TAXQNDMEC ANALWS 0F ELECTROPHORETIC BLOOD SERUM PATTERNS IN THE COTTON RAT. SEGMODON fhesis for the Degree of M. S. MECHiGAN STATE ’U‘NWERSITY PETER L. DALBY 1968 THESIQ ‘flmflhfiu "J BINDING av ‘3 ‘ ' HUAG & SflNS' ’ 800K BINDERY INS. ‘ LIBRARY mm: ISPIIT‘ U‘ '- .‘J ABSTRACT TAXONOMIC ANALYSIS OF ELECTROPHORETIC BLOOD SERUM PATTERNS IN THE COTTON RAT, SIGMODON. by Peter L. Dalby The blood serum protein patterns were analyzed from seven species of cotton rats, Sigmodon alleni, g, fulviventer, é, hispidus, §, leucotis, .§- melanotis, g. ochrognathus, and §, planifrons. In all, sera from l56 specimens were studied by means of the acrylamide disc electrophoresis procedure of Ornstein and Davis (I962), Davis (l96h) and modified by Wright and Mallmann (I966). The resulting protein patterns in the acrylamide gel and their densitometric tracings were compared to determine the amount of individual variation (between individuals of the same species from a single locality), geographic variation (between samples of the same species from different localities), and interspecific variation (between samples of different species). The electrophoretic serum patterns of some population samples within a single species were distinctive. Other samples could not be distinguished either because of the variability within individual popula- tion samples or because of close similarity between geographically-separated population sampleS. ’One case of transferrin polymorphism within a single population was noted, suggesting the occurrence of a limited gene exchange. Peter L. Dalby Between certain geographically-separated population samples of g. hispidus, transferrin polymorphism, consisting of five different phenotypes (transferrin patterns A,B,C,D and E) was observed to follow a specific arrangement which allowed for quick regional identification. No form of a species specific “species curve” or IIspecies pattern” was apparent in the wide ranging ubiquitous é, hispidus, but was present in s, leucotis, a spatially and ecologically restricted cotton rat. Differences between species were evidenced in the transferrins, some of the prealbumins, and to a lesser degree, in the postalbumin components. On this basis, §, hispidus, §, leucotis and §, ochrognathus could be distinguished from each other. However, Sigmodon fulviventer and g. melanotis appeared indistinguishable from each other and conspecific, as did g, alleni and g, planifrons. A table and key for electrophoretically differentiating the studied species and a diagram of their possible re- lationships are presented. TAXONOMIC ANALYSIS OF ELECTROPHORETIC BLOOD SERUM PATTERNS IN THE COTTON RAT, SIGMODON. BY Peter Lf‘balby A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology I968 ACKNOWLEDGMENTS I would like to express my appreciation to the members of the Michigan State University - The Museum field parties of I964, '65, '66 and '67, who, under the direction of Professor Rollin H. Baker and with financial assistance from the National Science Foundation (GB- 2227) and the M.S.U. Development Fund, collected all the Mexican samples of Sigmodon. Other specimens were generously contributed by mammalogists from different areas of the United States and Central America. I also acknowledge the valuable help of Larry Besaw, Michael K. Petersen and Professor G. L. Wright, who provided encouragement and guidance in various aspects of the electrophoretic procedure. Thanks also go to Professor H. A. Lillevik and Mrs. B. R. Henderson for their assistance in acquiring the necessary chemicals and equip- ment used in this project and, to Mrs. Elizabeth Rimpau for her technological assistance. Appreciation is expressed to those people too numerous to name but who also deserve recognition for their continuous help and advice. Special thanks go to the members of the Guidance Committee consisting of Professors R. H. Baker, H. A. Lillevik and R. A. Fennell, for their continual advice and encouragement throughout this project. I would also like to thank my wife Barbara, for assisting in any was she could throughout the study. CONTENTS Page I. INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . I A. Purpose. . . . . . . . . . . . . . . . . . . . . . . l B. Review of Literature. . . . . . . . . . . . . . . . I II. EXPERIMENTAL. . . . . . . . . . . . . . . . . . . . . . . . 6 A. Collecting Localities. . . . . . . . . . . . . . . . 6 8. Method of Bleeding. . . . . . . . . . . . . . . . . l0 C. Development of Procedure. . . . . . . . . . . . . . II D. Reagents. . . . . . . . . . . . . . . . . . . . . . l2 E. Equipment. . . . . . . . . . . . . . . . . . . . . . l2 F. Operational Procedure. . . . . . . . . . . . . . . . IS G. Controls. . . . . . . . . . . . . . . . . . . . . . 18 H. Analysis of Data. . . . . . . . . . . . . . . . . . l9 III. RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . 22 A. Individual Variation Within a Population Sample . . 22 B. Geographic or lntraspecific Variation. . . . . . . . 2h C. Specific Differences in Sigmodon. . . . . . . . . . . 27 IV. DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . 38 V. SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . . 96 VI. LITERATURE CITED. . . . . . . . . . . . . . . . . . . . . . 48 Table LIST OF TABLES Page Stock solutions. . . . . . . . . . . . . . . . . . . . . . l3 Working solutions. . . . . . . . . . . . . . . . . . . . . l3. Electrophoretic characteristics of Sigmodon blood serum proteins . . . . . . . . . . . . . . . . . . . . . . 37 LIST OF FIGURES Figure Page l.. Map showing the approximate geographical locations of Sigmodon hispidus used in this study. . . . . . . . . . 8 2a. Map showing the approximate geographical locations of Sigmodon fulviventer, S, melanotis, S, planifrons and S, alleni used in this study. . . . . . . . . . . . . . . 9 2b. Map showing the approximate geographical locations of Sigmodon leucotis and S, ochroqnathus used in this study. 9 3. Diagramatic representation of the position of the gels in the differential disc electrophoretic column. . . . . . l6 4. Schematic diagram of a Sigmodon densitometric tracing and accompanying gel and its nomenclature. . . . . . . . . 