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The first cytogenetic characterization of the poisonous black widow spider Latrodectus gr. curacaviensis from Brazil, with chromosomal review of the family Theridiidae (Arachnida, Araneae)
Micron 41 (2010) 165–168
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Short communication
The first cytogenetic characterization of the poisonous black widow spider Latrodectus gr. curacaviensis from Brazil, with chromosomal review of the family Theridiidae (Arachnida, Araneae) Douglas Araujo a,*, Ulysses Madureira Maia b, Antonio Domingos Brescovit c a Universidade Estadual de Mato Grosso do Sul – UEMS, Unidade Universita´ria de Mundo Novo, BR 163, Km 20.2, Bairro Universita´rio, CEP 79980-000, Mundo Novo, Mato Grosso do Sul, Brazil b Universidade do Estado do Rio Grande do Norte – UERN, Faculdade de Cieˆncias Exatas e Naturais – FANAT, Departamento de Cieˆncias Biolo´gicas, DECB, Campus Universita´rio Central, BR 110, Km 48, Rua Professor Antoˆnio Campos, s/n, Bairro Costa e Silva, CEP 59610-090, Mossoro´, Rio Grande do Norte, Brazil c Instituto Butantan, Laborato´rio de Artro´podes, Av. Vital Brazil, 1500, CEP 05503-900, Sa˜o Paulo, Sa˜o Paulo, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 July 2009 Received in revised form 3 October 2009 Accepted 3 October 2009
In this paper we present, for the first time, cytogenetical data on Latrodectus gr. curacaviensis (Theridiidae) from Brazil, as well as the first data on meiosis and sex chromosome system of this genus. Testes were submitted to colchicine, hypotonic, and fixation treatment, and chromosomal preparations were stained with Giemsa solution. The analysis showed 2n = 26 telo/acrocentric chromosomes in spermatogonial metaphases. Metaphase I exhibited 12 autosomal bivalents and two sex chromosome univalents (12II + X1X2). All bivalents revealed one terminal chiasma. Metaphases II confirmed the sex chromosome system, showing 12 autosomes or 12 autosomes plus two X chromosomes, respectively. Male karyotype prevailing in theridiids is formed by 2n = 22 chromosomes, including sex chromosome system X1X2 in all species. The Latrodectus species of the geometricus clade analyzed until now showed smaller diploid number (2n, = 16 and 2n, = 18) than the species of the mactans clade (2n, = 24 and 2n, = 26). Thus, according to the chromosome number, the examined Latrodectus species seems to be related to the mactans clade. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Latrodectinae Chromosome Meiosis Sex chromosome system Mitosis
1. Introduction Theridiidae (comb-footed spiders) is the fifth most species-rich family of spiders, including 2295 species and 109 genera (Platnick, 2009). From a cytogenetical point of view, only 29 species, belonging to 13 genera, were studied to date, revealing a high homogeneity of the karyotype. In 23 species, male karyotype was formed by 22 telo/acrocentric chromosomes, including an X1X2 sex chromosome system (SCS) (Table 1). Among the theridiids there are the widow spiders of the genus Latrodectus Walckenaer, 1805 (Latrodectinae), composed of 30 species (Platnick, 2009), with a worldwide distribution and notorious importance in medicine because of their neurotoxic venom (Garb et al., 2004). Interestingly, other theridiids potently venomous to vertebrates are those of the genus Steatoda Sundevall, 1833, another member of the subfamily Latrodectinae (Maretic et al., 1964; Diaz, 2004). Latrodectus is an exception to the
* Corresponding author. Tel.: +55 6739233187; fax: +55 6739233181. E-mail addresses: [email protected], [email protected] (D. Araujo), [email protected] (U.M. Maia), [email protected] (A.D. Brescovit). 0968-4328/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2009.10.002
chromosomal constancy observed among theridiids, with diploid number ranging from 2n = 16 to 2n = 28 in females of the four species analyzed so far (Table 1). According to Garb et al. (2004), there is great difficulty to distinguish between species of the genus Latrodectus based on morphological characters. Due to the high heterogeneity of diploid numbers within the genus, cytotaxonomy seems to be a promising approach to distinguish between Latrodectus species. In the present paper, a Latrodectus species from Brazil is chromosomally characterized, providing the first data on meiosis and SCS in the genus Latrodectus. In addition, a comparison between the cytogenetical and the cladistic data for the Latrodectus genus will be presented. 2. Materials and methods Five adult males and three adult females of Latrodectus gr. curacaviensis (Fig. 1) were collected manually, next to the roots of a carnauba wax palm, Copernicia prunifera, in the city of Ac¸u (58320 2300 S/368570 4500 W), state of Rio Grande do Norte, Brazil, in October of 2007. In contrast to females, gonads of males presented dividing cells suitable for analysis. Dissected specimens were
