Serological techniques have been used in systematic studies for about fifty years. At first their use was confined to those groups of animals offering plentiful supplies of blood, since blood was the tissue most widely used. Recent modifications that employ other tissues have now made possible similar study of groups of smaller animals

Cather, James N.

English

Southern Methodist University

FIELD & LABORATORY Volume XXIII January, 1955 Number 1 A Serological Technique for Gastropod Taxonomy1 James N. Cather 2 Serological techniques have been used in systematic studies for about fifty years. At first their use was con-fined to those groups of animals offering plentiful sup-plies of blood, since blood was the tissue most widely used. Recent modifications that employ other tissues have now made possible similar study of groups of smaller animals. Serological work in the invertebrates has in general been limited, but includes outstanding studies on the Crustacea by Boyden ('36, '39) and Leone (' 49) . Blood was the tissue used by these workers. Wilhelmi ('44) used other tissues of several invertebrates, including Busycon; desiccating them after freezing and then preparing a saline extract. Boyden points out ('42, '53) that serological data do not offer complete answers to taxonomic problems. They can, however, aid when morphological differentiation is incon-clusive, and thus help in establishing a more natural classi-fication of families and genera. Serologists interested in comparative morphology and physiology assume that ani-mal proteins are less rapidly changed in evolution than are most morphological features. Proteins, we should expect, suffer less change from environmental influences than do morphological features. The value of comparative serology in taxonomy has been proved in many instances, especially where its results have comported with those got by conven-tional comparative morphological methods. Such methods are often difficult to use at taxonomic levels above the genus, because of degeneracy, convergence, and parallel-ism. The quantitative and objective nature of comparative serology makes the method highly useful, when used with data obtained from comparative morphology. The Gastropoda notably require such study, since so much of our classification of them rests upon characteris-lThesis, submitted in partial fulfilment of the requiremen:s for the degree of M.S. in Biology, Southern Methodist University, 2With grateful acknowledgmen.s to Professors William B. Stallcup, E. P. Cheatum, and S. W. Geiser. 5 6 FIELD AND LABORATORY [Vol. 23 tics that show wide variation. Some taxonomists rely pri-marily upon reproductive structures as a basis for classi-fication; others depend upon shell characters which are more obvious to the average student and require less skill in their use. Shell characters have been used to advantage in many groups, even at the species level; but in a genus such as Physa, intergradation seems universal. A key based upon shell characters would be the only practical kind for '--------;L I I I I I I I I I I ' I ,,i' I ', I \ I \ I \ I \ I \ I ' I 1/ Figure 1. Relationships of the Gastropods studied. (Po, Pomacea; P, Physa; H, Helisoma; R, Rumina; L, Limax.) persons interested in this taxonomic group. Yet such keys may lead to confusion in certain families and genera, be-cause of wide variation in shell characteristics. There may be no solution of this problem of shell variation within a genus or species, when dealing with a plastic group like the gastropods, that reflect so much their environment-background. General Methods and Materials Most of the previous work in comparative serology, in which precipitin tests were used, employed whole sera as antigens. Martin & Leone ('52) and Stallcup ('54) showed that tissue extracts are satisfactory as antigens in serological testing with birds. Wilhelmi ('44) stated that lipid,s must be removed for ac-curate serology, but Stallcup's ('54) work showed this not 1955] SEROLOGICAL TECHNIQUE FOR GASTROPOD TAXONOMY 7 always necessary. In my work, removal of lipids was not at-tempted. The Gastropoda in these tests ( exclusive of Pomacea cali-ginosa) were collected in Dallas County, Texas, in March, 1955, and included the following: Subclass Prosobranchia Order Monotocardia Ampullariidae Pomacea caliginosa Subclass Pulmonata Order Bassommatophora Physidae Physa anatina Lea Planorbidae H elisoma trivolvis Say Order Stylommatophora Suborder Sigmurethra Holopoda Order Stylommatophora Suborder Sigmurethra Holopoda Achatinidae Rumina decollata (L.) Aulacopoda Limacidae Limax fiavus L. Antigen Preparation.