Electrophoretic properties of neotropical bat hemoglobin

Comp. Biochem. PhysioL, 1969, Vol. 30, pp. 117 to 122. Pergamon Press. Printed in Great Britain

ELECTROPHORETIC PROPERTIES OF NEOTROPICAL BAT HEMOGLOBIN* DARIO VALDIVIESO, J. R. T A M S I T T t and ENCARNITA CONDE-DEL PINO General Clinical Research Center, School of Medicine, San Juan, Puerto Rico 00905 (Received 9 December 1968)

Abstract--1. A single hemoglobin was found in five species of phyllostomatid and one species of molossid bats, and a double hemoglobin was found in the vespertilionid bat Eptesicus fuscus. 2. The phyllostomatid bats Monophyllus redmani, Artibeus jamaicensis, Stenoderma rufum and Erophylla bombifrons had indistinguishable hemoglobin morphs. 3. Chilonycteris parnellii differed from other phyllostomatid bats by having a slightly slower migrating hemoglobin. 4. The slow, almost isoelectric, hemoglobin of the molossid bat Molossus molossus differentiated it from phyllostomatid and vespertilionid bats. 5. Electxophoretic properties of neotropical bat hemoglobin do not distinguish closely related taxa but may be useful to confirm estimates of relationships derived from traditional criteria. INTRODUCTION BIOCHEMICAL characteristics have the same limitations as morphological ones in systematic studies and may be of value in the study of one group but not in another (Mayr, 1963; Dessauer, 1966). Electrophoresis of hemoglobin has been useful to detect hybrids in fish (Manwell, 1963) and in toads (Guttman, 1967) and to distinguish morphologically similar species of echinoderms (Manwell, 1966), fish (Barrett et al., 1966), amphibians, reptiles (Dessauer et al., 1957; Bertini & Rathe, 1962; Gorman & Dessauer, 1965), and rodents (Foreman, 1960, 1968). On the other hand, certain related species of birds cannot be identified on the basis of hemoglobin (Baker & Hanson, 1965), and mammal hemoglobin in general has shown limited use in taxonomy (Van Ros & Van Sande, 1965). Hemoglobins of only a few species of bats have been studied and only minor differences have been found (Manwell & Kerst, 1966; Mitchell, 1966). As only temperate species of bats have been studied thus far, we describe here the results * Research supported by the National Research Council of Canada, the Royal Ontario Museum and N.I.H. Clinical Research Center Branch Grant FR 63-06. Presented at the IVth Latin American Congress of Zoology, Caracas, Venezuela, November, 1968. t Present address: Department of Mammalogy, Royal Ontario Museum, Toronto 5, Ontario, Canada. 117

