Identification of a long-standing colony of Proechimys at the Instituto Evandro Chagas, Pará, Brazil, based on cytogenetic information

Identificação de uma colonia de longo prazo de Proechimys no Instituto Evandro Chagas, Pará, Brasil, com base em informações citogenéticas

Identificación de una colonia de largo plazo de Proechimys en el Instituto Evandro Chagas, Pará, Brasil, con base en informaciones citogenéticas

Nelson Antonio Bailão Ribeiro^I; Julio Cesar Pieczarka^II; Manoel do Carmo Pereira Soares^III; Cleusa Yoshiko Nagamachi^II

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ABSTRACT

The taxonomic classification of the genus Proechimys is complex because many of its species are morphologically similar but chromosomally different, with diploid (2n) values ranging from 14 to 62. The "Seção de Criação e Produção de Animais de Laboratório do Instituto Evandro Chagas" (The Division for Breeding and Production of Laboratory Animals, Instituto Evandro Chagas, Brazil) maintains a Proechimys colony for biomedical research. The colony members have been classified as P. guyannensis, which reportedly has 2n=40 and a fundamental number (FN) = 54. However, using karyotype analysis to aid in their taxonomic classification, we instead observed that a sample of the animals in this colony have 2n = 30 and FN = 56, with a medium-sized submetacentric X chromosome and a small acrocentric Y chromosome. Constitutive heterochromatin was distributed as follows: in the pericentromeric regions of chromosomes 6, 7, 9, 10, 11, 12, 13, 14 and X; on the distal short arms of chromosomes 3, 6, 10 and X; on the distal long arm of chromosome 12; on the long arm of the Y chromosome; and distally on both arms of chromosomes 7, 9 and 11. The nucleolar organizer regions (NORs) are located on the long arm of chromosome 9. This karyotype is consistent with that described previously for P. roberti, but not P. guyannensis, thus demonstrating the importance of using karyotyping for the taxonomic identification of Proechimys.

RESUMO

A classificação taxonómica do gênero Proechimys é complexa porque muitas de suas espécies são morfologicamente semelhantes, porém diferentes cromossomicamente, com números de diploide (2n) que variam entre 14 e 62. A Seção de Criação e Produção de Animais de Laboratório do Instituto Evandro Chagas mantém uma colónia de Proechimys para pesquisa biomédica. Os membros da colónia foram classificados como P. guyannensis, que possui 2n = 40 e um número fundamental (NF) = 54. No entanto, ao utilizar a análise do cariótipo para auxiliar em sua classificação taxonómica, observamos que uma amostra dos animais desta colónia possuem 2n = 30 e NF = 56, com um cromossomo X submetacêntrico de tamanho médio e um cromossomo Y acrocêntrico pequeno. A heterocromatina constitutiva foi distribuída da seguinte forma: nas regiões pericentroméricas dos cromossomos 6, 7, 9, 10, 11, 12, 13, 14 e X; na porção distal dos braços curtos dos cromossomos 3, 6, 10 e X; na porção distal do braço longo do cromossomo 12; no braço longo do cromossomo Y; e nas porções distais de ambos os braços dos cromossomos 7, 9 e 11. As regiões organizadoras nucléolos (NORs) localizam-se no braço longo do cromossomo 9. Este cariótipo é consistente com o descrito anteriormente para P. roberti, não para P. guyannensis, o que demonstra a importância do uso de cariotipagem para a identificação taxonómica de Proechimys.

Palavras chave: Cromossomos; Proechimys; Roedores; Cariotipagem.

