i s further divided into t r i b e s . The antigens were obtained from a number of species of the Natantia; however since they were not tested serologic-a l l y the c l a s s i f i c a t i o n w i l l not be outlined. The t r i b e s i n the suborder Reptantia are the Palinura, Astacura, Anomura, and the Brachyura. The two t r i b e s Palinura and Astacura are not represented i n the marine fauna of B r i t i s h Columbia. The Anomura are characterized by the f a i r l y large abdomen, by the lack of fusion of the carapace with the epistome, and by the external p o s i t i o n of the antennae with respect to the eyes. In the t r i b e Anomura there are three superfamilies found i n B r i t i s h Columbia coastal waters, the Galatheidea, the Thalassinidea, and the Paguridea. No members of the Galatheidea and the Thalassinidea are considered -19-i n the present study so th e i r c l a s s i f i c a t i o n w i l l not be outlined. Species of the superfamily Paguridea are recognized c h i e f l y by the asymmetrical abdomen which i s either soft and twisted, or bent under the thorax. There are two families i n the Paguridea, the Paguridae (hermit crabs) and the Lithodidae ("stone" crabs). The hermit crabs are known popularly by the i r habit of dwelling i n empty gastropod s h e l l s , but are recognized more cor r e c t l y by the reduced rostrum and soft twisted abdomen. The Lithodidae are characterized by the i r crab-like appearance and the reduced pair of f i f t h walking legs. The Brachyura or true crabs are recognized by the small abdomen, by the fusion of the carapace to the epistome, and the position of the antennae i n t e r n a l to the eyes. Unlike the Anomura, the t r i b e Brachyura i s divided into subtribes of which there are three. Only one subtribe, the Brachygnatha i s represented l o c a l l y , characterized by the square endostome and the normal size of the l a s t pair of walking legs. The Brachy-gnatha i s further divided into two superfamilies, the Oxyrhyncha and the Brachyrhyncha. The former includes the family Maiidae (spider crabs) which i s recognized by the narrow fore part of the carapace, and by the incomplete orb i t s of the eyes. In the superfamily Brachyrhyncha are found most of -20-thewelT.known crabs. Five families are found i n the marine fauna of B r i t i s h Columbia. The species of three families (Ateleeyelidae, Cancridae, Grapsidae) are tested i n the present study. The carapace i n the Ateleeyelidae and the Cancridae i s usually c i r c u l a r or oval i n shape. The two families are separated from each other by the length of the antennal f l a g e l l a . In the family Grapsidae the carapace i s square i n shape. -21-INTERPRETATION OF RESULTS Considering the success of Leone (1947£ and b) with protein extracts of insects i t i s surprising that reactions were not obtained with extracts of Crustacea. The writer had an opportunity to discuss the problem with Dr. C. A. Leone, and he reported the same experience i n h i s early t e s t i n g . He suggests that the enzymes present i n the extracts hydrolized the proteins. Consequently, i n s u f f i c i e n t protein remained to stimulate antibody formation i n the rabbit. The chemical merthiolate used as a bactericide apparently does not i n h i b i t enzyme a c t i v i t y . The use of 2% formalin i s suggested by Dr. Leone. The two t r i b e s Anomura and Brachyura have been estab-l i s h e d on the basis of anatomical characters. In the present comparisons Lophollthodes and Pagurus represented the Anomura, and Cancer productus. C. g r a c i l i s . Chionoeeetes. and Hemigrap-sus the Brachyura. In the seven reactions no serological r e l a t i o n s h i p was noted. The serological comparisons have shown that species representing the Anomura and Brachyura are d i s s i m i l a r , and has confirmed the placing of these species i n the two different t r i b e s on the basis of anatomical characters. Graham-Smith (1904) obtained a weak reaction between crab, Carcinus maenas, antiserum and the serum of a European hermit crab Pagurus bernhardus. However, Clark and Burnet (1942) found no relationship between an Australian hermit crab -22-Paguristes, and a brachyuran species Pseudocarcinus gigas. Within the t r i b e Anomura a relationship was shown between Pagurus and Lopholithodes. The re c i p r o c a l reaction i s needed here as a check on t h i s comparison. The one reaction has served to separate the two species representing the two families Paguridae and Lithodidae, yet has.revealed the f a i r l y close relationship between them, i n accord with the relationship based on anatomical characters. As far as known, no serological tests involving the Lithodidae have been per-former previously. Two superfamilies of the Brachyura are represented i n the B r i t i s h Columbia marine fauna, the Oxyrhyncha and Brachyrhyncha. In