20 S. Sketched densitometric tracings of the six Sigmodon hispidus from Oracle, Arizona to demonstrate minimal individual variation within a population sample. . . . . . 3O 6. Sketched densitometric tracings of the six Sigmodon hisgidus from Autlén, Jalisco, to demonstrate maximal individual variation within a population sample. . . . . . 3l 7. Sketched densitometric tracings from four specimens each of: Sigmodon hispidus from (a) Oracle, Arizona and (b) Autlén, Jalisco are superimposed to demonstrate the general similarity found within a population sample and between populations; S, alleni (c) and S, planifrons (d) are superimposed to demonstrate the similarity within a species. . . . . . . . . . . . . . . . . . . . . 32 8. Sketched densitometric tracings from four specimens each of (a) Sigmodog leucotis, (b) S, ochroqnathus, (c)‘S. melanotis, and (d) S. fulviventer, superimposed to demonstrate the similarity within.a.species. . . . . . . 33 9. Map showing the approximate geographical distribution of the five transferrin types (A through E) found in SlngdOD hiSpidUSo o o o o o o o o o o o o o o o o o o o 1+0 l0. A diagramatic sketch indicating possible lines of evolution in Sigmodon. . . . . . . . . . . . . . . . . . 45 LIST OF PLATES Plate Page la. The electrOphoresis apparatus and power supply. . . . . . IA lb. Destaining unit, consisting of (I. to r.) battery charger, destaining tank, filter system, and oscillating pump. 0 O O O O O O O O O I O O O O O O O O 0 11+ 2a. Davis and Ornstein 7.5% standard gels of (l. to r.) Sigmodon alleni, S, ochroqnathus, S, leucotis, S, planifrons, S, melanotis, S, fulviventer, and S.hispidus.......................31+ 2b. The resulting electrophoretic protein patterns from a specimen of Sigmodon hispidus, using various pore size separating gels. . . . . . . . . . . . . . . . . . . 3h 2c. ElectrOphoretic gel patterns of six Sigmodon hispidus from the Oracle, Arizona population sample. . . . . . . . 34 2d. Electrophoretic gel patterns of six Sigmodon hispidus from the Autlén, Jalisco population sample. . . . . . . . 34 3a. Electrophoretic serum protein patterns of Sigmodon hispidus from l6 geographic localities. . . . . . . . . . 35 3b. Electrophoretic serum protein patterns of the three Sigmodon ochrognathus from Boquilla, Durango. . . . . . . 35 3c. Electrophoretic serum protein patterns of Sigmodon leucotis, with (l. to r.) first four specimens from Hda. Coyotes, Durango, and last five specimens from lbarra, Guanajuato. . . . . . . . . . . . . . . . . . . . 35 ha. Electrophoretic serum protein patterns of the eight Sigmodon alleni from Capécuaro, Michoacan. . . . . . . . 36 Ah. ElectrOphoretic serum protein patterns of six Sigmodon fulviventer, . . . . . . . . . . . . . . . . . 36 4c. ElectrOphoretic serum protein patterns of the seven Sigmodon planifrons from Juchatengo, oaxaca. O O O O O O O O O O O O O O O O O O O O O O O O 36 Ad. Electrophoretic serum protein patterns of the six Sigmodon melanotis from La Barca, Jalisco. . . . . . . . 36 vi I. INTRODUCTION A. Purpose The objective of this study is to investigate the blood serum patterns of cotton rats (Sigmodon), using acrylamide disc electrophoresis, to determine: (I) non-geographical variation within a single population sample of a species from one locality; (2) geographic variation between population samples of the same species from different localities; and (3) interspecific variation between samples representing currently recognized species. If detectable differences in the serum protein patterns are observable at these three taxonomic levels, it might then be possible to use these findings to gain a greater knowledge of speciation in Sigmodon, the taxonomy of which is currently based entirely on morphological grounds. B. Review 2f Literature The concept that protein synthesis is dependent on rigid genetic control and is therefore a reflection of the genotype has provided a theoretical basis for utilizing physico-chemical characteristics of proteins in taxonomic studies. Moore (I945), using the Tiselius apparatus, described distinctive differences in blood serum protein electrophoretic patterns between such species as man, rhesus monkeys, swine, white rats, cats, guinea pigs and hamsters. In a later paper (I959). he reported that the serum patterns in the cotton rat and the guinea pig obtained in I957 had the same general characteristics as those obtained twelve years previously. I Auernheimer _£_gl, (I960) employed paper strip chromatography and compared the electrophoretic serum protein patterns of the cricetids Peromyscus, Sigmodon, and Neotoma, and concluded that there are generally marked differences between the species. Differences were most evident among the globulins although the albumin content varied over a wide range. He also noted that in several closely related species of Perognathus, no distinguishable characteristics were evident. Johnson and Wicks (I959) also employed paper strip chromatography and surveyed the serum proteins in eight orders, consisting of 74 species of North American mammals. Although the globulins were quite variable among individuals, these authors concluded that a standard species pattern existed, and that the prime taxonomic significance appeared at the generic and specific levels. In another paper (I964), Johnson and Wicks surveyed I7 mammalian orders consisting of 46 families, II2 genera and 206 species. They concluded that there is a generalized mammalian pattern consisting of albumin and from one to six globulin fractions. After they failed to separate such higher taxa as orders and families, Johnson and Wicks reiterated their former conclusion that the primary usefulness of electrophoresis in taxonomic work is at the generic and specific levels. Johnson (I968), employing paper electrophoresis of blood plasma proteins, studied twenty-five mammalian taxonomic relationships. In nineteen instances, he felt that clarification of relationships were obtained, but in six instances the proteins did not help. Petersen (I966, I968), employed acrylamide