Comparative Karyology of Brazilian Vampire Bats Desmodus Rotundus and Diphylla Ecaudata (Phyllostomidae, Chiroptera): Banding Patterns, Base-Specific Fluorochromes and FISH of Ribosomal Genes

Hereditas 134: 189-194 (2001)

Comparative karyology of Brazilian vampire bats Desmodus rotundus and Diphylla ecaudata (Phyllostomidae, Chiroptera): banding patterns, base-specific fluorochromes and FISH of ribosomal genes NEIDE SANTOS', VALERIA FAGUNDES2,3, YATIYO YONENAGA-YASSUDA, and MARIA JOSE DE SOUZA' Departamento de GenkticalCCB, Universidade Federal de Pernambuco, Cidade Universitaria, Recife, Pernambuco, Brazil Departamento de Cigncias BiologicaslCCHN, Universidade Federal do Espirito Santo, Vitbria, ES, Brazil Departamento de Biologia, Instituto de Biocigncias, Universidade de S6o Paulo, S6o Paulo, ,SP, Brazil Santos, N., Fagundes, V., Yonenaga-Yassuda, Y. and Souza, M. J. 2001. Comparative karyology of Brazilian vampire bats Desmodus rotundus and Diphylla ecaudata (Phyllostomidae, Chiroptera): banding patterns, base-specific fluorochromes and FISH of ribosomal genes.-Hereditas 134: 189-194. Lund, Sweden. ISSN 0018-0661. Received February 12, 2001. Accepted July 27, 2001

This paper provides new data on chromosomes of Brazilian vampire bats Desmodus rotundus and Diphylla ecaudata. These species were analyzed by GTG, CBG- and CB-DAPI banding, AgNO,/CMA, sequential staining, base-specific fluorochrome dyes and in situ hybridization with 18s rDNA probe. C-banding (CBG) revealed constitutive heterochromatin in the pericentromeric regions in all autosomes and the X and Y chromosomes appeared entirely heterochromatic in both species. CB-DAPI revealed a coincident banding pattern to that obtained by CBG. Triple staining CMA,/DA/ DAPI revealed an R-banding and a weak G-banding pattern in the karyotypes. Sequential AgNO,/CMA, staining showed a NOR located interstitially on the long arm of pair 8 in D. rotundus and on the short arm of pair 13 in D. ecaudata. FISH with a rDNA probe confirmed the location and number of NORs; a difference neither in intensity nor in size of hybridization signal was detected between homologues for both species. Neide Santos, Departamento de GeniticalCCB, Universidade Federal de Pernambuco, Cidade Universitaria, Recqe, Pernambuco, 50732-970, Brazil. E-mail: [email protected]

The New World leaf-nosed bats Phyllostomidae constitute a taxonomically diverse family and consist of approximately 46 genera and 140 species (KOOPMAN 1984). The subfamily Desmodontinae is represented by three species, Desmodus rotundus, Diphylla ecaudata and Diaemus youngi. These species are the only mammals whose diets consist solely of blood. Bats have been cytogenetically studied through Gbanding, C-banding and silver nitrate staining and are generally characterized by having a conservative rate of chromosomal evolution in which closely related species often have indistinguishable G- and C-banded karyotypes (BAKERand BICKHAM1980; S o u z and ~ ARAUJO1990). Despite the abundance of information available on the karyotype of the Phyllostomidae, D. rotundus, D. youngi and D. ecaudata, particularly, have been less investigated from the cytogenetic point of view (VARELLA-GARCIA et al. 1989). The use of fluorochromes in the comparative analysis of chromosomes has revealed differences within the chromatin and has been particular useful in characterizing different classes of heterochromatin (GC and AT-rich regions) in a wide variety of organisms, including humans, rodents, amphibians and fish

(SCHWEIZER 1980; BELLAand GOSALVEZ 1994; HERRERO et al. 1993; FONTANA et al. 1996; LISANTet al. 1996). This approach has seldom been applied to bats except for a few species of the family Vespertilionidae and Phyllostomidae (BICKHAM1987; RUEDASet al. 1990; SANTOSand S o u z ~1998a,b). Nucleolar organizer regions (NORs) are a suitable tool to study structural and functional organization of chromosomes, and they represent chromosome sites of the major rDNA genes. The NORs have usually been studied by silver nitrate and GC-specific fluorochrome dyes. More recently, fluorescent in situ hybridization (FISH) with rDNA probe, which reveaIs the presence of major rDNA sequences, has been used in many vertebrate species (BAKERet al. 1992; MAKINENet al. 1997; LOURENCO et al. 1998; MARTINSand GALETTI1998). In this paper, the chromosomes of D. rotundus and D. ecaudata were investigated using G-banding (GTG), C-banding followed by Giemsa staining (CBG) and DAPIIDA (CB-DAPI), the triple staining CMA,/DA/DAPI. The number and localization of NORs were determined by sequential staining by silver nitrate and chromomycin A, (AgNO,/CMA,), and in situ hybridization with rDNA probe. We