the present study, species of these two superfamilies are compared by one reaction between Chionoecetes and Cancer productus. This test separates the two species, yet demonstrates a c e r t a i n relationship (31$) between the two superfamilies. The relationships reported by Leone ( 1 9 4 9 , 1950, and 1 9 5 D are somewhat lower ( 9 - 3 0 $ ) • Species of families i n the superfamily Brachyrhyncha were compared; three species of Cancer, representing the Cancridae, Telmessus of the family Ateleeyelidae, and Hemi-grapsus of the Grapsidae. The reactions have separated the three f a m i l i e s , and revealed that the Cancridae are more closely related to the Ateleeyelidae than to the Grapsidae; t h i s confirms the relationship based on anatomical - 2 3 -eharacters. The reaction between Cancer g r a c i l i s and Hemigrapsus i s of int e r e s t because the percentage r e l a t i o n -ship found (26$) for the reaction between C. productus and Chionoecetes. In e f f e c t , t h i s test indicates a closer relationship between two species which are now placed i n two diff e r e n t superfamilies than between two species i n the same superfamily. However, since the r e c i p r o c a l reaction was not performed, the rela t i o n s h i p must be accepted with reserva-t i o n . The testing of the three species of Cancer has demonstrated the close inter-species relationship within the genus. An average value of 66$ has been established from the re c i p r o c a l t e s t s . Leone (1949) found a s l i g h t l y higher relationship of 70% among four species of Cancer, however, re c i p r o c a l tests were not performed i n a l l cases. The r e l a -tionship among the three species of Cancer i s greater than that between Cancer and other species of the Brachyura. The writer believes that the three species of Cancer have been separated e f f e c t i v e l y from each other, i . e . , they have been established as " v a l i d " species. The v a l i d i t y of the procedure used i n p l o t t i n g and interpreting the r e s u l t s should be considered. The concentra-tions of antigen are plotted along the abscissa using an arithmetic scale, although the concentration changes geometri-c a l l y (x2 for each d i l u t i o n ) . I f a geometrical scale were -24-used for p l o t t i n g these concentrations, the r e s u l t i n g curve would be inconvenient to graph. Instead of summing the ordinates, a c a l c u l a t i o n of the area under each curve might be considered. The areas of the curves of several reactions were calculated, but the differences i n the relationships did not change enough to warrant adopting t h i s procedure, at the present time there seems to be no alternate method of compar-ing serological reactions. There are p e c u l i a r i t i e s i n the shapes of several of the plotted curves. In Figure 7 the curves .of two heter-ologous reactions are skewed to the l e f t ; and i n Figure 4, the curves of the two heterologous reactions exhibit bimodality. The present testing was inadequate for duplica-t i o n of result s to determine errors i n technique. However, the same i r r e g u l a r i t i e s appear i n curves depicted i n the l i t e r a t u r e (Boyden, 1943; Leone, 1949). Bimodality i n these curves i s believed by Leone (1949) to be due to the presence of aggregates or numbers of "species" of protein molecules. At the present time the t h e o r e t i c a l knowledge of protein chemistry does not permit a f u l l appreciation of the sero-l o g i c a l t e s t i n g . This lack of t h e o r e t i c a l knowledge should not discourage further testing. The procedure seems sound and i s giving r e s u l t s by which the proteins of animals may be compared. Because of t h e i r s u i t a b i l i t y for serological t e s t -ing, the sera of the decapod Crustacea may be valuable for S . O r d e r N * t < i h » t i * P» I i n t r a P a I i H u K- (d a <L R o c k '/obs-feM* 8 s P Pa»-as^ac<'da*. I2<;p. ~ | | Pa tvu J7«S " f V i b e * Hi-achy ur* T r i b e s S t a n o p n V s A M W I V M C « i - i r f ( « 3 s p S t r o l o ^ i e i I o f D e c a p o d C r v » * + « e e j u h r f e r l ined S H - u d i e e l b y o H t 1 Fa i I re 1 L e u c o s i i d j * O c y p o d ' d o » - M a i i d a e Hat-ton . d a * X a n T n i d a t C a n c i - ^ a t /4- te l«cycf id«a ftr,nor^eridot G i a p " ' d » c QSX£2i± P o . + u ^ , A a i i+h - S C a . c e . T ^ r . . . ^ ' * a C * b S 3 s p - S^p 3»p-Figure 8. Diagram to i l l u s t r a t e s e rological research i n decapod Crustacea - 2 6 -protein research. As far as i s known no previous serological research has been conducted on the species which are considered i n the present study. Species representing the families Paguridae, Cancridae, Maiidae, and Grapsidae have been investigated by other workers. The two genera Chionoecetes and Hemigrapsus are here investigated for the f i r