disc electrophoresis to study the serum proteins in eighteen species of Peromyscus from the United States and México. On the basis of superficial characteristics such as number of bands, symmetry, general appearance, and slope of peaks, he demonstrated taxonomically significant differences at the species-group level. Since variation in the monor components was recognized in the previous taxonomic studies, the recognition of various species, Species groups, genera, and higher taxa has been based on the position of major globulin (5,9, transferrin) and albumin components. However, some workers have demonstrated polymorphism in these also, leaving some doubt as to their taxonomic significance and the reliability of Species curves or species patterns. Welser _£ 31. (I965) found albumin polymorphism in five forms of Peromyscus. Vertical starch-gel electrophoresis of the plasma proteins revealed five albumins of slightly different electro- phoretic mobilities, two of which frequently appeared in E, maniculatus bairdi. Data indicated that inheritance of these five albumins may be controlled by a single autosomal locus with multiple co-dominant alleles, each allele determining a different albumin. Later, Brown and Welser (I968) made a more detailed study of albumin polymorphism in seven species of laboratory and wild strains of Peromyscus. In E, leucopus populations, a great difference in polymorphism frequency was observed between two natural populations twenty-five miles apart. Albumin polymorphism was also found in natural populations of E, maniculatus, E, leucopus and 2, $5331. Brown and Welser noted that in E, maniculatus, the long-tailed, forest-dwelling forms of northern and western North America appeared to be monomonphic.while the shortétailed, grassland- inhabiting forms were polymorphic. Transferrin polymorphism was demonstrated by other investigators such as Ashton and Braden (l96l). Using starch-gel electrophoresis, they found three serum‘f9-globulin (transferrin?) types in different strains of laboratory house mice. They also noted that between individual mice of one inbred strain there was considerable variation in the staining intensity, .2,, relative quantity of protein within the i ,ég-globulins. Goodman _£‘gl. (I965), employing horizontal starch-gel electrophoresis, found ll molecular forms and 34 phenotypes of transferrin in 372 blood serum samples from six species of macaques. The tendency for polymorphism was observed to vary from species to species and from one local population to another. From this information they concluded that these macaques belong to a single highly-differentiated species rather than several species. Nadler and Hughes (I966) analyzed by two dimensional starch-gel electrophoresis the serum proteins of several species of Spermophilus (=Citellus). Although transferrin polymorphism was noted in one species, they found taxonomic significance at the population, subspecies, and species levels. Ahl (I968), using acrylamide disc electrophoresis, found four transferrin types in the blood plasma of Peromyscus maniculatus nebrascensis from three different altitudes. There were no differences in the plasma proteins between the three altitude groups which could be related to altitude or sex. It appeared that the four types were distributed in accord with geographic proximity of three altitude groups. Other protein components in blood serum may also vary. Russell and Semeonoff (I957), utilizing starch-gel electrophoresis, reported that evidence has been found for two loci which control the synthesis of separate subunits of a serum esterase isozyme system in Microtus agrestis. In the natural population studied, both loci appeared to be polymorphic. Russel and Ashton (I958) employed starch-gel electrOphoresis and found that horse serum exhibited individual variation in the prealbumins and [EB-globulins. Popp and Popp (I962) examined the blood serum by starch-gel electrophoresis of 2l strains and three partially inbred stocks of laboratory mice (M23). A single band of esterase was found in C57BL and CS7L mice. Two bands of slightly slower mobility were found in the other strains. Kristjansson (I963) used starch-gel electrophoresis and observed prealbumin polymorphism in domestic pigs. Shaw (I965) called attention to other papers which have demonstrated electrophoretic variations in enzymes and other blood components of 523, Peromyscus, and larger animals. In review, a variety of comparative studies on mammalian blood serum, using several electrOphoretic techniques, have been conducted at different taxonomic levels. Often laboratory strains which have gone through considerable breeding ”bottlenecks'I are used. The worker may have studied only a specific serum protein (2,3, albumins, transferrins, esterases) among certain animal groups. The physiological condition of the specimens is frequently overlooked. If field specimens are collected, they are often indiscriminately sampled from a small part of the animals range and/or consist of small sample sizes. However, when properly utilized, valuable information gathered by electrophoretic techniques has proved helpful in animal taxonomy. For this reason, the use of acrylamide disc electrophoresis as a taxonomic tool in studying cotton rat (Sigmodon) systematics is explored. II. EXPERIMENTAL A. Collecting Localities Seven species of Sigmodon (as designated by Hall and Kelson, I959; Baker and Greer, I962) were used in this study. Inbred (2-3 generations) laboratory specimens if used, are identified by an asterisk. The number which corresponds to the approximate collecting locality on Figures l, 2 a and b, the capture locality, and number of specimens are as follows: Sigmodon hispidus berlandieri Baird l. Durango: 8 km. SE ZaIvaIza, ll68 m., l 2. Jalisco: 2 km. NW La Barca, l535 m., 7 3 Nuevo Leon: 37 km. NNE Ciénega de Flores, 555 m., 4 4. San Luis Potosf: 2 km. N Santa MarTa del RIO, I677 m., 3 5. Zacatecas: 3 km. N Santa Rosa, ll74 m., 5 6. Texas: Lubbock Co., Lubbock., 3 Sigmodon hispidus chiriquensis J. A. Allen 7. Canal Zone: Chiva Chiva Road., 2 8. Canal Zone: