Comparative Serology
General Statement
The application of serological techniques to the problems of animal relationships has been attempted with varying degrees of success over a period of approximately fifty years. Few of the earlier studies were of a quantitative nature, but within the past decade, satisfactory quantitative serological techniques have been developed whereby taxonomic relationships may be estimated. The usefulness of comparative serology in taxonomy has been demonstrated in investigations of many groups wherein results obtained have, in most instances, been compatible with the results obtained by more conventional methods, such as comparative morphology. As Boyden (1942:141) stated, "comparative serology ... is no simple guide to animal relationship." However, the objectiveness of its methods, the fact that it has its basis in the comparisons of biochemical systems which seem to be relatively slow to change in response to external environmental influences, and the fact that the results are of quantitative nature favor, where possible, the inclusion of data from comparative serology along with that from more conventional sources when an attempt is made to determine the relationships of groups of animals.
The application of serological methods in ornithology has not been extensive. Irwin and Cole (1936) and Cumley and Irwin (1941, 1944) used two species of doves and their hybrids and demonstrated that a distinction between the red cells of these birds could be made by use of immunological methods involving the agglutinin reaction. McGibbon (1945) was able to distinguish the red cells of interspecific hybrids in ducks by similar methods. Irwin (1953) used similar techniques in his study of the evolutionary patterns of some antigenic substances of the blood cells of birds of the Family Columbidae. Sasaki (1928) demonstrated the usefulness of the precipitin technique in distinguishing species of ducks and their hybrids. This technique was used successfully also by DeFalco (1942) and by Martin and Leone (1952). Working with groups of known relationships, these investigators showed that the "accepted" systematic positions of certain birds were confirmed byserological procedures. The precipitin reaction, however, has never been applied to actual problems in avian taxonomy prior to the present study.
Preparation of Antigens
Although most previous work in comparative serology in which precipitin tests were used has involved the use of whole sera as antigens, Martin and Leone (1952) indicated that tissue extracts are satisfactory as antigens and that serological differentiation can be obtained with these extracts and the antisera to them. I decided, therefore, to use such extracts in these investigations, since the small sizes of the birds to be tested made it impracticable to obtain enough whole sera.
Most of the birds used were obtained by shooting, but a few were trapped and the exotic species were purchased alive from a pet dealer. When a bird was killed, the entire digestive tract was carefully removed to prevent the escape of digestive enzymes into the tissues and to prevent putrefaction by action of intestinal bacteria. As soon as possible (and within three hours in every instance) the bird was skinned, the head, wings, and legs were removed, and the body was frozen. Each specimen, consisting of trunk, heart, lungs, and kidneys, was wrapped separately and carefully in aluminum foil to prevent dehydration of the tissues. The specimens were kept frozen until the time when the extracts were made.
When an extract was to be prepared, the specimen was allowed to thaw but not to become warm. In the cold room with the temperature of all equipment and reagents at 2°C., the specimen was placed in a Waring blender with 0.9 per cent aqueous solution of NaCl buffered with M/150 K2HPO4and M/150 Na2HPO4to a pH of 7.0. The amount of reagent used was 75 ml. of saline for each gram of tissue to be extracted. The tissues were minced in the blender, allowed to stand at 2°C. for 72 hours, and the tissue residues removed by centrifugation in a refrigerated centrifuge. Formalin was added to a portion of the supernatant in the amount necessary to make the final dilution 0.4 per cent. This formolization was found to be necessary to inhibit the action of autolytic enzymes over the period of time required to complete the investigations. The effects of formolization on the antigenicity and reactivity of proteins are discussed later. It was necessary to sterilize and clarify the "native" (unformolized) extracts; this was done by filtration through a Seitz filter. These "native" substances were used only in the early stages of the investigation (see below). The filtrate was bottled and stored at 2°C. In the early stages of this investigation clarification of the formolized extract was accomplished by the same sort of filtration. It was determined, however, that centrifugation in a refrigerated centrifuge at high speeds (17,000g) served the same purpose and was quicker. The formolized extracts were bottled and also stored at 2°C. (although refrigerated storage of the formolized extracts does not seem necessary). For each extract the amount of protein present was determined colorimetrically by the method of Greenberg (1929) with a Leitz Photrometer.
