Karyotypes and heterochromatin variation (C-bands) in Melipona species (Hymenoptera, Apidae, Meliponinae)

Karyotypes and heterochromatin variation (C-bands) in Melipona species (Hymenoptera, Apidae, Meliponinae)

Marla Piumbini Rocha and Silvia das Graças Pompolo

Departamento de Biologia Geral, Universidade Federal de Viçosa, 36571-000 Viçosa, MG, Brasil. Send correspondece to S.G.P.

ABSTRACT

We describe the karyotypes of eight bee species of the genus Melipona and compare them in terms of heterochromatin content and location (C-banding technique). All species had 2n = 18 (females) and n = 9 (males) chromosomes, but a wide variation in heterochromatin content was detected among karyotypes. On the basis of these differences, the species were divided into two functional groups, one of them comprising species with a karyotype having a low heterochromatin content (M. bicolor bicolor, M. quadrifasciata, M. marginata, and M. asilvai), and the other species with a high heterochromatin content (M. seminigra fuscopilosa, M. capixaba, M. scutellaris, and M. captiosa). In the species with high heterochromatin content, heterochromatin occupied practically the entire extent of all chromosomes, with euchromatin being limited to the extremities, a fact that prevented observation of the centromere. In contrast, in the species with karyotypes having a low heterochromatin content, heterochromatin was visualized only in some chromosomes. In the chromosomes in which it was present, heterochromatin was located in the centromere or on the short arm. M. bicolor bicolor had the smallest heterochromatin content with only three chromosome pairs presenting heterochromatin in females. Increased heterochromatin content may be explained by interstitial and pericentromeric growth.

INTRODUCTION

Bees of the genus Melipona present a neotropical distribution. The first cytogenetic study of the genus was conducted by Kerr (1948) who determined 2n = 18 chromosomes for the species M. rufiventris and M. marginata. Ten species out of 42 described thus far have been analyzed cytogenetically, all of them presenting n = 9, 2n = 18 chromosomes (Kerr, 1948, 1952, 1972; Kerr and Silveira, 1972; Tambasco et al., 1979; Almeida, 1981; Hoshiba, 1988; Hoshiba and Imai, 1993), except for M. quinquefasciata which presented 2n = 20 (Pompolo, 1992). Most of these studies have been based only on the determination of chromosome number. The exceptions are a study by Tambasco et al. (1979), who applied the G-banding technique to M. quadrifasciata in an attempt to differentiate each chromosome in the haploid set, a study by Hoshiba (1988) who made morphometric measurements and classified the chromosomes of M. favosa into metacentrics, submetacentrics and subtelocentrics, and a study by Hoshiba and Imai (1993) who investigated C band distribution in this species.

Heterochromatin has been extensively studied. Although its functions are still not fully understood, heterochromatin is believed to have an important influence on the genome. At the molecular level, constitutive heterochromatin is characterized as an association of non-protein-encoding highly repetitive sequences (Pardue and Hennig, 1990). Heterochromatin corresponds to the late-replicating fragments and forms the more condensed part of interphase chromatin, thus being considered inactive. There is a relation between chromatin condensation and inactivation (John, 1988). Cytogenetically, it can be detected by C banding (Sumner, 1990). Its distribution and content in chromosomes vary among different organisms, and heterochromatin is considered to play an important role in karyotype differentiation.

The objective of the present study was to describe the karyotypes of several species of the genus Melipona and to determine their relationships on the basis of heterochromatin content and location.

MATERIAL AND METHODS

Eight species of bees of the genus Melipona from different localities were analyzed cytogenetically: M. bicolor bicolor, Viçosa (MG); M. quadrifasciata, Viçosa (MG); M. marginata, Caeté (MG); M. asilvai, Pedra de Maria da Cruz (MG); M. seminigra fuscopilosa, Rio Branco (AC); M. capixaba, Venda Nova do Imigrante (ES); M. scutellaris, Lençóis (BA); M. captiosa, Rio Branco (AC).

Thirty individuals (27 females and 3 males) of each species and 10 cells per individual were analyzed on average.

Metaphase chromosomes were obtained by the technique of Imai et al. (1988). The cerebral ganglion was placed in a colchicine-citrate solution for 10 min to 2 h.