Empire Range., I 9. Canal Zone: France Field., 4 Sigmodon hispidus cienegae A. B. Howell IO. Arizona: Pinal Co., Oracle., 6 Sigmodon hispidus furvus Bangs ll. Honduras: Puerto Cortés., 5 Sigmodon hispidus ischyrus Goodwin l2. Guerréb: 7 km. SE Cuajinicuilapa, 9l m., 6 Sigmodon hispidus komareki Gardner I3. Tennessee: Anderson Co., Oak Ridge., 4 I4. South Carolina: Aiken Co., Savannah River Project., 3 Sigmodon hispidus littoralis Chapman l5. Florida: Palm Beach Co., Boca Raton., l Sigmodon hispidus major V. Bailey l6. Nayarit: 27 km. SE Tuxpan, I46 m., 6 l7. Sinaloa: 3 km. N Santa Lucia, I296 m., l Sigmodon hispidus mascotensis J. A. Allen l8. Jalisco: IO km. sw Autlan, I342 m., 6 l9. Colima: 8 km. NW Santiago, I8 m., I Sigmodon hispidus texianus (Audubon and Bachman) 20. Arkansas: Benton Co., 6 km. E Rogers, 422 m., 7 2l. Oklahoma: Marshall Co., 2 km. E Willis, I92 m., 6 22. Texas: Colorado Co., Eagle Lake, 52 m., 8 Sigmodon hispidus toltecus (Saussure) 23. Tamaulipas: Hda. Acufia, 808 m., 4 24. Veracruz: Isla del Toro, 6 m., 3 Sigmodon alleni V. Bailey I. Micthén: IO km. W Capécuaro, 2059 m., 8 Sigmodon fulviventer fulviventer J. A. Allen 2. Durango: 9 km. NNW Canatlan, I952 m., I 3. Durango: Hda. Coyotes, 2475 m., 4 4. Durango: Hda. de Atotonilco, 2037 m., 3 Zacatecas: I3 km. S Villanueva, 2090 m., I U'1 6. Guanajuato: 8 km. SW Ibarra, 2500 m., 6 .>U:um m_;u c_ pom: m:n_mm_: copOEm_m mo mco_umoo_ .mo_;amcmoom oumE_xocaam on“ mc_zo;m am: ._ 0L:m_m V. III I I III I III I I fl I I I I O \ 20.52.93 ($2-333 1:52.? Eon?) mam»l.>0._( on! Own qufi .Iuo ”no uiv INN mwi_} 0.90. -10 qu 90' our i x4 *ooooooomfigggym L... I I P“ I I T i a I J B'modon fulvl‘venter -------- o w- _____ , Sigmodon melanotis ........ * . _. »_ . I I Sigmodon planifrons ........ 0 ' ' ‘ _ ;,.,II,.;‘-‘, - ~ ‘ .‘ ‘4. “I ‘Kfikr _ -, TI“ . 3 8W“! 0.. 00000000 00‘ "‘I " . 3,). -"I K . ”a \II 'I I I L... I I . I l 5‘ | C . if if ‘ I .0 ‘ ""“T ‘ V"\ I . I I . . r , I I I" 2 "PL“? 39° "L55 x» t I F ' 260 Y 460 Iutounms - . - ‘ ‘ I \\ ,IIL ‘M' ___ #00“ Im' _ ‘ ‘0’. , ‘8' .2 fl ’ -, "0' ‘ \‘ Figure 2a. Map showing the approximate geographical locations of Sigmodon fulviventer, S. melanotis, S. planifrons and S, alleni used in this study. u . . 7 . 'g‘ ' \ I ‘F \ ‘ : ‘ C ' A I 8‘ ’ I~\ ."\ J L I I I r1 ~"v ‘ ‘ I I \ o ' \ \ . ' ' - k 'v-y I L _. \ I I ,. .-§ I L l C. .i - I ‘ ’ .I 1" v . \. - \ J . s 1" ,I :1 y, I I.’ A‘ ‘ I . . r I I- I, | I “r - f. L43 81' I r, x _ ‘ o\ I ‘I‘ ‘ - .-\ I‘ S S ' - 1 .’ '\ .I ' ,I ‘ v r $ \ 2, v ' O _ s ._ ‘ ~. ‘ 1 z \ . ‘ I ’\ . x.‘ = _, 9 ‘ A I ‘ 3‘ J .‘ I g n v ‘ o n. L I T I .1 N ' \ "‘ I'-" I E I — "v ‘ . I ‘1 a, ‘ no i K“ ,. Ir I I I \‘ . I ‘\ (I . , . T, T ‘ I .I I 4" V on 290 390mm“ ' 260 ' ‘OOKILOUETERS &=:__bt__, LL -? __L__L.w,-z , _w .w s, Figure 2b. Map showing the approximate geographical locations of Sigmodon leucotis and S, ochroqnathus used in this study. IO Sigmodon fulviventer minimus J. A. Allen 7. Chihuahua: Gallego, I366 m., 3 8. Durango: ll km. NNE Boquilla, I952 m., 3 Sigmodon leucotis V. Bailey I. Durango: Hda. Coyotes, 2477 m., 4 2. Guanajuato; l3 km. SW lbarra, 2592 m., 5 Sigmodon melanotis V. Bailey l. Jalisco: 2 km. NW La Barca, l525 m., 6 Sigmodon ochrognathus baileyer. A. Allen I. Texas: Davis Co., 3 km. NW Fort Davis, l6IO m., 2 2. Durango: 3 km. NE Boquilla, I952 m., 3 3. Durango: lI km. NE Boquilla, I952 m., 7 Sigmodon planifrons planifrons Nelson and Goldman I. Oaxaca: l3 km. SSW Juchatengo, I92l m., *7 2. Oaxaca: 8 km. ESE Rio Grande, 30 m., 2 B. Method of Bleeding To evaluate properly the taxonomic significance of electrophoretic studies of blood serum proteins, the effects of development and physiological variables, such as age, health, and sexual condition (Engle and Woods, I960; Petermann, I960; Moore, l959)uena recognized. To keep such effects at a minimum, all cotton rats used were acclimated to captivity, following standard laboratory procedures, for a minimum of one month. Subsequently,a specimen was bled if it was a breeding adult (non-pregnant, if female) and in good health. Bleeding was accomplished by using the orbital bleeding technique described by Riley (I960). This technique of rupturing the ophthalmic ll venous plexus was varied by slightly etherizing the specimen for better control and by using a 6-inch Pasteur pipette instead of a capillary tube for obtaining blood. The blood was allowed l5-20 minutes to clot, then inserted into an International Clinical Model Centrifuge, and spun at 3200 r.p.m. (l6l0 x g) for I5 minutes. The serum was drawn off, sealed, and frozen at -I2°C. Infrequently, serum collected for storage by this method would show some hemolysis upon thawing which in extreme cases noticeably affected the protein mobility and resolution. In several other instances, serum samples were whitish after centrifugation. This had little effect on the results when compared with other samples devoid of the whitish substance. When these samples were centrifuged at higher speeds, the white material would gather at the top of the serum, suggesting a lipid material. C. Development 2j_Procedure The ease with which pore size can be varied with polyacrylamide gels is a distinct advantage over other gel media. Wright and Mallmann (I966) developed a new procedure by which two consecutive different pore size separating gels gave better separation and resolution of certain animal serums than that of the standard 7-5% separating gel 0f Ornstein and Davis (I962). The new method was termed ”differential disc electro- phoresis.