Species for which extracts were prepared and the protein values of the extracts are listed in Table1. Extracts of some species were used throughout most of the experiment; extracts of others were used only when needed for purposes of comparison.
Table 1.—Species from Which Extracts Were Prepared and Injection Schedules for Extracts Against Which Antisera Were Produced
Series 1: Subcutaneous, 0.5, 1.0, 2.0, and 4.0 ml.
[A]Series 1: Intravenous, 0.5, 1.0, 2.0, and 4.0 ml.
[A]Series 2: Subcutaneous, 0.5, 1.0, and 2.0 ml.
Intraperitoneal, 8.0 ml.
Series 1:Intravenousand subcutaneous, respectively, 0.5 and 0.5 ml., 1.0 and 1.0 ml., 3.0 and 1.0 ml., 5.0 and 3.0 ml.
Series 2: Subcutaneous, 0.5, 1.0, 2.0 and 4.0 ml.
Series 1: Intravenous, 0.5, 1.0, 2.0, and 4.0 ml.
Series 2: Intravenous, 0.5, 1.0, 2.0, and 4.0 ml.
Series 3: Subcutaneous, 0.5, 1.0, 2.0, and 4.0 ml.
[A]Antiserum prepared against formolized antigen.
[A]Antiserum prepared against formolized antigen.
Preparation of Antisera
All antisera were produced in rabbits (laboratory stock ofOryctolagus cuniculus). Three methods of injection of antigen were used in various combinations: intravenous, subcutaneous, and intraperitoneal. Injection schedules used in the production of each antiserum are listed in Table1. Both formolized and "native" antigens were used. Each rabbit received one or more series of four injections, each injection being administered on alternate days and doubling in amount: 0.5 ml., 1.0 ml., 2.0 ml., and 4.0 ml. In all but two instances more than one series of injections was necessary to produce a useful antiserum. More than two series, however, resulted in little or no improvement of the reactivity of the antiserum.
The injection-series were separated by intervals of eight days. On the eighth day after the last injection of each series, 10 ml. of blood were withdrawn from the main artery of the ear of the rabbit, and the antiserum was used in a homologous precipitin test to determine its usefulness. If the antiserum contained sufficient amounts of antibodies to conduct the projected tests, the rabbit was completely exsanguinated by cardiac puncture, by using an 18-gauge needle and a 50 ml. syringe. The whole blood was placed in clean test tubes and allowed to clot. It was allowed to stand at 2°C. for 12 to 18 hours so that most of the serum would be expressed from the clot. The serum was then decanted, centrifuged to remove all blood cells, sterilized in a Seitz filter, bottled in sterile vials, and stored at 2°C. until used.
Methods of Serological Testing
The precipitin reaction is the most successful of the serological techniques thus far devised for systematic comparisons. The reaction occurs because antigenic substances introduced into the body of an animal cause the formation of antibodies which precipitate antigens when the two are mixed. The antisera which are produced show quantitative specificities in their actions; therefore, when an antiserum containing precipitins is mixed with each of several antigens, the reaction involving the homologous antigen (that used in the production of the antiserum) is greater than those reactions involving the heterologous antigens (antigens other than those used in the production of the antiserum). Furthermore, the magnitudes of the reactions between the antiserum and the heterologous antigens vary according to the degrees of similarity of these antigens to the homologous one.
The method of precipitin testing follows that outlined by Leone (1949). The Libby (1938) Photronreflectometer was used to measure the turbidities developed by the interaction of antigen and antiserum. With this instrument parallel rays of light are passed through the turbid systems being measured. Light rays are reflected from the suspended particles to the sensitive plate of a photoelectric cell; this generates a current of electricity which causes a deflection on a galvanometer. The deflection is proportional to the amount of turbidity developed and readings may be taken directly from the scale of the instrument.