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compared their karyotypes in an attempt to understand the structural chromosome organization among species of vampire bats. MATERIAL AND METHODS Chromosome analyses were carried out on eight specimens of D. rotundus (6 females and 2 males), and eight specimens of D. ecaudata (6 females and 2 males). Specimens were captured in the localities of Agua Preta, S2o LourenCo da Mata (Reserva Ecologica de Tapacura) and Toritama, the state of Pernambuco, northeastern Brazil. Metaphase chromosome preparations were obtained from bone marrow cells according to conventional procedures. G- (GTG) and C-banding after Giemsa (CBG) and DAPI-DA (CBDAPI) staining were obtained using routine cytogenetic techniques, according to SEABRIGHT(1971), SUMNER(1972) and SANTOSand S o u z ~(1998a), respectively. Triple staining CMA,/DA/DAPI was performed according to SCHWEIZER (1980) with some modifications (SANTOSand S o u z ~1998a). In the sequential staining (AgNO,/CMA,), the slides were stained with silver nitrate according to HOWELLand BLACK (1980), and after photographing, the slides were destained (GUERRA1991) and stained with CMA,/ DA (SCHWEIZER 1980). In situ hybridization using a probe containing the 18s rDNA gene of Xenopus laevis was carried out according to MARTINSand GALETTI(1998) with some modifications. Briefly, slides were treated with RNAse and pepsin, denatured (70 YOformamide/2 x SSC at 70°C for 5 min), and 200 ng of biotinylated probe (50 pl) in a hybridization mixture (50 % formamide/2 x SSC, 10 YO dextran sulfate) was applied on the slides. Hybridization was performed overnight in a moist chamber at 37°C. FITC-avidin and biotinylated anti-avidin antibody (Vector) were used to detect the probe hybridization signals. After detection, slides were mounted in an antifade (Vectashield) staining solution containing propidium iodide (0.5 pl/ml) and DAPI (0.8 pl/ml). Metaphases were analyzed using a Zeiss Axiophot microscope equiped with a dual band pass filter and photographed with 400 I S 0 film (Fuji). RESULTS In the karyotypes of Desmodus rotundus (2n = 28, NF = 52) and Diphylla ecaudata (2n = 32, N F = 60) the G-banding pattern allowed the precise identification of all chromosome pairs (Fig. l a and Ib). Comparative banding analysis suggested a homeology of pairs 1, 2, and 3 between the two species. In addition,

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the chromosome 7 of D. rotundus was identifiable in the karyotype of D. ecaudata, corresponding to chromosome 8. C-banding (CBG) revealed constitutive heterochromatin in the pericentromeric regions in all autosomes and in the X chromosome of both species. D. rotundus presented small pericentric blocks (Fig. 2a) in contrast to D. ecaudata which revealed conspicuous pericentric blocks in addition to faintly stained interstitial blocks in some autosomes (Fig. 2c). In addition, the arms of the X chromospme were darker when compared with the euchromatic of the other chromosomes, and the Y appeared entirely heterochromatic for both species. CB-DAPI treated chromosomes showed a similar pattern to that obtained by CBG-technique (Fig. 2b and 2d). Triple staining CMA,/DA/DAPI revealed an Rbanding and a weak G-banding pattern in the karyotype of both species. However, when DAPI staining was applied alone, the G-banding pattern was enhanced (results not shown). The number and localization of NORs were first analyzed by silver nitrate staining. Only one interstitially located pair of NORs was observed in both species: on the long arm of pair 8 of D. rotundus (Fig. 3a) and on the short arm of chromosome 13 of D. ecaudata (Fig. 30. Hybridization with rDNA probe confirmed the location and number of NORs. The same intensity of signals was detected on the pair.8 of D. rotundus (Fig. 3b, 3c) and on the pair 13 of D. ecaudata (Fig. 3g and 3h). Sequential AgNO,/CMA, staining showed signals that were coincident on chromosome 8 in D . rotundus (Fig. 3d and 3e) and 13 in D. ecaudata (Fig. 3i and 3j), indicating that the NORs in these species are CMA, positive (GCrich). DISCUSSION The subfamily Desmodontinae is characterized by having karyotypes of 2 n = 3 2 in Diphylla ecaudata and Diuemus youngi, and 2n = 28 in Desmodus rotundus. Vampire bat chromosomes were studied using AgNO, staining, base specific fluorochromes and the constitutive heterochromatin patterns. The G-banding patterns of D. rotundus and D. youngi have been previously described, and homeology were observed in the pairs 1, 2, 3, 4 and 5 between both species (VARELLA-GARCIA et al. 1989). While these authors found homeology between some pairs of D. rotundus and D. youngi, our comparative GTG-banding analyses also show homeology in some pairs between D. rotundus and D. ecaudata. However, although BASS (1978) found at least seven unique chromosomal rearrangements that unify the three genera of vampire