“ In a preliminary investigation of Sigmodon serum proteins, various specimens and gel alterations were tested. The electrophoretic results from employing the standard gel on a representative serum sample from each species are shown in Plate 2a. In Plate 2b, is pictured the result of a serum sample that was electrophoresed using the standard gel and various combinations of differential pore size gels. The results I2 demonstrated that the ”differential disc electrophoresis” method, using 4.75% and I0% separating gels, gave improved separation of the prealbumin and transferrin components over the standard gel. D. Reagents The latest modifications (Davis, I964) in the stock and working solutions were employed. The stock solution, however, was changed to a 4.75% and a IO% acrylamide monomer stock. The reagents are listed in Tables I and 2. E. Equipment The disk electrophoresis apparatus deveI0ped by Lawrence Besaw (per. comm.) was employed. As shown in Plate Ia, each tube has its own upper buffer reservior and current supply. This allowed for independent removal of a gel tube without disturbing the others. The lower common buffer tank holds I400 ml., the same total volume as for the eight upper buffer reserviors. The electrodes are composed of 20 gauge platinum wire. Current was supplied by a Spinco Duostat Model RD regulated D. C. power ‘supply. After the electrophoresis procedure, the acrylamide gels were inserted into a plexiglass gel holder (Plate lb) capable of simutaneously holding I4 gels, and immersed for two hours in a staining tray containing a solution of I gm. Amido Black IOB dye to each I00 ml. of 7% acetic acid. The electrolytic destaining unit (Plate lb) contained 7% acetic acid as the electrolyte and two stainless steel plates as electrodes. Powered by a I2 volt, I I/2 amp. battery charger, a normal load of 8 gels was I3 TABLE I. Stock solutions (A) (B) I N HCL 48 ml I N HCL approx. 48 mlI TRIS 36.6 gm TRIS 5.98 . TEMED 0.23 ml TEMED 0.46 ml DHOH to I00 ml (pH 8.9) DHOH to IOO ml (pH 6.” D (C) A757. 10% . ( ) Acrylamide l9 gm 40 gm Acrylamide I0.0 gm BIS 0.4 gm 0.4 gm BIS 2.5 gm DHOH to IOO ml to I00 ml DHOH to ICC ml (E) (F) Riboflavin 4 mg Sucrose 40 gm DHOH to IOO ml DHOH to IOO ml (G) Ammonium persulfate 0.I4 gm DHOH to I00 ml IpH adjusted by titrating with l N HCL TRIS tr15(hydroxymethyl)aminometbane TERMED N, N, N, N - tetramethylethylenediamine BIS N, N - methylenebisacrylamide DHOH deionize water TABLE 2. Working solutions Small-pore Small-pore Large-pore Stock buffer' solution #I solution #2 solution solution f r reservoirs l part A I part A l part B TRIS 2 parts C IO% 2 parts C 4.75% 2 parts 0 Glycine 28.8 gm I a t DHOH a DHOH I part E DHOH to l liter 3H 8.95(8.8-9.0) $158596 (8.8-9.0) 4 parts F pH 8.3 pH 6.7 (6.6-6.8) Diluted Izlo before use 'One to three runs were made before the buffer was discarded. Plate Ia. Plate lb. The electrophoresis apparatus and power supply. This apparatus allows for removal of each electrophoresis tube independently of the others. Different gel lengths may also be electrophoresed concurrently, as shown by adjusting the position of the rubber strip (rubber tubing cut in cross section) circling the upper buffer reservoir. Destaining unit, consisting of (I. to r.) battery charger, destaining tank, filter system,and oscillating pump. The destaining tank has one stainless steel electrode removed to show the plexiglass gel-holder With two gels in position. PLATE I l5 destained in 2 hours. The 7% acetic acid, as it accumulated dye from the gels, was continously circulated by a rheostat-controlled Dynalab oscillating pump (Plate lb) through a 6 cm. x 25 cm. tubular plexiglass filter system. The filter system contained from t0p to bottom: a piece of glass wool, l6 cm. of activated charcoal, a perforated plastic divider, 3 cm. of glass beads and, another piece of glass wool. At the termination of all the electrOphoretic runs, selected specimen gels were scanned with a Joyce Loebl Double-Beam Recording Densitometer, Model MK III C, to produce densitometric curves representing protein zones and the proportions of each. F. Operational Procedure Pyrex tubes, (5 mm. i.d. x l2 cm.) were etched circularily with a glass tube cutter at 2 cm., 4 cm., and 7.5 cm. distances, starting from the top (Figure 3). The marked tubes were thoroughly cleaned inside with a small test tube brush, rinsed well in tap water, followed by distilled water, and lastly, stored under a solution of I part Kodak Photo Flo to 200 parts deionized water. Just before use, the tubes were drained and dried in an oven at l05°C for IS minutes. The tubes were then inserted vertically into base supports (vaccine-bottle stoppers) and filled to the 7.5 cm. mark with the l0% acrylamide working solution. Next, the 4.75% acrylamide working Solution was carefully layered to the 4 cm..mark, layered over with 5 mm. deionized water and allowed to polymerize for 40 minutes. The latter acrylamide solution and the water layer were introduced with a bent-tipped 3.5 inch 22 gauge needle attached to a 2 ml. plastic syringe barrel topped by a squeeze bulb. After polymerization, spacer gel l6 5 mm. inside diameter 0.cm SAMPLE GEL 2 cm. SPACER GEL 4 cm. 4.75% SEPARATING GEL 7.5 cm. l0% SEPARATING GEL l2 cm. Figure 3. Diagramatic representation of the position of the gels in the differential disc electrophoretic column. monomer (large-pore working solution) was first added as a rinse, decanted, then re-added to the 2 cm. mark, layered over with 3 mm. of water, and photopolymerized for IS minutes. During this time each sample gel solution was prepared in a 5 ml. vial by mixing 8 microliters of serum from a l0 microliter syringe with 0.4 ml. of spacer gel monomer. After the spacer gel was polymerized, spacer gel monomer was added as a rinse, and decanted. Sample gel solution was then introduced on top of the spacer gel and photopolymerized for 30 minutes. Following photopolymerization, the tubes were removed from their bases and the sample gel ends were inserted into the rubber stoppers at the bottom of the empty upper buffer reserviors. The buffer solution, containing a few drops of concentrated bromophenol blue as a tracking dye, was next gently poured into each upper tank. Electrophoresis was started at 2 mA per gel tube, and continued until the bromophenol blue tracking front, approximately one hour later, reached the separating gel (near 4 cm.).