Constitutive heterochromatin was visualized by the barium/saline/Giemsa (BSG) method of Sumner (1972), with adaptations for bees. Three days after preparation, the slides were processed as follows:

- immersion in 0.2 N HCl for 6 min;

- a rapid wash in distilled water;

- 5% Ba(OH)₂ at 60^oC in a waterbath for 13 min;

- washing with 0.2 N HCl for approximately 30 s;

- a rapid wash in distilled water;

- 2 x SSC, pH 7.0, at 60^oC in a waterbath for 15 min.

After these steps, the slides were stained with Giemsa and 8% Sörensen buffer, pH 6.8.

The metaphases of best quality obtained by standard staining and by C banding were photographed and the karyotypes were mounted by pairing the chromosomes in decreasing order of size.

RESULTS AND DISCUSSION

The chromosome number of the eight species was 2n = 18 for females and, for the species in which it was possible to study males, it was n = 9 (M. bicolor bicolor, M. quadrifasciata and M. seminigra fuscopilosa). There was a wide variation in heterochromatin content between the karyotypes of the species studied, a fact that had not been reported previously.

The karyotype of M. bicolor bicolor presented metacentric and submetacentric chromosomes and one acrocentric pair (Figure 1A). Only one chromosome pair of metacentric morphology presented well-visible centromeric heterochromatin by C banding, whereas small blocks probably of pericentromeric location were observed in two other pairs (Figure 1B).

Figure 1 -
Karyotypes of M. bicolor bicolor (A, standard staining; B, C banding), M. quadrifasciata (C, standard staining; D, C banding), M. marginata (E, standard staining; F, C banding) and M. asilvai (G, standard staining; H, C banding). Bar = 5 mm.

Melipona quadrifasciata mostly presented metacentric and submetacentric chromosomes, and one acrocentric pair (Figure 1C). Four chromosome pairs presented well-visible heterochromatin blocks. In metacentric, submetacentric and acrocentric chromosomes, heterochromatin was located in the centromeric and pericentromeric region, and in the sixth pair (acrocentric) it was present on the short arm. A small deeply stained region, but not as clearly visible as in the four previous pairs, was detected in two pairs, probably around the centromere (Figure 1D). The male karyotype (n = 9) of this species was similar to that of the female (2n = 18), both in terms of chromosome morphology and heterochromatin distribution.

In M. marginata most chromosomes presented submetacentric morphology (Figure 1E). C banding was positive in six chromosome pairs, and was of pericentromeric location and more clearly visible in three of them (Figure 1F).

In M. asilvai, most chromosomes were submetacentric (Figure 1G). Heterochromatin was present in a pericentric position in the first, second, fourth and fifth pairs. The fourth pair, as well as the first, showed a large heterochromatin block. In the sixth pair, which was acrocentric, heterochromatin was located on the short arm (Figure 1H).

Standard staining permitted a clear definition of the centromeric region of each chromosome of the above species, which were similar in heterochromatin content and distribution. The first, second and fourth pairs also presented the most clearly visible heterochromatin of pericentromeric location. Another important characteristic of the group was the presence of a heterochromatin block in one of the chromosomes of the first pair, demonstrating C-banding polymorphism (Figure 1).

The karyotypes in Figure 1 are arranged in increasing order of heterochromatin content (Figure 1B,D,F,H). M. bicolor bicolor has smallest heterochromatin content (Figure 1B).

The morphology of the chromosomes of M. seminigra fuscopilosa, M. capixaba (a species recently described by Moure and Camargo, 1994), M. scutellaris and M. captiosa (Figure 2) was difficult to define with standard staining due to the high extent of chromosome condensation, which did not permit visualization of the centromere region. Heterochromatin was distributed almost throughout the chromosome, with euchromatin limited to the ends in most chromosomes (Figure 2B,D,F and G). The last chromosome pair of M. capixaba (Figure 2D) and M. captiosa (Figure 2G) had the smallest heterochromatin content. Due to the high heterochromatin content of these species, the time of colchicine treatment for obtaining metaphase chromosomes was reduced to as little as 10 min, to avoid problems with excessive chromosome condensation.

Figure 3 shows the variation in heterochromatin content and distribution in interphase nuclei and metaphase chromosomes in four Melipona species. In the species with a high heterochromatin content, an association between chromosomes was observed on the ends of the arms, which correspond to the euchromatin regions, including non-homologous chromosomes (Figure 3D, large arrow), a fact that was not observed in the species with low heterochromatin content.