-From 6 to 30 grams of living snails were placed in a Waring blender and minced with 100 ml. of unbuffered 8 percent NaCl solution. The mixtures were refrigerated for 48 hours at 2° C. to permit extraction of proteins. The tissue residues were then centrifuged out, and the extracts stored at 2° C. Protein content of the ex-tract was determined by the biuret method, using the Bausch & Lomb "Spectronic 20" colorimeter. Antisera Preparation.-All antisera were produced in domestic rabbits. A series of 4 injections were given each rabbit, with an interval of 48 hours between injections. The following methods of injection were used: (1) 0.5 ml. intravenously, (2) 0.5 ml. intravenously; 0.5 ml. subcutaneously, (3) 1.0 ml. intravenously; 1.0 subcutaneously, (4) 1.0 ml. intravenously; 3.0 ml. subcutaneously. On the eighth day after the final injection, 10 ml. of blood were withdrawn from the main artery of the ear, and the antiserum was used in a homologous precipitin test (against the antigen which produced the antiserum) to determine whether antibodies were present in sufficient amounts. If enough were present, the rabbit was bled to death by cardiac puncture, using an 18-gauge needle and a 50 ml. syringe. The whole blood was placed in test tubes and allowed to 8 FIELD AND LABORATORY [Vol. 23 clot. The blood was allowed to stand until the serum was expressed. Both serum and clots were then centrifuged, and the serum decanted. This was then sterilized by filtration • I J I I ~ i! ~ i : I . ~ ~ l I j J ~ J !I i l. , .. 3.0 2.2 1.0 o.o l. ,13.5 ..o 10.S a.o ... 2. ,.o 3.0 2., l.5 o.o 2. 1.s.1 a.o 0.3 •• o ... 3. ,., 3.0 2.0 2.0 0.2 .. 15.0 7.6 l.5 •• o ... ! .. 3.5 3.0 l.5 1.0 0.2 j 5. 2.5 2.2 l.5 1.0 0.2 ! .. u.o ... 2-0 s.o 4.0 I .. 7o5 ,.o 2.0 •• o 2., !/ ,. 1., 2.0 o.5 1.0 O.l g 7. 1.0 1.0 o., o.5 o.o Ii ! 6. ,.2 2.s ,.0 ,.o 1.a ! 7. 2.7 ... 2.0 2., 0.5 ~ .. ·o.7 o.5 0.5 o., o.o .. i., 2.0 2.2 2., 0.2 .. o.7 o., o., o.5 o.o .. 1.0 2.0 1., 2., o.2 10. l.O o.5 o.o o.a o.o 10. 1., 1.0 1., 2.0 0., rotal 29.2 1l!o7 ll.7 9.7 M Tota1 7~.9 ... o .... .... "'·· ' d ! I I 1 J I i i ii ! I I j J I !I :Z, 1. a.2 2.0 7.5 7.0 ,.o 1. "'·· 16.2 11.0 18.0 1., .. 3.7 2.0 7.0 ... 4.0 2. z,.2 6.7 ,.0 ... 2., .. 14.4 1.0 6.5 , .. ,.o .. 9.7 6.0 4.0 l.6 ... ! .. "'·5 1.0 ,.o ,.0 2.5 ! .. ... ,.0 . .. ... ,.0 ~ ~ ,. 21.1 1.0 ,.o ,.o 2.0 I .. 7.2 3.0 1., 1., 2.0 !/ 6. 14.l 0.2 •• 0 2.3 2.0 !/ .. 4.7 2., 1.0 2.0 l.O j 7. a.o 0 .. 2., 1.2 1.0 I 7. 3.7 1.s 1.a 1.0 1.0 Ii .. 6.6 o.5 2.0 1.2 1.0 .. 2., 0., 1.;:, o., ... .. l.B o.o 1.0 1.0 2.0 .. l.9 0.2 1.0 0.0 ... 10. 0~7 o.o l.O 1.0 1.0 10. o.g 1.0 1.0 0.0 1.0 Total <;)~.1 a.5 ;,,9.5 ::56.'1 23.5 ,,,,.,_ 61 0 8 41.6 27.6 27.5 15.s Figures 2-5. Turbidity values of precipitin Tests with antisera of Limax (2), Helisoma (3), Rumina (4), and Pomacea (5). through a Seitz filter. It was then transferred to sterile 5 ml. bottles with sterile pipettes, and stored at 2° C. until used. Method of Testing. -Antigenic substances introduced into the body of an animal cause the production of anti-bodies, which in turn cause precipitation of the antigen when the two are mixed. When an antiserum is added to some of the antigen that caused its production (homologous antigen), maximum precipitation occurs. When the anti-serum is added to the other antigens (heterologous anti-gens), a smaller amount of precipitate is formed. The 1955] SEROLOGICAL TECHNIQUE FOR GASTROPOD TAXONOMY 9 amount of precipitate produced corresponds to the degree of similarity of the antigen in question, to the homologous antigen. Thus we are able to infer serological relationships. The method of testing follows that used by Leone ('49), and requires the use of the Libby ('39) photronreflectometer. This instrument. measures the turbidities produced by the interaction of antigen and antiserum. Biuret tests were run to determine the amount of protein in each mixture, then enough 8 percent salt solution was added to give an initial dilution of 1 part of protein to 500 parts of salt solution. Tests were run in Kolmer test-tube racks with ten tubes for each test. The first tube contained an initial dilution of 1 ' b J j ! u i i ! j l. 11.2 , .. o.o •• o ... .. ... , .. o.o •• o ..o .. s.1 s.2 1.0 5,0 ,.o ! .. ,.o ... l,O ... ..o ~ .. s.2 ,.2 2,0 2.• 2.0 A I .. ... 2.s 1.0 1.0 2.2 ~ ,. ... l.8 1.0 1.0 1.0 ll .. 2.• 2.0 1.0 o.• o •• .. 1.6 l.8 o.• o.s 1.0 10. 1.2 1.0 o.s 0.2 2.0 , . ..,_ ... , '°·" 8.1 24.5 ..., Figure 6. Turbidity values of precipitin tests withPhysa antiserum. part of protein in 500 parts salt solution, and each successive tube contained a protein concentration only half of that of the preceding tube. The photronreflectometer was made to use a volume of 2 ml., and this quantity was used in all tests. The amount of antiserum was held constant at 0.3 ml., but the concentration of antigenic extract was varied. The volume of antigen was always 1. 