I 18

DARIOVALDIVIESO,J. R. TAMSITTANDENCARNITACONDE-DEL PINO

of hemoglobin e]ectrophoresis of neotropical Puerto Rican bats of the families Phyllostomatidae, Vespertilionidae and Molossidae. MATERIALS AND METHODS Hemoglobin samples were obtained in May 1968 from forty bats--one Parnell's mustached bat, Chilonycteris parnellii portoricensis Miller; six Jamaican long-tongued bats, Monophyllus redmani portoricensis (Miller); eight Jamaican fruit-eating bats, Artibeus jamaicensis jamaicensis Leach; twelve brown flower bats, Erophylla bombifrons bombifrons (Miller) and two Wetmore's big brown bats, Eptesicusfuscus wetmorei Jackson, from Aguas Buenas; three red fig-eating bats, Stenoderma rufurn darioi Hall & Tamsitt, from the Luquillo Experimental Forest; and eight mastiff bats, Molossus molossus fords (Miller), from Isla Verde Airport, San Juan, Puerto Rico. Bats were collected in "mist" nets, and identifications were made by the senior authors. Blood was obtained by heart puncture with sterile syringes rinsed with heparin, transferred to 0"5 ml tubes and centrifuged at 3000 rev/min for 20 min. Plasma was separated from the packed cells, and the buffy coat was removed by aspiration with a micropipette. The hemolysate was prepared by adding 2 vol. of distilled water to the packed cells. After thorough mixing, the solution was frozen and thawed three times. Immediately after the final thawing, the hemolysates were centrifuge at 4000 rev/min for 5 min, and the supernatant was used for analysis. Electrophoresis was carried out in the Model R-101 Microzone Cell (Beckman Instrument Co., Fullerton, California) with Sepraphore III polyacetate membranes (Gelman Instrument Co., Ann Arbor, Michigan) and barbital buffer pH 8"6, ionic strength 0"075. With this method eight hemolysates could be compared simultaneously, and results were comparable to those obtained with lengthy gel methods. The sample (0"25/xl) was applied to the membrane with a Beckman applicator for 20 sec, and separation was accomplished at 250 V for 20 min. Human hemoglobin A, C and S were run simultaneously with bat hemoglobin as controls. Immediately after electrophoresis hemoglobins were visualized by staining with Ponceau S. RESULTS Examination of the hemolysates at p H 8.6 showed hemoglobin to consist of two electrophoretic bands in E. fuscus (Fig. 1A). For convenience the hemoglobins of the big brown bat are referred to as H b - E F 1 and Hb-EF2, in order of decreasing mobility. A single component was found in C. parnellii (Hb-CP), M. redmani ( H b - M R ) , A. jamaicensis (Hb-AJ), E. bombifrons (Hb-EB), S. rufum (Hb-SR) and M. molossus ( H b - M M ) (Figs. 1 and 2). T h e human controls are designated Hb-A, H b - C and H b - S . H b - A migrated faster than H b - C , and H b - S at a rate between H b - A and H b - C but only slightly faster than the latter. Hb-EF~, the slowest component of E.fuscus hemoglobin, migrated at a rate appreciably slower than Hb-C, whereas the faster component, H b - E F 1, had a mobility isoelectric with H b - S (Fig. 1A). At p H 8.6 ManweU & Kerst (1966) found a slowly moving and a more rapidly moving anodal zone in hemoglobin of the big brown bat (E. f. fuscus) and the little brown bat (Myotis lucifugus lucifugus) from Illinois, whereas at the same pH, Mitchell (1966) found the hemoglobins to be homogeneous in Missouri representatives of the same taxa. T h e s e discrepancies in results are difficult to understand. T h e populations of E. f. fuscus and M. l. lucifugus studied by Mitchell were only approximately 350

FIG. 1. Cellulose acetate electrophoresis of bat erythrocyte hemolysates at pH 8.6. The arrow marks the origin. From left to right the samples are: A, Artibeus jamaicensis, Monophyllus redmani, Erophylla bombifrons, Eptesicus fuscus (a and b), human CA, Chilonycteris parnellii, E. bombifrons and E. fuscus (a and b) ; B, human CA, E. bombifrons, Stenoderma rufum, A. jamaicensis, M. redmani and C. parnellii.

B

C 3 +

D 1

2345678

FIG. 2. Cellulose acetate electrophoresis of bat erythrocyte hemolysates The arrow marks the origin. A. Monophyllus redmani: l-3, males; 4, 5, human SA; 6-8, females. S. Artibeus jamaicensis: 14, females; 5, 6-8, males. C. Erophylla bombifrons: 1-3, males; 4, human SA; 5, 6-8, females. D. Mo/ossu.~ molossus: 1-3, females; 4, human SA; 5, 6-8. males.

at pH human human human human

X.6. CA; CA; CA; CA;