Analysis of the karyotypes of the eight species of the genus Melipona illustrated in Figures 1, 2 and 3 showed wide variation in heterochromatin content among them, a fact that permitted us to separate them into two functional groups, one of them comprising species with karyotypes having a low heterochromatin content, and the other species with a high heterochromatin content. The first group includes the species M. bicolor bicolor, M. quadrifasciata, M. marginata and M. asilvai (Figure 1) and the second the species M. seminigra fuscopilosa, M. capixaba, M. scutellaris and M. captiosa (Figure 2).

The absence of C bands in some chromosomes of the species in the first group (M. bicolor bicolor, M. quadrifasciata, M. marginata and M. asilvai) may be due to the fact that small heterochromatin segments were not detected by the BSG method.

Several hypotheses have been raised to explain the function of heterochromatin in the genome of species. The hypothesis defended by Imai et al. (1986, 1988, 1994) and by Imai (1991) is that heterochromatin has the function of recovering the stability of the telomere after the mechanism of fission. Heterochromatin accumulation is often found on one of the chromosome arms in karyotypes of ants with high chromosome numbers. The same has been observed in some wasp and bee species (Hoshiba and Imai, 1993; Gomes et al., 1995; Pompolo, 1992, 1994; Pompolo and Campos, 1995). However, in the Melipona species studied here heterochromatin did not seem to have this function since its location was interstitial. The species have the same chromosome number and very wide variations in heterochromatin content were detected.

The presence of a high heterochromatin content in the Melipona species of the second group (M. seminigra fuscopilosa, M. capixaba, M. scutellaris and M. captiosa) compared to the species in the first group may be explained by heterochromatin addition at the interstitial and pericentromeric level. The species M. asilvai (Figure 1G,H) and M. favosa (Hoshiba and Imai, 1993) presented an intermediate heterochromatin content. The karyotypes of the species in the first group seem to differ in terms of heterochromatin content, but the same is not true for the species in the second group. The use of new cytogenetic techniques as well as the study of other Melipona species may better clarify the role of heterochromatin in karyotype differentiation in these species.

ACKNOWLEDGMENTS

Research supported by CNPq, CAPES and FAPEMIG. M.P.R. is the recipient of a Scientific Initiation Fellowship from CNPq-PIBIC UFV. We are grateful to Dr. L.A.O. Campos and to L.F. Gomes for criticism and suggestions, and to Prof. E.F. Morato (UFAC) for supplying some Melipona species. We are indebted to the Department of Plant Biology, UFV, for permitting the use of their photography equipment.

RESUMO

Este trabalho descreve os cariótipos de oito espécies de abelhas do gênero Melipona, relacionando-os por meio do conteúdo e localização da heterocromatina (técnica de banda C). Todas as espécies estudadas apresentaram 2n = 18 (fêmea) e n = 9 (macho) cromossomos, entretanto encontrou-se uma grande variação no conteúdo de heterocromatina entre seus cariótipos, o que permitiu separá-los em dois grupos funcionais: um reunindo espécies de cariótipo com baixo conteúdo de heterocromatina (M. bicolor bicolor, M. quadrifasciata, M. marginata e M. asilvai) e o outro com cariótipo de alto conteúdo heterocromático (M. seminigra fuscopilosa, M. capixaba, M. scutellaris e M. captiosa). Nas espécies com alto conteúdo de heterocromatina, esta ocupa quase toda a extensão de todos os cromossomos, ficando a eucromatina restrita às extremidades, o que impossibilitou a observação do centrômero. Contrariamente, nas espécies de cariótipos com baixo conteúdo, a heterocromatina foi evidenciada somente em alguns cromossomos. Naqueles em que ela está presente, sua localização foi centromérica ou foi evidenciada no braço curto. A espécie que apresentou menor conteúdo heterocromático foi M. bicolor bicolor, sendo que na fêmea apenas três pares de cromossomos apresentaram heterocromatina. O aumento no conteúdo de heterocromatina pode ser explicado pelo crescimento intersticial e pericentromérico.

REFERENCES

Almeida, M.G. (1981). Estudo sobre o número de cromossomos e contagem de espermatozóide na abelha Melipona scutellaris Latreille, 1811. Ciên. Cult. 33: 539-542.