The reaction-cells of the photronreflectometer are designed to operate with a volume of 2 ml.; therefore, this volume was used in all testing. In every series of tests the amount of antiserum was held constant and the amount of antigen was varied. The volume for each antigen dilution was always 1.7 ml., and to this was added 0.3 ml. of antiserum to make up a volume of 2 ml.
Table 2.—Percentage values obtained from analyses of precipitin reactions. Numerals represent relative amounts of reaction between antigens and antisera. Homologous reactions are arbitrarily valued as 100 per cent, and heterologous reactions are expressed accordingly.Comparisons are meaningful only if made within each horizontal row of values.
Antigens were diluted with 0.9 per cent phosphate-buffered saline solution. Tests were run in standard Kolmer test-tube racks, each test consisting of 12 tubes. Each dilution was made on the basis of the known protein concentration of the antigen. The first tube contained an initial dilution of 1 part protein in 250 parts saline and each successive tube contained a protein dilution one-half the concentration of the preceding tube, ranging up to 1:512,000. Saline controls, antiserum controls, and antigen controls were maintained with each test to determine the turbidities inherent in these solutions. These control-turbidities were deducted from the total turbidity developed in each reaction-tube, the resultant turbidity then being considered as that which was caused by the interaction of antigens and antibodies. The turbidities were allowed to develop over a 24-hour period. In the early stages of this investigation the reactions were allowed to take place at 2°C. in order to inhibit bacterial growth.Later tests were carried out at room temperatures, and bacterial growth was prevented by the addition to each tube of 'Merthiolate' in a final dilution of 1:10,000.
Experimental Data
Corrected values for the turbidities obtained were plotted with the turbidity values on the ordinate and the antigen dilutions on the abscissa. The homologous reaction was the standard of reference for all other test reactions with the same antiserum. By summing the plotted turbidity readings, numerical values are obtained which are indices serving to characterize the curves. Such values were converted to percentage values, that of the homologous reaction being considered 100 per cent. These values, plus the curves, provide the data by means of which the proteins of the birds may be compared. Plots representative of the precipitin curves are presented in Figs.10to21. For convenience each plot represents only several of the 10 curves obtained with each antiserum.
A summary of the serological relationships of the birds involved in the precipitin tests is presented in Table2, in which percentage values are presented. Since the techniques involved in testing were greatly improved as the investigation proceeded, the summary is based solely on those tests run in the later stages of the investigation. For reasons which will become apparent in later discussion, it should be emphasized that in Table2comparisons may be made only within each horizontal row of values.
Discussion of the Serological Investigations
One of the problems met early in this investigation was instability of the proteins in the extracts that were prepared. Extracts in which no attempt was made to inactivate the enzymes present proved unsatisfactory. It was necessary to maintain the temperature of the "native" antigens at 2°C, and all work with such antigens had to be performed at this temperature. This arrangement was inconvenient; furthermore, inactivation of the enzymes was not complete even at this low temperature, and some denaturation of the proteins took place as evidenced by the gradual appearance of insoluble precipitates in the stored vials.
The preservatives, 'Merthiolate' and formalin, were used in an attempt to inhibit the autolytic action of the enzymes present. Formalin, when added to make a final dilution of 0.4 per cent, proved to be the more satisfactory of the two preservatives and was used throughout most of the work. Formalin caused slight denaturation of some of the proteins, but this effect was complete within a few hours, after which any denatured material was removed by filtration or centrifugation. The proteins remaining in solution were stable over the period necessary to complete the investigations.