ELECTROPHORETIC PROPERTIES OF NEOTROPICAL BAT HEMOGLOBIN

119

and 475 miles from the population studied by Manwell & Kerst, and although hemoglobin differences could be the result of selection on isolated populations, differences in electrophoretic technique might be a more feasible explanation. Mitchell used Sepraphore III and may have overlooked the minor component revealed by the starch-gel technique used by Manwell & Kerst. In E. f. wetmorei we found the anodal component to be only slightly less in concentration than the cathodal component (Fig. 1A), whereas in E. f. fuscus Manwell & Kerst found the fastest component to be only a trace when compared to the major slower moving component. Hb-MR, Hb-AJ, Hb-SR and Hb-EB were isoelectric and consisted of a single major anodal zone migrating at a rate equal to Hb-S and Hb-EF 1 (Fig. 1A, B). Hb-CP migrated at a rate slightly slower than Hb-MR, Hb-AJ, Hb-SR and Hb-EB and at a point intermediate between Hb-C and Hb-S (Fig. 1B). The slowest moving homogeneous hemoglobin was Hb-MM, with a mobility corresponding to Hb-EF2 but considerably behind Hb-C (Fig. 2D). We found no sexual, individual or intraspecific differences, and hemoglobin polymorphism seems to be absent in these bats (Figs. 2 A-D). DISCUSSION Three hemoglobin electrophoretic patterns were seen in Puerto Rican bats (Fig. 3). One pattern, found in Chilonycteris, Monophyllus, Artibeus, Stenoderma and Erophylla (family Phyllostomatidae), consisted of a single rapid modal band. Indistinguishable hemoglobin morphs were seen in Monophyllus (subfamily Glossophaginae), Erophylla (subfamily Phyllonycterinae), Artibeus and Stenoderma (subfamily Stenoderminae). Although the Glossophaginae and Phyllonycterinae are nectar, soft fruit pulp or juice feeders, and the Stenoderminae are fruit pulp crushers, identical hemoglobins in bats of these subfamilies reinforce Miller's (1907) view that these taxa represent natural assemblages of closely related genera and species. A variant of the phyllostomatid pattern was seen in Chilonycteris (subfamily Chilonycterinae), which was distinguished by its slightly slower hemoglobin. Miller (1907) considered Chilonycteris and related genera sufficiently distinct in morphology from other phyllostomatids to merit subfamilial status. After a study of ectoparasite-host relationships, Machado-Allison (1967), on the other hand, suggested that the chilonycterines might form a distinct family. The pulses of their orientation sounds resemble those of sac-winged bats (family Emballonuridae) more closely than those of other phyllostomatids (Griffin, 1958; Novick, 1963), and they differ also from other subfamilies of the family in hemoglobin as well as karyotype characteristics (Baker, 1967). These differences indicate that the evolutionary relationship of the Chilonycterinae, a small group of insectivorous species systematically associated with a large number of nectofrugivorous bats, needs a reappraisal. A second pattern was found in Eptesicus (family Vespertilionidae), in which the hemoglobin was heterogeneous and consisted of a slower major and a slightly

120

DARIO VALDIVIESO,J. R. TAMSITTAND ENCARNITACONDE-DEL PINO

faster minor zone. This pattern has also been found by Manwell & Kerst (1966) in the North American vespertilionids E. fuscus, Myotis lucifugus and Pipistrellus subflavus and by Mitchell (1966) in Plecotus townsendii.

HUMAN

AS

FAMILY PHYLLOSTOMATIDAE SUBFAMILY CHILONYCTERINAE

Chilonycteris par nellii SUBFAMILY GLOSSOPHAGINAE

Monophyllus redmani SUBFAMILY STENODERMINAE

Artibeus jamaicensis

stenoderma rufum SUBFAMILY PHYLLONYCTERINAE Erophylla bombifrons FAMILY VESPERTILIONIDAE

II I I I I I I I i

Eptesicus fuscus FAMILY MOLOSSIDAE Molossus molossus

o FIG. 3. Cellulose acetate electrophoresis of bat erythrocyte hemolysates at pH 8.6. The circle represents the origin.