* At this time the current was changed to 4 mA per tube until the tracking front, approximately 3/4 hours later, was flush with the bottom of the gel tube. At that time the current was shut off and the tube removed. Following electrophoresis, the gels were carefully removed by inserting between the gel and the glass tube wall a blunt-tipped 2-inch, 22 gauge needle mounted on a IO cc. water-filled syringe to provide constant *It should be noted that if two gels tubes are run in parallel the current required for effective separation will be double that required for one tube alone. Likewise, for 3 tubes, three times the current will be required. The voltage will be the same, irrespective of the number of tubes employed (Sargent, I964). Ilubrication. Following their extrusion from the glass tubes, the gels were stained, destained, and analyzed densitometrically as previously mentioned (p.l5). G. Controls Human blood serum was used as a control throughout the experimental work. In this way any differences or deviations in the electrophoretic technique or the stock solutions were likely to be detected. Of the eight gel tubes which could be run simultaneously, three were used for each of two Sigmodon blood serum samples and the remaining two tubes were for the human serum. Specimens from one sample or species were not usually electrophoresed at any one time, but randomly inserted throughout the electrophoretic period. From using the human blood serum control and this practice, one could reason- ably assume that any distinguishable similarities observed within a population or species were real and perhaps of taxonomic significance. Approximately one-third of all the serum samples, irrespective of the species, were rerun at the termination of the three-month electrophoretic analysis period. This served as a final check and for the purpose of resolving certain questionable observations on protein fractions. Several times during the study, Sigmodon Specimens from the laboratory colony were bled by graduate students during the absence of the investigator and the serum was given to him to see if he could determine by gel electrophoresis the species or possibly the original geographical location of the specimen. Several species were also bled again at a later date to confirm previous results. l9 H. Analysis gf_Data The densitometric tracing of a stained gel was magnified by the densitometer approximately I.5 times on mm. ruled graph paper, and the resulting tracing interpreted the protein components (bands or zones) in the gel as peaks (Figure 4). Although certain mammalian serum proteins such as prealbumins, albumins, and transferrins are commonly found in acrylamide gels (Ornstein and Davis, I962) at the relative position represented in Figure 4, no chemical tests were performed to confirm these protein identities. It was very possible (as certain electrOphoretic runs suggested) that minor protein components were over- shadowed by the presence of major components which had migrated the same distance. However, for simplicity the zone identification terminology given in Figure 4 is followed. Analysis of the densitometric tracing was made by first measuring (in mm.) the x and y coordinates for every peak, using as the origin the boundary between the spacer and the separating gels. Variance and Rm (relative mobility) values, where on the densitometric tracing R =dlstance in mm. from origin to center of a given peak distance in mm. from origin to center of the albumin pea * were calculated for those components which were considered to be of taxonomic importance. The mean relative distance, Dm, within a given number of specimens is equal to 2E(Rm2'le) where (RmZ‘RmI) is the difference n *The albumin component was used as a reference point because of its stability within the species of Sigmodon studied. 20 ..mc_c_mum uc_mm Lo E:_voE .uocoumcnmmoLo.mmc_c_mum omcouc_ .mucmn v__om .cac mo c_m.LoIm_..mc._3no_mI m_I IN. .mc_ueommcmcuu __I um .mc_E:n_mumoaIwI um .c.E:n_mI: .mc_E:n_mucaI :m _. .ocaum_ocoeoc mu. vcm _om mc_>cmaEOoom pcm mc_omcu o_cuoEou.mcov dammamflw m *0 Em.cmm_u o.umEo;om : ocsm_u . . v . 0 O . . . '. '4 O .‘ . 7‘ . O O C 0 v .0 ,0 I I V m: 2 22.122 : o; me: e m 21 between the relative mobilities of any two peaks (Rm2 and le) within a specimen; §:(Rm2-le) is the sum of the differences in a series of specimens and; n is the number of specimens involved. The height (Ymax) of a densitometric peak was assigned to a unit (unit=5 mm.) on the y-axis if it was halfway or more through the unit and still one mm. or more in width. This procedure was followed to reduce error due to background interference. Maximum height of the peaks (3.3. the albumin) was 26 units. To correlate densitometric peaks with their respective protein bands in the gels, each gel and its corresponding densitometric tracing were viewed together before any measurements were taken. This was especially necessary where 2-4 bands could be visually observed in the destained gel but appeared together in the densitometric tracing. III. RESULTS A.. Individual Variation Within §_Population Sample l. Sigmodon hispidus Twenty-four p0pulation samples of g, hispidus totaling 97 individuals were available for examination. The samples ranged in size from single specimens from each of six localities to eight specimens from each of two localities (pp.6,7) Electrophoretic results indicate that between individuals within a sample from one locality some blood serum protein components varied in mobility, number, and relative volume (as measured visually and by the height of individual peaks) while other serum components showed little variation. Minimal individual variation is observable within a sample of six specimens from 0racle,Arizona (Plate 2c, Figure 5). To demonstrate best the general relationships in the sample without losing the identity of each densitometric tracing, four of the six tracings are shown super- imposed in Figure 7a. The transferrins, the albumin and the prealbumin adjacent to the albumin (Pal) are similar in amplitude and mobility in all specimens. Other blbod serum protein components varied as follows: a. \The calculated Rm values in two specimens'for several postalbumin peaks overlap with the values calculated for closely neighboring peaks on other specimens. However, these peaks maintain similar respective positions to each other in all the densitometric tracings of this sample. b. In four specimens (the last four shown in Plate 2c, and Figure 5c,d,e,f), one or two prealbumins are present in addition to the stable prealbumin (Pal). 