RESUMEN

La clasificación taxonómica del género Proechimys es compleja porque muchas de sus especies son morfológicamente semejantes, aunque diferentes cromosómicamente, con números de diploide (2n) que varían entre 14 y 62. La Sección de Cría y Producción de Animales de Laboratorio del Instituto Evandro Chagas mantiene una colonia de Proechimys para investigación biomédica. Los miembros de la colonia fueron clasificados como P. guyannensis, que posee 2n = 40 y un número fundamental (NF) = 54. Sin embargo, al utilizar el análisis del cariotipo para auxiliar en su clasificación taxonómica, observamos que una muestra de los animales de esta colonia tenía 2n = 30 y NF = 56, con un cromosoma X submetacéntrico de tamaño mediano y un cromosoma Y acrocéntrico pequeño. La heterocromatina constitutiva fue distribuida de la siguiente forma: en las regiones pericentromérica de los cromosomas 6, 7, 9, 10, 11, 12, 13, 14 y X; en la porción distal de los brazos cortos de los cromosomas 3, 6, 10 y X; en la porción distal del brazo largo del cromosoma 12; en el brazo largo del cromosoma Y; y en las porciones distales de ambos brazos de los cromosomas 7, 9 y 11. Las regiones organizadoras nucleolares (NORs)se localizan en el brazo largo del cromosoma 9. Este cariotipo es consistente con lo descrito anteriormente para P. roberti, no para P. guyannensis, lo que demuestra la importancia del uso de cariotipado para la identificación taxonómica de Proechimys.

Palabras clave: Cromossomos; Proechimys; Roedores; Cariotipificación.

INTRODUCTION

The number of Proechimys species identified in the literature ranges across different authors and papers: Tate⁸ recognized 46 species, Gardner and Emmons¹ identified 32, Ellerman⁹ reported 21, Moojen⁵ found 15, and Cabrera¹⁰ recognized only 12. Patton^11' used bacular and skull data to define 59 species of Proechimys organized into nine groups:

three monotypic groups (decumanus, canicollis, and simonsi) and six polytypic groups (semispinosus, longicaudatus, goeldii, cuvieri, trinitatus and guyannensis). According to this classification, the guyannensis group was composed of the species P. guyannensis, P. cherriei, P. roberti, P. vacilator, P. oris, P. warreni, P. boimensis, P. arescens, P. riparum, and P. arabupu.

These species range from the coastal lands of the Guyanas to the Rio Negro basin and the eastern half of the Amazon basin. Some populations are also found in Goiás and Minas Gerais in Brazil. From this study, the geographic distribution of many species of this genus was defined, such as P. guyannensis, which was found in the left banks of the Amazon River, and P. roberti, which was found in the right banks. This was confirmed by other authors^2,12,15,16. However, despite this extensive classification, the number of species in each group remains unclear. More recently, Emmons and Feer¹² reported that there are probably no more than 20 or 30 species of Proechimys, whereas Nowak² found 32 species. Clearly, additional lines of evidence, such as skull structure and karyotype, must also be analyzed concurrently to devise a more precise strategy for identifying the species within Proechimys¹¹.

Based on studies of the sequence of the mitochondrial gene Cytochrome b, Lara et al¹³ suggested elevating Trinomys, a former subgenus of Proechimys, to the genus level. Leite and Patton¹⁴ analyzed the sequences of Cytochrome b, as well as the 12S and 16S genes, which provided support for the proposal of Lara et al¹³. These data show the potential of molecular studies to elucidate the taxonomy and phylogenetic relationships of this group of rodents.

As Proechimys has huge karyotypic variability, with a diploid (2n) number ranging from 14¹⁷ to 62¹⁸, cytogenetic analysis appears to be particularly well-suited for resolving the taxonomic problems of this genus. Weksler et al¹⁹ used cytogenetic data and other information to evaluate the taxonomic status of the species P. roberti Thomas, 1901 and P. oris Thomas, 1904, and concluded that P. oris is actually a junior synonym of P. roberti. The authors combined their data with that of previous reports to compile a table of the 48 karyotypes described for Proechimys (Table 1), organized according to the species groups proposed by Patton¹¹. The karyotypic characterization of P. roberti was later confirmed by Machado et al²⁰, using samples obtained from Piauí, Tocantins, and Mato Grosso States in Brazil.