A third pattern, an almost isoelectric, homogeneous hemoglobin, was evident in Molossus (family Molossidae). Although the molossid bat Tadarida brasiliensis has a similar hemoglobin (Johnson & Wicks, 1959), hemoglobin of other species needs study before it can be concluded that the pattern is a familial characteristic. Bats of this family show the greatest modification of the forelimb for flight, and on morphological considerations, are considered to have evolved from vespertilionid bats (Winge, 1941). Characteristics of the serum proteins do not differentiate the Molossidae from the Vespertilionidae (Johnson & Wicks, 1959), but hemoglobin and karyotype differences (Wainberg, 1966; Baker & Patton, 1967) support Miller's (1907) interpretation that these two groups of insectivorous bats are sufficiently distinct to merit familial status.

ELECTROPHORETIC PROPERTIES OF NEOTROPICAL BAT H E M O G L O B I N

121

In their evolution phyllostomatid and molossid bats have retained a single form of hemoglobin, whereas vespertilionid bats have evolved two different molecules (Fig. 3). Although multiple hemoglobins occur frequently in mammals (Gratzer & Allison, 1960), the reason for their existence is not clear, and a physiological significance is not yet associated with them (Riggs, 1965). A gain or loss of hemoglobin polypeptide chains is a relatively simple evolutionary event, and it is well known that multiple hemoglobins of closely related taxa may have more than one basis (Ingrain, 1963). Selection pressure applied to other genes linked to the hemoglobin or genetic drift as a result of isolation, could account for the difference between a single or double hemoglobin. Whether single or multiple hemoglobins are related to selective pressures acting on bat populations is a question of considerable interest, but to date, data are not yet sufficiently complete to even approach this question. Most mammals are polymorphic for hemoglobin (Lush, 1966; Tenth European Conference on Animal Blood Groups and Biochemical Polymorphisms, 1966), and the absence of polymorphism in bat hemoglobin is unusual. A single non-varying hemoglobin occurred in the phyllostomatid and molossid bats for which series were available, and homogeneity may be a specific character rather than sampling of homozygotes for a hemoglobin polymorphism. Although not revealing the definitive relationship among closely related species, genera and, in some instances, subfamilies, bat hemoglobins are constant throughout the greatest range of variation of other protein and morphological characteristics and do not reflect labile genetic characters which are found in mammals showing polymorphism. Certainly this constancy should be helpful in grouping higher taxonomic categories and could be useful to interpret the broad evolutionary affinities of bats. Acknowledgements--We wish to thank Dr. A. A. Cintr6n-Rivera, Director, General Clinical Research Center, School of Medicine, San Juan, for provision of laboratory facilities; Miss Alma Annexy and Mrs. Luisa Lindsey for laboratory assistance; the Puerto Rico Nuclear Center for co-operation and the use of facilities at the E1 Verde field station; and Dr. R. L. Peterson, Dr. J. C. Barlow and Mrs. Diana Young for reading the manuscript. The illustrations were prepared by Mrs. Sophie Poray, Mr. Lee Warren and Mr. Alan McColl, Royal Ontario Museum, Toronto. REFERENCES BAKERC. M. A. & H~gSONH. C. (1965) Molecular genetics of avian proteins--VI. Evolutionary implications of blood proteins of eleven species of geese. Comp. Biochem. Physiol. 17, 997-1006. BAKERR. J. (1967) Karyotypes of bats of the family Phyllostomidae and their taxonomic implications. Southwest. Nat. 12, 407--428. BAKERR. J. & PATTONJ. L. (1967) Karyotypes and karyotypic variation of North American vespertilionid bats. J. Mammal. 48, 270--286. BAaagrr I., JOSEPH J. & MOSER G. (1966) Electrophoretic analysis of hemoglobins of California rockfish (genus Sebastodes). Copeia 1966, 489-494. BEaTXNIF. & RaTrm G. (1962) Electrophoretic analysis of hemoglobin of various species of anurans. Copela 1962, 181-185.