22 C. 23 In two specimens (the first two shown in Plate 2a and in Figure 5a,b), only one globulin, instead of two, is present. Although most samples were intermediate in variation, the extreme amount found was observed in the six specimens from Autlén, Jalisco (Plate 2d and Figure 6; with four of the densitometric tracings superimposed in Figure 7b). The prealbumin adjacent to the albumin (Pal), the albumin, and two transferrins farthest from the origin are similar in amplitude and mobility in all specimens. Other blood serum protein components varied as follows: a. It components In two specimens (Figure 6e,f), five transferrins occurred instead of four. This resulted in a lighter series of. three components nearest the origin occupied by two transferrins in the other four specimens. This was the only case in the Sigmodon studied which suggested transferrin polymorphism within a population sample. One specimen (Figure 6a) has three globulins present; the other five specimens have two globulins. The number of postalbumins varied from four to seven components. One specimen (Figure 6b) has one additional prealbumin other than Pa]. is known (Longsworth, I959) that the presence of nearby protein may influence the ionic environment. The possibility of a protein-protein interaction may also exist. In both instances this could influence the protein mobilities. However, no extreme changes in 24 Rm values occurred in this instance or others which might give evidence of the above phenomena affecting the electrOphoretic results. 2. Comments 92 the other species 9j_Sigmodon. Individual variation within a population sample in the number of prealbumins (excluding the one nearest the albumin) and globulins occurred in S, alleni, S, ochrognathus and §, planifrons. The number of globulins and also the number and position of the postalbumins and prealbumins varied in S, melanotis and §, fulviventer. B. Geographic or lntrgspecific Variation l. Sigmodon hispidus A single gel representative of S, hispidus from each of l6 population samples is shown in Plate 3a. Gels l-7 are from specimens within the United States, gels 8-15 are from México, and gel I6 is from the Canal Zone. Representative gels of cotton rats from South Carolina, Honduras and the Mexican states of Guerrero, Nuevo Leon, Sinaloa, and San Luis Potosf were not available when the photograph was taken. Geographic or intra- specific variation in g. hispidus is characterized as follows: a. Prominent in the 97 specimens of S. hispidus is the presence of a prealbumin adjacent to the albumin (Pal) where the mean Rm=l.08 and 52:.000l, mean Yma;= 5.6 units, and 5%.I.9. b. Except for the Lubbock, Texas sample, representing a subspecies which inhabits the open, arid country of the southwestern United States and Mexico's Central Plateau, all the samples from the United States were 25 characterized by having the transferrin nearest the origin widely separated from the remaining two confluent transferrins by a mean relative distance, Dm=.065. Termed transferrin pattern A, it is represented in the first six gels of Plate 3a, and in the densitometric tracings of the Oracle sample, shown separately in Figure 5, and superimposed in Figure 7a. The Mexican forms,with two exceptions, are characterized by four transferrins (pattern B), the two nearest the origin separated from each other more than the next two which are slightly confluent. The four transferrin characteristic is represented in gels 7-l5 of Plate 3a, and some of the densitometric tracings of the Autlén sample, shown separately in Figure 8b. One exception to this was in the Autlén sample, as mentioned previously. The other exception was in the Guerrero sample, which is characterized by having the transferrin nearest the origin widely separated from the remaining three confluent transferrins by a mean relative distance, Dfi=.O6O (pattern C). The Canal Zone samples are characterized by three con- fluent transferrins (pattern D), represented in gel l6 of Plate 3a. The Honduran sample is characterized by having the transferrin nearest the origin widely separated from 26 the remaining four confluent transferrins by a mean relative distance, Dm=.O70 (pattern E). The five basic transferrin patterns (A,B,C,D, and E) found in S, hispidus are regionally represented in Figure 9. Using laboratory specimens of S. hispidus, the five regional patterns (A-E) are readily discernible. To identify specimens from any one region to their respective localities is more difficult. Of the population samples studied, approxi- mately one-third could be distinguished with certainty, based on the characteristic postalbumin patterns. For instance, even though there is considerable variability in the postalbumins of the Autlén S, hispidus (Plate 2d and Figures 6,8b), the presence of a very prominent postalbumin identified this population sample from other Mexican population samples. However, specimens from the population samples of S, hispidus from Arkansas, Florida, and Oklahoma (represented in gels 2-4 of Plate 3a) are not readily distinguishable from each other. 2. Comments 22.£h£.2£h§£ species gfi Sigmodon. Since the electrophoretic results of the S, leucotis, S, ochrognathus, and S, planifrons population samples within each species were essentially identical, differences attributable to geographic or intraspecific variation were not observable. In S, fulviventer, 'the degree of variation within a sample in the pre- and postalbumins made it impossible to identify a specimen to locality. Because population samples were available from only one locality for S.,melanotis and S, alleni, the amount of geographic or intraspecific variation was not determinable. 