s t time. No previous research has been recorded for the families Lithodidae and Atelecyclidae. In Figure 8, the system of c l a s s i f i c a t i o n for the decapod Crustacea i s outlined, and the serological research to date i s indicated. The present study has shown that the sera of the B r i t i s h Columbia marine decapod Crustacea may be compared s e r o l o g i c a l l y , and that the r e s u l t s may be used i n taxonomic study. The result s though l i m i t e d seem promising enough to warrant further study. An extension of the tes t i n g to include smaller decapod species i s possible now by the use of a tech-nique described by Leone and Pryor ( 1952) . They extracted sera from penaeid shrimps, by making an Incision i n the ventral haemocoel at the junction between the cephalothorax and the abdomen, and allowing the animals to flounder i n buffered physiological saline. I t should now be possible to compare s e r o l o g i c a l l y the representatives of most taxonomic categories within the order Decapoda. - 2 7 -SUMMARY 1. Serological techniques are outlined i n d e t a i l . 2. Serological testing of protein extracts was not successful and consequently the result s are based on larger species, from which sera were obtained. 3. No serological relationship was demonstrated between the Anomura and the Brachyura. 4. Within the Anomura the two f a m i l i e s Paguridae and Lithodidae were found to be closely related. 5. Serological testing of families of the Brachyura has shown that the family Cancridae i s c l o s e l y related to the family Ateleeyelidae, and more d i s t a n t l y related to the families Maiidae and Grapsidae. 6. Three species i n the genus Cancer are more clos e l y related to each other than to other species of the Brachyura. The three species can be distinguished s e r o l o g i c a l l y . -28-LITERATURE CITED A l l i s o n , J. B. and W. H. Cole, 1940. The nitrogen, copper and hemocyanin of the sera of several Arthropods. Jour. B i o l . Chem., 135: 259-265. Borradaile, L. A., 1907. On the c l a s s i f i c a t i o n of the decapod Crustaceans. Ann. Mag. Nat. Hist., 19_: 457-486. Boyd, W. C , 1937. Cross r e a c t i v i t y of various haemocyanins with special reference to the blood proteins of the black widow spider. B i o l . B u l l . , 22s I8I-I83. Boyden, A. A., 1942. Systematic Serology: A c r i t i c a l apprecia-t i o n . P hysiol. Zool., 1£: 109-145. Boyden, A. A., 1943. Serology and animal systematics. Amer. Nat., 12% 234-255. Clark, E., and F. M. Burnet, 1942. The application of serologic-a l methods to the study of Crustacea. Austral. Jour. Exp. B i o l , and Med. S c i . , 20: 89-95. Dungern, E. F. Von., 1903. Bindungs-Verhfilt-nisse bei der P r a z i p i t i n Reaktion. Z e n t r a l b l . f . Bakt., ^4: 355. Evans, A. C , 1922. A buffered physiologic s a l t solution. Jour. Infec. Dis., ^0: 95-98. Graham-Smith, G. S., 1904. Blood rel a t i o n s h i p amongst the lower Vertebrata and Arthropoda, as indicated by 2,500 tests with p r e c i p i t a t i n g antisera. Section VIII i n : N u t t a l l , Blood immunity and blood r e l a t i o n s h i p . Cambridge. Haurowitz, F., 1952. The mechanism of the immunological response. B i o l . Rev. 2£: 247-280. Hourston, W. R., 1949. The s e r o l o g i c a l r elationships of some P a c i f i c coast salmonoid f i s h e s . MA Thesis, Univ. of B r i t . Col. Landsteiner, K., 1945. The s p e c i f i c i t y of s e r o l o g i c a l re-actions. S p r i n g f i e l d . Leone, C. A., 1947 a. Systematic serology among certa i n insect species. B i o l . B u l l . , 23: 64-71. -29-Leone, C. A., 1947 b. A serological study of some Orthoptera. Ann. Entom. Soc. Amer., 40: 417-433. Leone, C. A., 1949. Comparative serology of some brachyuran Crustacea and studies i n hemocyanin correspondence. B i o l . B u l l . , 21* 273-286. Leone, C. A., 1950 a. Serological relationships among common brachyuran Crustacea of Europe. Pubbl. d e l l a Staz. d i Napoli, 22: 273-282. Leone, C. A., 1950 b. Serological systematics of some palinuran and astacuran Crustacea. B i o l . B u l l . , £8: 122-127. Leone, C. A., 1951. A serological analysis of the systematic relationship of the brachyuran crab Geryon quinquedens. B i o l . B u l l . , 100: 4 4 - 4 8 . Leone, C. A. and C. W. Pryor, 1952. Serological correspondence among three species of penaeid Crustacea. Jour. E l i s h a M i t c h e l l S c i . S o c , 68: 27-31 . Libby, R. L., 1938. The photronreflectometer - an instrument for the measurement of turbid systems. Jour. Immun., .34* 71-73. N u t t a l l , G. H. F., 1904. Blood Immunity and blood Relation-ship. Cambridge. Wilhelmi, R. W., 1942. The appli c a t i o n of the p r e c i p i t i n technique to theories concerning the o r i g i n of vertebrates. B i o l . B u l l . , 8 2 : 179-189. 

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