122

DARIO VALDIVIESO,J. R. TAMSITT AND ENCARNITACONDE-DEL PINO

DESSAUERH. C. (1966) Taxonomic significance of electrophoretic patterns of animal Hera. Serol. Mus. Bull. 34, 4-8. DESSAUER H. C., Fox W. & RAMIREZJ. R. (1957) Preliminary attempt to correlate paperelectrophoresis migration of hemoglobins with phylogeny in Amphibia and Reptilia. Archs Bioehem. Biophys. 71, 11-16. FOREMAN C. W. (1960) Electromigration properties of mammalian hemoglobins as taxonomic criteria. Am. Midl. Nat. 64, 177-186. FOREMAN C. W. (1968) Hemoglobin ionographic properties of Peromyscus and other mammals. Comp. Biochem. Physiol. 25, 727-731. GORMAN G. C. & DESSAUERH. C. (1965) Hemoglobin and transferrin electrophoresis and relationships of island populations of anolis lizards. Science 150, 1454-1455. GRATZER W. B. & ALLISONA. C. (1960) Multiple hemoglobins. Biol. Rev. 35, 459-506. GRIFFIN D. R. (1958) Listening in the Dark. Yale University Press, New Haven. GUTTMAN S. I. (1967) Transferrin and hemoglobin polymorphism, hybridization and introgression in two African toads, Bufo regularis and Bufo rangeri. Comp. Biochem. Physiol. 23, 871-877. INGRAM V. M. (1963) The Hemoglobins in Genetics and Evolution. Columbia University Press, New York. JOHNSON M. L. & WICKS M. J. (1959) Serum protein electrophoresis in mammals--taxonomic implications. Syst. Zool. 8, 88-95. LUSH I. E. (1966) The Biochemical Genetics of Vertebrates except Man. North-Holland Publishing, Amsterdam. MACHADO-ALLISONC. E. (1967) The systematic position of Desmodus and Chilonycteris, based on host-parasite relationships (Mammalia; Chiroptera). Proc. Biol. Soc. Wash. 80, 223-226. MANWELL C. (1963) The blood proteins of cyclostomes; a study in phylogenetic and ontogenetic biochemistry. In The Biology of Myxine (Edited by BRODALA. & FANGE R.), pp. 372-455. Universitetsforlaget, OHio. MANWELL C. (1966) Sea cucumber sibling species: polypeptide chain types and oxygen equilibrium of hemoglobin. Science 152, 1393-1395. MANWELL C. & KERST K. V. (1966) Possibilities of biochemical taxonomy of bats using hemoglobins, lactate dehydrogenase, esterases and other proteins. Cutup. Biochem. Physiol. 17, 741-754. MAYR E. (1963) Animal Species and Evolution. Bellknap Press, Cambridge, Mass. MILLER G. S. (1907) The families and genera of bats. U.S. natn. Mus. Bull. 57, 1-282. MITCHELL H. A. (1966) Multiple hemoglobins in bats. Nature, Lond. 201, 1067-1068. NOVlCK A. (1963) Orientation in neotropical bats--II. Phyllostomatidae and Desmodontidae. J. Mammal. 44, 44-56. R i o t s A. (1965) Functional properties of hemoglobin. Physiol. Rev. 45, 619-673. Tenth European Conference on Animal Blood Groups and Biochemical Polymorphisms (1966) Polymorphismes Biochimiques des Animaux. Institut National de la Recherche Agronomique, Paris, France. VAN Ros G. & VAN SANDE M. (1965) La comparaison des hemoglobines animales par electrophorese de zone en milieux g61ifi6s et son interet en systematique zoologique. Bull. Soc. Roy. Zool. D'Anvers 35, 19-54. WAINBERG R. L. (1966) Cytotaxonomy of South-American chiroptera. Arch. Biol. 77, 411--423. WINGE H. (1941) The Interrelationships of the Mammalian Genera, Vol. I: Monotremata, Marsupialia, Insectivora, Chiroptera, Edentata. Reitzels, Copenhagen. Key Word Index--Haemoglobin; bats; electrophoresis; Molossus molossus; Eptesicus fuseus ; Monophyllus redmani ; Artibeus jamaicensis ; Stenoderma rufum ; Erophylla bombifrons; Chilonycterus parnellii.