MATERIAL AND METHODS

The karyotypes of four animals (two males and two females) of Proechimys belonging to the SACPA-IEC colony were studied. The chromosomes were obtained by direct extraction from bone marrow³³ and analyzed by conventional staining using Giemsa, G-banding³⁴, C-banding³⁵ and Ag-NOR staining³⁶. The metaphase chromosomes were photographed under Carl Zeiss III and Axiophot Zeiss microscopes, and karyotypes were mounted based on chromosome morphology, in order of decreasing size. This work was performed in accordance with the Brazilian national ethic rules (Law no. 11.794/08; Arouca Law).

RESULTS

The four animals were found to have 2n = 30 , fundamental number (FN) = 56. The autosomes comprised 13 pairs of biarmed chromosomes, ranging in size from large to small, and one pair of subtelocentrics. The X chromosome was found to be a medium-sized submetacentric chromosome, whereas the Y was a small acrocentric chromosome (Figure 2A). The determination of the chromosome pairs was made by G-banding pattern analysis (Figure 2B).

C-banding (Figure 3A) revealed the presence of small blocks of pericentromeric constitutive heterochromatin (CH) in nine autosome pairs (6, 7, 9, 10, 11, 12, 13, 14, and X). CH was also found on the distal short arm of chromosomes 3, 6, 10, and X, on the distal long arm of chromosome 12, on the long arm of the Y chromosome, and distally on both arms of chromosomes 7, 9, and 11.

The nucleolar organizer regions (NORs) were located in the interstitial region of the long arm of chromosome 9 (Figure 3B).

When the rodent colonies were initially established at the Instituto Evandro Chagas, the Proechimys were classified as P. guyannensis. The published cytogenetic data on this species show karyotypic formulas of 2n=40, FN = 56⁷ for a sample from Balta (Peru), and 2n=40, FN = 54²² for samples from Cayenne and Saul (French Guyana). In sharp contrast, we found that the sample from the SACPA-IEC colony had 2n = 30, FN = 56. Comparison of our data (karyotypic formula, G- and C-banding, and Ag-NOR staining patterns) with those found in the literature for Proechimys shows that our cytogenetic results match those described by Weksler et al¹⁹ and Machado et al²⁰ for P. roberti. Additionally, these animals were originally collected in the southwestern region of the Para state. This further suggests that the tested samples must belong to the P. roberti species because previous studies^2,12,15,16 demonstrated that P. roberti is found only at the right banks of the Amazon River, where there are no records of P. guyannensis.

Based on these results and those of other studies^19,20, the karyotype of the species P. roberti does not have numerical variation, with all tested samples having 2n = 30. However, there are morphological differences involving chromosome 14, the last autosome pair. Populations can be found with a homomorphic, acrocentric pair (FN = 54), a heteromorphic pair with one acrocentric chromosome and a bi-armed chromosome (FN = 54-56), and a homomorphic pair with two bi-armed chromosomes (FN = 56). As previously defined^19,20, such chromosomal differences are typical in populations with a defined geographic distribution. This makes them useful to identify populations with biogeographical and phylogenetic similarities.