The addition of formalin reduces the reactivity of the extracts when they are tested with antisera prepared against "native" antigensand causes changes in the nature of the precipitin curves. This effect has been pointed out by Horsfall (1934) and by Leone (1953) in their work on the effects of formaldehyde on serum proteins. Their data indicate, however, that even though changes in the immunological characteristics of proteins are brought about by formolization, the proteins retain enough of their specific chemical characteristics to allow consistent differentiation of species by immunological methods. In the tests which I performed, the relative positions of the precipitin curves, whether native or formolized extracts were involved, remained unchanged (Figs.10,11).All data used in interpretation of the serological relationships were obtained from tests in which formolized antigens of equivalent age were used.
Only three antisera were produced against formolized antigens, all others being produced against "native" extracts. The formolized antigens seemed to have a greater antigenicity, in most instances, than did those which were unformolized, and precipitin reactions involving antisera produced against formolized antigens developed higher turbidities. The antisera produced against formolized antigens were equal to but no better than those prepared against "native" extracts in separating the birds tested (Figs.12,13).
The rabbit is a variable to be considered in serological tests. Two rabbits exposed to the same antigen, under the same conditions, may produce antisera which differ greatly in their capacities to distinguish different antigens. It is logical to assume, therefore, that two rabbits exposed to different antigens may produce antisera which also differ in this respect. This explains the unequal values of reciprocal tests shown in Table2. Thus, in the test involving the antiserum to the extracts ofRichmondena, a value of 71 per cent was obtained forSpizaantigen, whereas in the test involving anti-Spizaserum, a value of 98 per cent was obtained forRichmondenaantigen. In Table2, therefore, comparisons may be made only among values for the proteins of birds tested with the same antiserum.
Since the amount of any one antiserum is limited, there is, of necessity, a limit as to the number of birds used in a series of serological tests. Therefore, although the results reveal the actual serological relationships of the individual species, interpretation of the relationships of the taxonomic groups must be undertaken with the realization that such an interpretation is based on tests involving relatively few species of each group. It is reasonable to assume, however, that a species which has been placed in a group on the basis of resemblances other than serological resemblance would show greaterserological correspondence to other members of that group than it would to members of other groups. Specifically, in the Fringillidae and their allies, there seems to be little reason to doubt that genera, and even subfamilies, are natural groups. This is illustrated in tests involving closely related genera:RichmondenaandSpiza(Figs.14,15,18),EstrildaandPoephila(Fig.21),SpinusandCarpodacus(Figs.12,17,19,20). In each of these tests the pairs of genera mentioned show greater serological correspondence to each other than they do to other kinds involved. This point is illustrated further by a test (not illustrated) involvingZonotrichia querula(the homologous antigen) andZonotrichia albicollis. Although this test was one of an earlier series in which difficulties were encountered (the data, therefore, were not used), it is of interest that the two species were almost indistinguishable serologically.
The serological homogeneity of passeriform birds is emphasized by the fact that the value of every heterologous reaction was more than 50 per cent of the value of the homologous reaction, except in the test involving the anti-Richmondenaserum andMyiarchus(Fig.13) in which the value of the heterologous reaction was 45 per cent. Because most ornithologists consider these genera to be only distantly related (they are in different suborders within the Order Passeriformes), the relatively high value of the heterologous reaction emphasizes the close serological correspondence of passerine birds and indicates that small consistent serological differences among these birds are actually significant. The possibility that some of the serological correspondence is due to the "homologizing" effect of formalin on proteins should not be excluded. I think, however, that this effect is not entirely responsible for the close correspondence observed here.
An additional point to consider in interpretation of the serological tests is that the techniques used tend to separate sharply species that are closely related whereas species that are distantly related are not so easily separated. In other words, comparative serological studies with the photronreflectometer tend to minimize the differences between distant relatives and to exaggerate the differences between close relatives.
In analyzing the serological relationships of the species used in this study, it becomes obvious that two or more series of tests must be considered before the birds can be placed in relation to each other. For example, the data presented in Fig.14indicate thatSpizaandMolothrusshow approximately the same degree of serological correspondence toRichmondena. This does not imply necessarilythatSpizaandMolothrusare closely related. If Fig.15is examined, it can be determined thatRichmondenashows much greater serological correspondence toSpizathan doesMolothrus. Thus, an analysis of both figures serves to clarify the true serological relationships of the three genera. By reference to other series of tests involving these three birds a more exact determination of their relationships may be obtained.