Gomes, L.F., Pompolo, S.G. and Campos, L.A.O. (1995). Cytogenetic analysis of three species of Trypoxylon (Hymenoptera, Sphecidae, Larrinae). Rev. Bras. Genet. 18: 173-176.

Hoshiba, H. (1988). Karyological analysis of a stingless bee Melipona favosa (Apidae, Hymenoptera). Cytologia 53: 153-156.

Hoshiba, H. and Imai, H.T. (1993). Chromosome evolution of bees and wasps (Hymenoptera, Apocrita) on the basis of C-banding pattern analyses. Jpn. J. Ent. 61: 465-492.

Imai, H.T. (1991). Mutability of constitutive heterochromatin (C-bands) during eukaryotic chromosomal evolution and their cytological meaning. Jpn. J. Genet. 66: 635-661.

Imai, H.T., Maruyama T., Gojobori T., Inoue Y. and Crozier, R.H. (1986). Theoretical bases for karyotype evolution. The minimum interaction hypotheses. Am. Nat. 128: 900-920.

Imai, H.T., Taylor, R.W., Crosland, M.W.J. and Crozier, R.H. (1988). Modes of spontaneous chromosomal mutation and karyotype evolution in ants with reference to the minimum interaction hypothesis. Jpn. J. Genet. 63: 159-185.

Imai, H.T., Taylor, R.W. and Crozier, R.H. (1994). Experimental bases for the minimum interaction theory. I. Chromosome evolution in ants of the Myrmecia pilosula species complex (Hymenoptera: Formicidae: Myrmeciinae). Jpn. J. Genet. 69: 137-182.

John, B. (1988). The biology of heterochromatin. In: Heterochromatin: Molecular and Structural Aspects (Verma, R.S., ed.). Cambridge University Press, Cambridge, pp. 1-147.

Kerr, W.E. (1948). Estudos sôbre o gênero Melipona. Anais Esc. Sup. Agric. "Luiz de Queiroz" 5: 181-276.

Kerr, W.E. (1952). A variação do número de cromossomas na evolução dos Hymenoptera. Sci. Genet. 4: 182-190.

Kerr, W.E. (1972). Numbers of chromosomes in some species of bees. J. Kans. Entom. Soc. 45: 11-122

Kerr, W.E. and Silveira, Z.V. (1972). Karyotype evolution and corresponding taxonomic implications. Evolution 26: 197-202.

Moure, J.S. and Camargo, J.M.F. (1994). Melipona (Michmelia) capixaba, uma nova espécie de Meliponinae (Hymenoptera, Apidae) do sudeste do Brasil. Rev. Bras. Zool. 11: 289-296.

Pardue, M.L. and Hennig, W. (1990). Heterochromatin: Junk or collectors item? Chromosoma 100: 3-7.

Pompolo, S.G. (1992). Estudos citogenéticos em Meliponinae. Anais do Encontro Brasileiro de Biologia de Abelhas e Outros Insetos Sociais. Naturalia (número especial) 62-66. Editora UNESP, São Paulo, SP, Brasil.

Pompolo, S.G. (1994). Análise dos cariótipos de 19 gêneros de abelhas da subfamília Meliponinae. Anais do 1° Encontro sobre Abelhas, Ribeirão Preto, SP, Brasil, 1: 143-146.

Pompolo, S.G. and Campos, L.A.O. (1995). Karyotypes of two species of stingless bees, Leurotrigona muelleri and Leurotrigona pusilla (Hymenoptera, Meliponinae). Rev. Bras. Genet. 18: 181-184.

Sumner, A.T. (1972). A simple technique for demonstrating centromeric heterochromatin. Exp. Cell. Res. 75: 304-306.

Sumner, A.T. (1990). Chromosome Banding. Unwin Hyman Ltd., London, UK.

Tambasco, A.J., Giannoni, M.A. and Moreira, L.M.A. (1979). Analyses of G-bands in chromosomes of the Melipona quadrifasciata anthidioides. Lepeletier (Hymenoptera, Apidae, Meliponinae). Cytologia 44: 21-27.