Weksler et al¹⁹ analyzed samples from Goiás and from Pará, Maranhão and Tocantins in the Amazonian region. In the north of Tocantins and Maranhão, they found karyotypes with FN = 54, in which both copies of chromosome 14 were acrocentric. In the other localities, the entire sample had FN = 56, with both copies of chromosome 14 being bi-armed. This is in agreement with the data of Gardner and Emmons¹ and Leal-Mesquita²¹ for samples collected in Curuá-Una, Pará State, and with the data of Barros¹⁷ for samples of the Transamazonia Road in Pará. The Proechimys colony from SACPA - IEC originated from the Barros collection¹⁷. Thus, our data for this colony are in agreement with Barros. Apart from the homomorphic states of the chromosome 14 pair (acrocentric or bi-armed), Weksler et al¹⁹ identified some karyotypes with FN = 56 and FN = 54-56 in the same locality, meaning the chromosome 14 pair is heteromorphic. According to the authors, this is a consequence of a pericentric inversion in one of the homologues. These karyotypic patterns (NF = 56 and NF =54-56) were found in populations of the Cavalcante Farm, northeastern Goiás, and Primavera Farm, northeastern Pará. Machado et al²⁰ also analyzed karyotypes of P. roberti from six regions of three Brazilian states where they found karyotypes with FN = 56 and bi-armed chromosome 14 homologues: the Ecological Station at Uruçuí-Una, Piauí; Parana and Peixe, Tocantins; Cláudia, Gaúcha do Norte and Vila Rica, Mato Grosso.

From the geographic and karyotypic analyses of P. roberti, the existence of two populations can be noted: a western population, in Pará, north of Tocantins, Goiás and Mato Grosso, with FN = 56 and a homomorphic, bi-armed chromosome 14 homolog pair; and an eastern population, located in Maranhão, south of Tocantins, with FN = 54 and an acrocentric chromosome 14 homolog pair. However, Weksler et al¹⁹ found karyotypes with FN = 54-56 (with heteromorphic copies of chromosome 14) in populations from the Cavalcante farm, northeastern Goiás, and Primavera Farm, northeastern Pará. However, it is possible that these are hybrid populations, resulting from the mix of the homomorphic groups (one-armed in the east and bi-armed in the west), as the heteromorphic populations are observed in the contact region of both the homomorphic types (northeast of Para and Goiás). This region may thus constitute a potential hybridization zone.

CONCLUSION

This mistake in classification is very understandable given all the problems related to classifying members of genus Proechimys, such as the relative lack of morphological variability among species and the intrapopulational variations in some traits typically used for mammal systematics. At the time of the colony's initial classification four decades ago, taxonomic identification was made exclusively using morphology.

Since many biomedical studies deal with the species-specific relationships among vertebrate reservoirs and pathogenic species (viruses, bacteria, fungi, protozoa, helminthes, etc.) that have been established over thousands of years of co-evolution, with different vertebrate species having different reactions to pathogens, it is very important that model animals be correctly characterized. Due to the morphological similarities among species of Proechimys, these rodents should be accurately identified using techniques beyond morphological examination. Here, we show that cytogenetic studies, in conjunction with other data, are very useful tools for the precise definition of members of Proechimys.

By comparing the cytogenetic data of the sample of P. roberti from SACPA-IEC with published samples, we concluded that the SACPA-IEC sample belongs to the largest populational group of this species, with FN = 56 and a geographical distribution ranging from Pará to Mato Grosso. This suggests that the sample shares a similar evolutionary origin with that group and therefore must have the same genetic and biological properties that allow its distribution and survival in a forest environment characterized by a particular complement of pathogenic species. The SACPA-IEC P. roberti samples may thus represent an ideal model system in which to study species-specific relationships with pathogens.

ACKNOWLEDGMENTS

We thank Dr. Regina Barros (UFPa) for her expertise in the field of cytogenetics, Doctor Reinaldo Carvalho (chairman of the SACPA-IEC at the time of this study) for permission to collect samples, the Instituto Evandro Chagas and Universidade Federal do Pará for the use of laboratory facilities, and CNPq and CAPES for financial support.

FINANCIAL SUPPORT

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Universidade Federal do Pará, Instituto Evandro Chagas.

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Correspondência /Correspondence / Correspondencia:
Nelson Antonio Bailão Ribeiro
Rua dos Pariquís
Alameda Gonçalo Duarte n^o 56 ]]> Bairro: Jurunas
CEP:66030-040
Belém-Pará-Brasil
E-mail:nelsonribeiro@iec.pa.gov.br

Recebido em: 9/9/2010
Aprovado em: 4/5/2011