To illustrate this point by a hypothetical example, two species might seem equidistant, serologically, from a third species. Additional testing should indicate if the first two species are equidistant in the same direction (therefore, by implication, close relatives) or in opposite directions (therefore, distant relatives). A single test supplies only two dimensions of a three dimensional arrangement.
It is impossible to interpret and to picture the serological data satisfactorily in two dimensions; therefore, a three-dimensional model (Figs.22,23) was constructed to summarize the serological relationships of the birds involved. Each of the eleven kinds used consistently throughout the investigation is represented in the model. By use of the percentage values (Table2), each bird was located in relation to the other birds. Where possible, averages of reciprocal tests (Table3) were used in determining distances between the elements of the model. In this way seven of the birds were accurately located in relation to each other. Lacking reciprocal tests, the positions of the other birds were determined by the values of single tests (Table4). Although these birds were placed with less certainty, at least four points of reference were used in locating each species. At least one serological test is represented by each connecting bar in the model. The lengths of the bars connecting any two elements were determined as follows: a percentage value (Table3and Table4) representing the degree of serological correspondence between two birds was subtracted from 100 per cent; the remainder was multiplied by a factor of five to increase the size of the model and the product was expressed in millimeters; a bar of proper length connects the two elements involved.
From the model it is observed that,MolothrusandPasserexcluded, the birds fall into two distinct groups: one includesPiranga,Richmondena,Spiza,Junco, andZonotrichia; the other includesEstrilda,Poephila,Carpodacus, andSpinus.
Table 3.—Reciprocal Values Used to Determine Distances Between Elements of the Model; Each Value Represents the Average of Serological Tests Between the Species Involved
Table 4.—Single Values Used to Determine Distances Between Elements of the Model; Each Value Represents a Single Test Between the Species Involved
key
Figs. 10-13.Graphs of precipitin reactions illustrating effects of formalin on antigenicity and reactivity of the extracts. For further information, see text, pp.190-193.
Fig. 10.Reactions of unformolized antigens ofRichmondena,Zonotrichia, andMolothruswith anti-Richmondenaserum.Fig. 11.Reactions of formolized antigens ofRichmondena,Zonotrichia, andMolothruswith anti-Richmondenaserum.Fig. 12.Reactions of anti-Richmondenaserum prepared against native antigen with antigens ofRichmondena,Zonotrichia,Carpodacus, andSpinus.Fig. 13.Reactions of anti-Richmondenaserum prepared against formolized antigen with antigens ofRichmondena,Zonotrichia,Poephila,Spinus, andMyiarchus.
key
Figs. 14-17.Graphs of precipitin reactions illustrating serological relationships. For further explanation, see text, pp.190-193.
Fig. 14.Serological relationships ofRichmondena,Spiza, andMolothrus.Fig. 15.Serological relationships ofRichmondena,Spiza, andMolothrus.Fig. 16.Serological relationships ofCarpodacuswith the richmondenine-emberizine-thraupid assemblage.Fig. 17.Serological relationships ofCarpodacusandSpinuswithRichmondenaandJunco.
key
Figs. 18-21.Graphs of precipitin reactions illustrating serological relationships. For further explanation, see text, pp.190-193.
Fig. 18.Serological relationships ofSpinusandPoephilawith the richmondenines.Fig. 19.Serological relationships ofCarpodacusandSpinuswithRichmondenaandPiranga.Fig. 20.Serological relationships ofPoephilaand Richmondena with the carduelines.Fig. 21.Serological relationships ofRichmondenaandSpinuswith the estrildines.
Relationship ModelRelationship Model
Fig. 22.Two views of a model illustrating serological relationships among fringillid and related birds. For further explanation, see text, pp.193-194.