Karyotypic analysis in species of the genus Dasyprocta ( Rodentia : Dasyproctidae ) found in Brazilian Amazon

A total of 30 animals of the genus Dasyprocta were cytogenetically studied. They belong to the following species:D. prymnolopha (N=20), D. leporina (N=6), D. fuliginosa (N=1) andDasyprocta sp. (N=3) (Dasyproctidae, Hystricognathi). Cell suspensions were obtained by peripheral blood culture, besides bone marrow and spleen cells, fromD. prymnolopha andD. leporina. The diploid number was 64 /65 for all samples. The karyotypes showed similarity, and chromosomal polymorphism was not detected by Giemsa conventionalstaining and G banding. The constitutive heterochromatin distribution at the pericentromeric region of all the chromosomes was similar in all species. D. prymnolopha, D. leporina andDasyprocta sp. presented variation in the heterochromatical block size at one of the homologues of the A18 pair. D. fuliginosa presented the heterochromatin uniformly distributed in all chromosomes. There was not variation in the NORs pattern in the species studied.

and Yonenaga- Yassuda 1984).Lima (1993) studied D. leporina and D. fuliginosa species through G and C banding standards and through Nucleolus Organizer Regions (NORs).Both species present well-defined constitutive heterochromatin located in the pericentromeric region of all the autosome chromosomes, with variable intensity.The X chromosome presents pericentromeric C banding and the Y chromosome of D. leporina is almost totally heterochromatic.In both species, theAg-NOR is located in the telomeric region of only one acrocentric pair.
Therefore, the presented research has the proposal of characterize karyotypes of D. leporina, D. prymnolopha, D. fuliginosa and Dasyprocta sp. in specimens from the Brazilian Amazon, using G (GTG) and C (CBG) bandings and NOR techniques Species and number of animals specified by sex, using preparations obtained with different tissues.

MATERIALS AND METHODS
Twenty specimens of D. prymnolopha, six of D. leporina, one of D. fuliginosa and three of Dasyprocta sp. were cytogenetically analyzed.These specimens were maintained at the vivarium of the Federal University of Pará (UFPA) and Museu Paraense Emílio Goeldi Zoobotanic Park.The progeny from couples kept in the vivarium of the UFPA are included in this sample.Most of these animals are from unknown origin, including those from MPEG.All the animals were subjected to peripheral blood collection: eight to bone marrow and five to spleen.Table I shows species, number and genus of animals analyzed by peripheral blood, bone marrow and spleen.To obtain metaphasic chromosomes from lymphocytes culture, the modified methodology described by Moorhead et al. (1960) was used, where the mitogenic agent utilized was the Pokeweed.To obtain chromosomes from bone marrow, the methodology described by Baker et al. (1982) with modifications was used.

Live Animals
The animals were sedated with Ketalar (Parke-Davis & Co., Aché Laboratórios Farmacêuticos S.A. Brasil) and Rompum (Bayer do Brasil S.A. Veterinária, Brasil) by intramuscular way in proportions of 0.3 and 0.1 ml/kg of body weight, respectively.The bone marrow cells collection was done in the region of femur-tíbio-patelar joint with syringe and BD (Luerlok) 30 × 12 special needle with mandril.The collected material was transferred to the centrifuge's tube that contained 5ml of medium HAM-F10, added of 20% of foetal serum and 0,025 ml of colchicine at 0.02% being incubated at 37 • C during 40-60 minutes.

Sacrificed Animals
The animals were sacrificed with an excess dosis of Ketalar and Rompum associated with ether or chloroform inhalation.Four hours before the sacrifice, a proportion of 1ml to 1000g of the body weight of colchicine at 0.02% was injected by intramuscular way.In addition to the bone marrow cells collected, the spleen was removed.To obtain metaphasic cells from spleen, the technique described by Ford and Hamerton (1956) was used.To obtain the G and C banding and NOR the methodologies described by Seabright (1971), Sumner (1972) and Howell and Black (1980) were used.The karyotypes were set up in decreasing size order in according to Hungerford and Snyder (1964) model.

RESULTS
All analyzed specimens presented a diploid number of 64/65 chromosomes (Figures 1A to 3B).This variation was observed with a frequency of about  Cienc (2003) 75 (1) 70% in cells with 2n = 64 and 30% in cells with 2n = 65, in the three different tissues: peripheral blood in all species; bone marrow and spleen in D. prymnolopha and D. leporina (Tables II, III  and IV).The lineage 2n = 65 presents a supernumerary metacentric chromosome with size ranging between medium and small (Figures 1B,2B and 3B) in D. prymnolopha, D. leporina and Dasyprocta sp., whereas in D. fuliginosa it seems to be a submetacentric (Figure 4).Through the conventional Giemsa stain, the chromosomes of the lot A (2n = 64) showed the same morphology in all studied species.The karyotypes were characterized by the following model: Group A -18 pairs of metacentric chromosomes being the A1 pair the largest metacentric and the A18 pair, the smallest, with other pairs varying in size from large to small.The A18 pair presents, in the short arm, a morphology similar to a lower degree of spiralization looking like a secondary constriction, in conventional stain (Figure 2A).Group B -nine pairs of submetacentric chromosomes, ranging from large to small.Group C -three pairs of subtelocentric ones, pairs C1 and C2 of medium size, and C3 pair with small size.