7 ml., but increasing dilu-tions were made by adding 8 percent saline. Each test to de-termine the inherent turbidity was read as soon as it was completed. The turbidities were allowed to develope over a 24-hour period, when.a second reading was taken. The dif-ference between the two readings is the turbidity caused by antigen-antibody reaction ( Figs. 2-6) . The tests were car-ried out at room temperature. "Merthiolate" (Thimerosal, 10 FIELD AND LABORATORY [Vol. 23 Lilly) in a final dilution of 1 :10,000 was added to prevent bacterial reaction. Serological Relationships Serological evidence showed H elisoma and Physa to be the most closely related genera studied (Figs. 1; 7). This would be expected from the classification given on page 7; and both paleontology and morphology give evidence that the Bassommatophora arose from the Stylommatophora. As these genera have probably undergone similar evolu-tions from a common ancestor, we should expect similar proteins. 7 m . ~ i ~ i ~ ::1 " ! i ... . ! . I j ~ ,:l /;: i Pomacea calignosa Physa a.ne.tina 47 He11eoaa. tr1Vol v1:S 43 68 Rum1na decollata "'- "' 22 L1JnaX. navus 11 55 34 "' Figure 7. Percentage values obtained from analyses of precipitin reactions. Pomacea shows a more closely related serological kin-ship to Physa and Helisoma than to Rumina; but Limax is apparently closer to Physa than to either Pomacea or Rum-ina. At the outset this differs from what one would expect, but it may reflect origins. The Monotocardia are believed to be ancestral to the Pulmonata (because of the retention of the single auricle by the Pulmonata) ; the first evolved mem-bers of the Pulmonata were the Stylommatophora. If this be true, then Pomacea and Rumina should be relatively close in serological relationship. While this does not seem to be the case at first sight, our findings indicate that Pomacea is closer to Rumina than Rumina is to any of the other snails tested (Fig. 7). Thus we may infer that Rumina lies to one side of the line ancestral to Physa and H elisoma. 1955] SEROLOGICAL TECHNIQUE FOR GASTROPOD TAXONOMY 11 The genus Pomacea and the genus Limax show the widest divergence in serological relationship of all genera studied. Rumina is also more remotely related to Limax than are H elisoma and Physa. This may mean that Physa and Limax are related, and it is possible that an emergence took place a second time, and that Limax descended from some bassommatophoran. The Limacidae show variability within the family, and perhaps are polyphyletic in origin. Whether this is true or not, it is logical to infer that a family that evidences so much evolutionary change in mor-phology would undergo more extensive serological change than the more conservative snails studied in these investi-gations. SUMMARY Serological techniques which have been applied to vertebrates may be of value in gastropod taxonomy. The results of the present study appear to support the theory that the Pulmonata arose from the Monotocardia. From the results we can infer that Rumina lies to one side of the main path of the evolution of the Bassommatophora, and that Limax has undergone considerable protein change since its presumed early origin from the Monotocardia. LITERATURE CITED BOYDEN, ALAN (1936). Serological study of the relationships of some common inver-tebrata. Ann. Rept. Tortugas Lab., Carneg. Inst. Wash., Yearbook, 1936, p. 82. ----(1939) Ibid., 1939, pp. 219-20. ----(1942) Systematic serology; a critical appreciation. Physiol. Zool. 15:109-45. ----(1953) Fifty years of systematic serology. System Zool. 2:19-31. CHEATUM, E. P. (1955). Fresh-water and land gastropods of Texas. (Unpubl.) LEONE, CHARLES A. (1949). Comparative serology of some brachyuran crustacea, and studies in hemocyanin correspondence. Biol. Bull. 87:96-105. LIBBY, R. L. ( 1938). The photonreflectometer - an instrument for measurement of turbid systems. Jour. lmmun. 34:71-73. MARTIN, E. P. & C. A. LEONE (1952). Serological relationships among domestic fowl, as shown by the comparisons of protein preparations from corresponding organ systems. Trans. Kans. A cad. Sci. 55: 439-41. PILSBRY, HENRY A. (1939-48). Land Mollusca of North America. Academy of Natural Sciences of Philadelphia. STALLCUP, WILLIAM B. ( 1954). Myology and serology of the avian family Fringillidae, a taxonomic study. Univ. Kansas, Publ. Mus. Nat. Hist. 8:157-211. WILHELMI, R. W. (1944). Serological relationships between the mollusca and other invertebrates. Biol. Bull. 87: 96-105. 

A Serological Technique for Gastropod Taxonomy

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A Serological Technique for Gastropod Taxonomy

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