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Fig. l a and b. GTG-banding pattern. a Desmodus rotundus. b Diphylla ecaudata. Bars = 10 pm.

bats (BAKERand BICKHAM1980), the GTG-banding technique did not allow us to identify the chromosomal rearrangements that should be responsible for the karyotype evolution and occurrence of two diploid numbers in the Desmodontinae. On the other hand, a high-resolution banding may allow more precise chromosome analysis and the establishment of extensive homeology in the karyotypes of these species. The constitutive heterochromatin patterns detected by CBG-banding showed a complete coincidence with CB-DAPI blocks in both species analyzed. Coincident patterns of the CBG- and CB-DAPI banding have been already reported for other species of Phyllostomidae bats, Carollia perspicillata, Artibeus lituratus, A . jamaicensis, A . cinereus and Phyllostomus discolor (SANTOS and S o u z ~1998a,b). The CBDAPI pattern, in most of these species, contrasts with those obtained with DAPI alone, where only the G-banding pattern is visualized. Interstitial blocks

detected in some autosomes of D. ecaudata (Fig. 2c) were not seen in the karyotype of D. rotundus (Fig. 2a). Our data are in contrast to those described by VARELLA-GARCIA et al. (1989) who observed interstitial blocks in some autosomes of D. rotundus. The lack of these blocks in our specimens of D. rotundus could be due to variation among populations. The differential staining in the arms of the X chromosome of D. rotundus (Fig. 2a-b) and D. ecaudata (Fig. 2c-d) by CBG and CB-DAPI was also observed in three species of Artibeus genus (SANTOS and S o u z ~1998b). This differential staining could be explained through the presence of chromatin structurally different from the pericentromeric chromatin. Chromosomes of many mammals exhibit G- and R-banding patterns and heterochromatic positive blocks after triple staining CMA,/DA/DAPI. In the karyotype of D. rotundus and D. ecaudata triple staining exhibited an R-banding pattern when CMA,

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Fig. 2a-d. C-banded metaphases from Desmodus rotundus (a-b) and Diphylla ecaudata (c-d). In a and c standard C-banding with Giemsa (CBG). In b and d CB-DAPI patterns in male metaphase. Arrows and arrowheads show X and Y chromosomes, respectively. Bars = 10 l m .

was applied and a weak G-banding was visualized after DAPI. However, a better definition of G-banding was detected when DAPI was used alone. On the other hand, neither AT nor GC constitutive heterochromatin was detected after triple staining, which has also been observed in three species of Artibeus. In contrast, triple staining in chromosomes of C. pevspicillata showed euchromatic chromosome bands (Rand G-band) and a heterochromatin heterogeneity (CMA, positive, DAPI positive, and CMAJDAPI neutral) and in Phyllostomus discolor it showed DAPI positive blocks in the pericentromeric regions of some chromosomes (SANTOS and S o u z ~1998a,b). Such

differential response to GC- and AT-specific fluorochromes, in several Phyllostomidae species, indicates an existence of variability in the composition of heterochromatin within the family. In the species analyzed here the base pairs might be so intercalated that the chemical affinity between the fluorochromes and GC- and AT-pairs is blocked. The sequential staining AgNOJCMA, applied to the karyotypes of D. rotundus and D. ecaudata exhibited only one pair of NORs that was CMA, positive, indicating a GC-rich region. Label of NORs by GCspecific fluorochromes has been also shown in Artibeus lituratus and A . jumaicensis, but NORs were

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Fig. 3a-j. Metaphases of Desmodus rotundus (a-e) and Diphylla ecaudata (f-j). AgNO, staining (a,f). Partial metaphases of in situ hybridization with 18s rDNA genes (b,g) and DAPI counterstaining (c,h). Sequential staining AgNO,/CMA, (d-e) and (i-j) of the same metaphases. Arrows show NORs sites. Bars = 10 pm.

CMA, neutral in C. perspicillata and P. discolor, indicating a differential composition of NORs between species of the family Phyllostomidae (SANTOS and S o u z ~1998a,b). Results of in situ hybridization with rDNA probe of Brazilian species (D. rotundus and D. ecaudata) are coincident with those from El Salvador (BAKERet al. 1992). Our data obtained after AgNO,, CMA, and FISH (Fig. 3) showed a perfect correspondence in the number and location of constitutive and active NORs. Neither difference in intensity nor size of hybridization signal was detected between homologues for both species. Our cytogenetic data in D. rotundus and D. ecaudata show that banding patterns are conservative in these species. VARELLA-GARCIA et al. (1989) argued

that vampire bats are monophyletic. Our data support this view. ACKNOWLEDGEMENTS We thank A. Langguth for the taxonomic identification of the specimens. Additional thanks are extended to Mrs. Francisca Tavares for her technical assistance in this work. Research supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Funda&o de Amparo a Pesquisa do Estado de SBo Paulo (FAPESP) and FundaqBo de Amparo a CiCncia e Tecnologia do Estado de Pernambuco (FACEPE).

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