(Received February 17, 1997)

• Almeida, M.G. (1981). Estudo sobre o número de cromossomos e contagem de espermatozóide na abelha Melipona scutellaris Latreille, 1811. Cięn. Cult. 33: 539-542.
• Gomes, L.F., Pompolo, S.G. and Campos, L.A.O. (1995). Cytogenetic analysis of three species of Trypoxylon (Hymenoptera, Sphecidae, Larrinae). Rev. Bras. Genet. 18: 173-176.
• Hoshiba, H. (1988). Karyological analysis of a stingless bee Melipona favosa (Apidae, Hymenoptera). Cytologia 53: 153-156.
• Hoshiba, H. and Imai, H.T. (1993). Chromosome evolution of bees and wasps (Hymenoptera, Apocrita) on the basis of C-banding pattern analyses. Jpn. J. Ent. 61: 465-492.
• Imai, H.T. (1991). Mutability of constitutive heterochromatin (C-bands) during eukaryotic chromosomal evolution and their cytological meaning. Jpn. J. Genet. 66: 635-661.
• Imai, H.T., Maruyama T., Gojobori T., Inoue Y. and Crozier, R.H. (1986). Theoretical bases for karyotype evolution. The minimum interaction hypotheses. Am. Nat. 128: 900-920.
• Imai, H.T., Taylor, R.W. and Crozier, R.H. (1994). Experimental bases for the minimum interaction theory. I. Chromosome evolution in ants of the Myrmecia pilosula species complex (Hymenoptera: Formicidae: Myrmeciinae). Jpn. J. Genet. 69: 137-182.
• Kerr, W.E. (1948). Estudos sôbre o gęnero Melipona. Anais Esc. Sup. Agric. "Luiz de Queiroz" 5: 181-276.
• Kerr, W.E. (1952). A variaçăo do número de cromossomas na evoluçăo dos Hymenoptera. Sci. Genet. 4: 182-190.
• Kerr, W.E. (1972). Numbers of chromosomes in some species of bees. J. Kans. Entom. Soc. 45: 11-122
• Kerr, W.E. and Silveira, Z.V. (1972). Karyotype evolution and corresponding taxonomic implications. Evolution 26: 197-202.
• Moure, J.S. and Camargo, J.M.F. (1994). Melipona (Michmelia) capixaba, uma nova espécie de Meliponinae (Hymenoptera, Apidae) do sudeste do Brasil. Rev. Bras. Zool. 11: 289-296.
• Pardue, M.L. and Hennig, W. (1990). Heterochromatin: Junk or collectors item? Chromosoma 100: 3-7.
• Pompolo, S.G. (1992). Estudos citogenéticos em Meliponinae. Anais do Encontro Brasileiro de Biologia de Abelhas e Outros Insetos Sociais Naturalia (número especial) 62-66. Editora UNESP, Săo Paulo, SP, Brasil.
• Pompolo, S.G. (1994). Análise dos cariótipos de 19 gęneros de abelhas da subfamília Meliponinae. Anais do 1° Encontro sobre Abelhas, Ribeirăo Preto, SP, Brasil, 1: 143-146.
• Pompolo, S.G. and Campos, L.A.O. (1995). Karyotypes of two species of stingless bees, Leurotrigona muelleri and Leurotrigona pusilla (Hymenoptera, Meliponinae). Rev. Bras. Genet. 18: 181-184.
• Sumner, A.T. (1972). A simple technique for demonstrating centromeric heterochromatin. Exp. Cell. Res. 75: 304-306.
• Sumner, A.T. (1990). Chromosome Banding Unwin Hyman Ltd., London, UK.
• Tambasco, A.J., Giannoni, M.A. and Moreira, L.M.A. (1979). Analyses of G-bands in chromosomes of the Melipona quadrifasciata anthidioides Lepeletier (Hymenoptera, Apidae, Meliponinae). Cytologia 44: 21-27.

Figure 2 - Karyotypes of M. seminigra fuscopilosa (A, standard staining; B, C banding), M. capixaba (C, standard staining; D, C banding), M. scutellaris (E, standard staining; F, C banding) and M. captiosa (G, C banding). Bar = 5 mm.

Figure 3 - C banding of interphase nuclei (small arrow) and metaphase chromosomes in M. bicolor bicolor (A), M. marginata (B), M. quadrifasciata (C) and M. seminigra fuscopilosa (D). The larger arrow corresponds to the association between euchromatins. Bar = 5 mm

Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  Mar 1998

History

• Received
  17 Feb 1997