An Acad Bras
Group D -only one acrocentric pair with large size.
X Chromosome -submetacentric with large size.
Y Chromosome -small submetacentric, smaller than the B9 pair.This chromosome was observed in D. prymnolopha, D. leporina and Dasyprocta sp.; however only one female of D. fuliginosa was karyotyped.
G band pattern was similar in all analyzed samples (Figure 4).C banding technique allows the identification in all species, in the different analyzed tissues, of constitutive heterochromatin in the pericentromeric region of the most of the autosomes, including the supernumerary chromosomes (Figure 5A).
In the X chromosome, the constitutive heterochromatin was restricted to pericentromeric re-gion, whereas in the Y chromosome, was observed variation in the distribution of this chromatin.In the D. prymnolopha the constitutive heterochromatin was limited to pericentromeric region (Figure 5).In D. leporina and Dasyprocta sp., the Y chromosome was almost totally heterochromatic (Figures 5B and C).The A6 pair in D. prymnolopha, D. leporina and Dasyprocta sp.presented polymorphism in the amount of constitutive heterochromatin.One of the chromosomes showed the heterochromatic block twice the size of the other homologous of the pair (Figures 5A, B and C).In D. fuliginosa karyotype no polymorphism was observed (Figure 6).The A18 pair in D. prymnolopha and Dasyprocta sp.karyotypes showed constitutive heterochromatin in only one of the homologues (Figures 5A and C).This pair, in the D. leporina and D. fuliginosa karyotypes did not show variation (Figures 5B and 6).D. fuliginosa karyotype showed uniformity in the standard distribution of constitutive heterochromatin in all of chromosomes (Figure 6).
The NORs were observed in the telomeric region of D1 acrocentric pair short arm, in the karyotypes obtained using different tissues of all specimens studied (Figure 7).

DISCUSSION
All specimens of D. prymnolopha, D. leporina, Dasyprocta sp. and D. fuliginosa presented two cellular lineages, with 2n = 64 and 2n = 65 chromosomes.No animal showed only the basic karyotype of 2n = 64, as described in the literature (Kasahara and Yonenaga-Yassuda 1984, Lima 1993, Lima and Langguth 1995, 1998).This variation is because of the presence of one supernumerary chromosome metacentric in the D. prymnolopha, D. leporina and Dasyprocta sp. and submetacentric in D. fuliginosa.Supernumerary chromosomes are considered common among the Hystricognathi, mainly in the Chinchillidae, Octodontidae, Caviidae, Ctenomyidae, Hydrochaeridae and Echimyidae (George and Weir 1974, Kasahara and Yonenaga-Yassuda 1984, Leal-Mesquita 1991a, b, An Acad Bras Cienc (2003) Gallardo 1991, 1992, Fagundes and Yassuda 1995, Moreira et al. 1995).The presence of lineage 2n = 65, with one supernumerary chromosome in our sample, can characterize a geographic variation, since all constants species of literature were derived from different South America regions other than the Northern region.In D. prymnolopha, D. leporina and D. fuliginosa we detected a small variation in the A18 pair in relation to Dasyprocta sp.due to the fact that 100%    of the analyzed cells showed one of the homologues with a secondary constriction and about 40% from the cells of both lineages present this morphology in two homologues.However, there is a necessity of a more detailed research to confirm this characteristic.The marking character of A18 chromosome of our samples was first described here.The remaining chromosome complement agrees with Lima (1993), Lima andLangguth (1995, 1998) researches in D. leporina and D. fuliginosa.
The X chromosome of the four species here studied is submetacentric, different from the one found in D. leporina and D. fuliginosa described as large metacentric (Kasahara and Yonenaga-Yassuda 1984, Lima and Langguth 1995, 1998).The Y chro-mosome of D. prymnolopha, D. leporina and Dasyprocta sp. is a small submetacentric, similar to the one described by D. variegata (Hungerford and Snyder 1964), but different from the described small metacentric chromosome of D. leporina (Kasahara and Yonenaga-Yassuda 1984, Lima 1993, Lima and Langguth 1995, 1998).
G banding comparison between species showed several chromosomes similarities (Figure 8), suggesting that these species are conserved at the chromosome level.This finding is similar to that found in four species of Ctenomys (C.flamarioni, C. mendocinus, C. porteousi and C. australis), in relation to 1, 2, 4-8, 11-14, 16, 18, 21 and 23 pairs (Freitas 1994), and in two species of Cavia (C.aperea and  C. aperea pamparum) that present similarity in the G banding standard in most of the autosomes (Maia 1984).
In spite of the difficulties in obtaining banded trypsin-Giemsa karyotypes of D. leporina and D. fuliginosa, we presented an idiogram (Figure 9) where we suggest the interpretation of positive (dark) and negative (clear ) bands distribution like an essay of G banding standard in Dasyprocta.
In C banding analysis, it was evident the pericentromeric constitutive heterochromatin is all chro-mosomes, including the supernumerary chromosome and X andY sexual pair.This pattern is similar to the one found in other Hystricognathi as Cavia porcellus (Natarajan and Raposa 1974); Clyomys laticeps laticeps (Souza and Yonenaga-Yassuda 1982) and P. longicaudatus (Maia et al. 1988).D. leporina and Dasyprocta sp.presented the Y chromosome almost totally heterochromatic, as found in D. leporina (Lima 1993, Lima and Langguth 1995, 1998) and in some species from Ctenomys that presented Y chromosome varying from totally hete-

Fig. 5 -
Fig. 5 -Banding C (CBG) standard in the lineages 2n=64 and 2n=65.A) and C) the arrows point to one of the homologues from the pair A18, lacking the heterochromatical block (D. prymnolopha and Dasyprocta sp., respectively.);A), B) and C) one of the homologues from the pair A6 had an increasing of the heterochromatical block (D. prymnolopha, D. leporina and Dasyprocta sp., respectively); A) and C) the supernumerary chromosome with a constitutive heterochromatin in the pericentromeric region (D.prymnolopha and Dasyprocta sp., respectively).B) and C) the arrows point to the Y chromosome, almost totally heterochromatical (D. leporina and Dasyprocta sp., respectively).