27 C. Specific Differences in Sigmodon. With the intra— and interpopulation information obtained up to this point, the variability and stability of certain protein components in S, hispidus is acknowledged and may be applied to characterize the remaining six species as follows: I. Sigmodon ochrognathus Twelve specimens from three localities were studied. Plate 3b and Figure 7b show representative gels and densitometric tracings of three specimens from Boquilla, Durango, since the Specimens from the other localities were not available at the time the photograph was taken. This species may be identified by two confluent transferrins (eight specimens have a lesser third confluent component with the transferrin farthest from the origin), and a third near the origin separated from its neighboring transferrin with Dh=.057. A prealbumin (Pal) is present next to the albumin with a mean Rd=l.07. Four distinct postalbumins are present. 2. Sigmodon leucotis Nine specimens from two localities more than 450 km.apart were studied. Plate 3c and Figure 7a give representative gels and densitometric tracings. This species may be identified by the presence of four confluent transferrins. The components are separated in gels 3 and 4 for observational purposes. Another more conspicious characteristic of this species is the presence of two distinct prealbumins, with Rmzl.06, and l.ll, respectfully. Four distinct postalbumins are present. 28 3. Sigmodon alleni Eight specimens from one locality were studied. Plate 4a and Figure 8c give representative gels and densitometric tracings. This species may be identified by three transferrins. The one nearest the origin is separated from the others by a Dm:.035. The prealbumin nearest the albumin does not differ from that of S. hispidus. A dense component precedes the three transferrins, where Rm=.725 and the.mean Ymaxsl0.7 units. Four to five postalbumins are present. 4. Sigmodon planifrons Nine specimens from two localities were studied. Plate hc and Figure 8d give representative gels and densitometric tracings of Juchatengo specimens since the specimens from Rio Grande were not available at the time the photograph was taken. This species is very similar to S, alleni in relation to the transferrin and prealbumin components. Five to seven postalbumins are present. .5. Sigmodon fulviventer Twenty-one specimens from seven localities were studied. Plate 4b and Figure 7d give representative gels and densitometric tracings with the exception of specimens from Hda. Altotonilco, which were not available at the time the photographs were taken. This species may be distinguished by its three dense, confluent transferrins 22g a lesser component confluent with the transferrin farthest from the origin. The prealbumin nearest the albumin, when compared with S, hispidus, has a mean Yma£=2'ls cm., and a greater variance, 52:2.63; also, Rdsl.l2, and . 2 . . its variance, 5 =.002,are greater. Two to four distInct postalbumlns 29 are present. 6. Sigmodon melanotis Six specimens from one locality were studied. Plates 4d and 7c give representative gels and densitometric tracings. This species could not be distinguished from S. fulviventer. Two to three distinct post- albumins are present. The blood serum electrophoretic characteristics of the seven species of cotton rats are summarized in Table 3. 30 NJ A m Figure 5. Sketched densitometric tracings of the six Sigmodon hispidus from Oracle, Arizona to demonstrate minimal individual variation within a population sample. 3l W .. 0 l ffi va fl F‘l . v w -fi "“7 cw +— T v vvvvv Vt Figure 6. Sketched densitometric tracings of the six Sigmodon hispidus from Autlén, Jalisco, to demonstrate maximal individual variation within a population sample. ' .mucocoasou Ezcmm too—n m:o_Lm> ocu mo co_u_mon ago mc_umo_pc_ oomeE vcm twosome Lospst m_ mohooam ozu Louum_ ocu Eocm mcmomcu oco .mo_omam m c_;u_3 >u_cm._e_m mg“ oomcumcoeon op oomoaewumasm oLm nov.mmmuMHdmam aw vcm on _co__m aw mmco_um_:a0a coozuon ocm o_aEmm co_um_:a0a m c_cum3 peso» >u_cm__E_m .mLocom 0:“ oummecoEov o» voman_LoQ:m ocmndm__mw .cm_p:< Any vcm mco~_c< .o_omco Amv Sou» m:n_om_; couoamflw "mo Lump mcoE_ooam Lao» soc» mmc_omcu uhcao50u_mcoo oozouoxm .m mL:m_u 32 Plate 2a. Plate 2b. Plate 2c. Plate 2d. 3h Davis and Ornstein 7.5% standard gels of (l. to r.) Sigmodon alleni, S, ochroqnathus, S, leucotis, S. planifrons, S, melanotis, S, fulviventer, and S, hispidus. The resulting electrophoretic serum protein patterns specimen of Siqmodon hispidus, using various pore size separating gels. These are (l. to r.) Davis and Ornstein 7.5% standard gel; 7.S%-]0% gel; 10% gel; 4.75% gel; 7.5% gel; 4.75%-l0% gel. Electrophoresis was terminated in each gel when the tracking front reached the bottom of the electrophoresis tube. Electrophoretic gel patterns of six Sigmodon hispidus from the Oracle, Arizona population sample. Electrophoretic gel patterns of six Sigmodon hispidus from the Autlén, Jalisco population sample. Iii PLATE 2 . O ‘C UV V‘Utuvu .l I.)- all a .' '03-” ;- a. I I I it ‘7 3:" fit a asymfl" “Va J v. “I ‘E‘. 35 Plate 3a. Electrophoretic serum protein patterns of Sigmodon hispidus from 16 geographic localities. Gels 1-7 are from the United States, gels 8-15 are from México, gel 16 is from the Canal Zone. Plate 3b. Electrophoretic serum protein patterns of the three Sigmodon ochrognathus from Boquilla, Durango. Plate 3c. Electrophoretic serum protein patterns of Sigmodon leucotis, with (l. to r.) first four specimens from Hda. Coyotes, Durango, and last five specimens from lbarra, Guanajuato. The last two gels from Hda. Coyotes were stopped before the tracking front reached the bottom of the electrophoresis tube. Their normal patterns were like the remaining gels. PLATE 3 36 Plate ha. Electrophoretic serum protein patterns of the eight Sigmodon alleni from Capécuaro, Michoacan. Plate hb. ElectrOphoretic serum protein patterns of six Sigmodon fulviventer. From (1. to r.) Hda. Coyotes, Durango (first two gels); Boquilla, Durango; Canatlan, Durango; Gallego, Chihuahua; lbarra, Guanajuato. Plate he. Electrophoretic serum protein patterns of the seven Sigmodon planifrons from Juchatengo, Oaxaca. Plate 4d. Electrophoretic serum protein patterns of the six Sigmodon melanotis from La Barca, Jalisco. PLATE 1+ H-‘flifi I l H4"! mg. 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