D. Araujo et al. / Micron 41 (2010) 165–168
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Table 1 List of species of the family Theridiidae studied cytogenetically so far. Valid names extracted from Platnick (2009); cited as = species name used in the original paper; 2n = male diploid number (without parentheses), or female diploid number (between parentheses); SCS = sex chromosome system. S = supernumerary chromosome; T = telocentric; A = acrocentric. Valid name
Cited as
2n
SCS
Chromosome morphology
Collection site
Bibliography
Argyrodinae Argyrodes cyrtophorae Tikader, 1963 Argyrodes gazedes Tikader, 1970 Argyrodes gazingensis Tikader, 1970 Argyrodes sp. Argyrodes sp. Ariamnes cylindrogaster Simon, 1889 A. cylindrogaster Simon, 1889
Argyrodes cyrtophore – – – – – –
22 22 24 22 22 22 22
X1X2 X1X2 X1X2 X1X2 X1X2 X1X2 X1X2
– – – – – – 20A + X1X2A
India India India India India – Japan
Datta and Chatterjee Datta and Chatterjee Datta and Chatterjee Datta and Chatterjee Datta and Chatterjee Suzuki (1950) Suzuki (1954)
– – –
22 (18) (28)
X1X2 – –
20A + X1X2A – –
Finland South Africa South Africa
Suzuki (1954) Martindale (1980) Martindale (1980)
– – –– – – –
(24) (26) (16) 22 22 22
– – – X1X2 X1X2 X1X2
– – – 20A + X1X2A 20A + X1X2A –
Australia USA South Africa Finland Russia USA
Martindale (1980) Martindale (1980) Martindale (1980) Hackman (1948) Gorlov et al. (1995) Tugmon et al. (1990)
Enoplognatha japonica Theridium ovatum
22 22
X1X2 X1X2
20A/T + X1X2A/T –
Japan –
Kageyama and Seto (1979) Sokolov (1960)
– Chrysso venusta Theridion japonicum
22 24 22
X1X2 X1X2 X1X2
– 22A/T + X1X2A/T 20A/T + X1X2A/T
India Japan Japan
Datta and Chatterjee (1983) Kageyama and Seto (1979) Kageyama and Seto (1979)
Theridium tepidariorum
24
–
–
USA
Montgomery (1907)
Theridium tepidariorum Theridium tepidariorum Theridium tepidariorum Theridion tepidariorum Achaearanea tepidariorum Achaearanea tepidariorum Achaearanea tepidariorum Theridium sterninotatum
22 22 22 22 22 22 22 22
X1X2 X1X2 X1X2 X1X2 X1X2 X1X2 X1X2 X1X2
20A + X1X2A 20A + X1X2A 20A + X1X2A 20A + X1X2A 20A/T + X1X2A/T 20A/T + X1X2A/T – 20A + X1X2A
Finland Japan – – Japan China Taiwan Japan
Hackman (1948) Suzuki (1954) Diaz and Saez (1966) Igarashi and Kondo (1977) Kageyama and Seto (1979) Youju et al. (1995) Chen (1999) Suzuki (1954)
Theridion chikunii Theridion latifolium –
22 22 22
X1X2 X1X2 X1X2
20A/T + X1X2A/T 20A/T + X1X2A/T –
Japan Japan India
Kageyama and Seto (1979) Kageyama and Seto (1979) Srivastava and Shukla (1986)
Enoplognatha foliicola
22
X1X2
20A + X1X2A
Japan
Suzuki (1954)
– – – – – – –
22 22 22 22 22 22 22 + 1S
X1X2 X1X2 X1X2 X1X2 X1X2 X1X2 X1X2
20A/T + X1X2A/T 20A + X1X2A – 20A + X1X2A 20A + X1X2A 20A + X1X2A 20A + X1X2A
Japan Ecuador Ecuador Ecuador Ecuador Ecuador Ecuador
Kageyama and Seto (1979) Avile´s and Maddison (1991) Avile´s et al. (2000) Avile´s and Maddison (1991) Avile´s and Maddison (1991) Avile´s and Maddison (1991) Avile´s and Maddison (1991)
Latrodectinae Crustulina guttata (Wider, 1834) Latrodectus geometricus C.L. Koch, 1841 Latrodectus indistinctus O.P.-Cambridge, 1904 Latrodectus mactans (Fabricius, 1775) Latrodectus mactans (Fabricius, 1775) Latrodectus rhodesiensis Mackay, 1972 Steatoda bipunctata (Linnaeus, 1758) Steatoda grossa (C.L. Koch, 1838) Steatoda triangulosa (Walckenaer, 1802) Pholcommatinae Enoplognatha caricis (Fickert, 1876) Enoplognatha ovata (Clerck, 1757) Theridiinae Achaearanea budana Tikader, 1970 Chrysso scintillans (Thorell, 1895) Parasteatoda japonica (Bo¨senberg & Strand, 1906)a Parasteatoda tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a Platnickina sterninotata (Bo¨senberg & Strand, 1906)b Takayus chikunii (Yaginuma, 1960) Takayus latifolius (Yaginuma, 1960) Theridion sp. Remaining theridiids Anelosimus crassipes (Bo¨senberg & Strand, 1906) A. crassipes (Bo¨senberg & Strand, 1906) Anelosimus domingo Levi, 1963 A. domingo Levi, 1963 Anelosimus eximius (Keyserling, 1884) Anelosimus jucundus (O.P.-Cambridge, 1896) Anelosimus studiosus (Hentz, 1850) A. studiosus (Hentz, 1850)
(1983) (1983) (1983) (1983) (1983)
a The species Parasteatoda japonica and Parasteatoda tepidariorum were included in Theridiinae because they were transferred from Achaearanea Strand, 1929, a theridiine genus according the phylogenetic analyses of Agnarsson (2004) and Arnedo et al. (2004), only recently by Yoshida (2008) and Saaristo (2006), respectively. b The genus Platnickina was included in Theridiinae because it was transferred from Keijia Yoshida, 2001, a theridiine genus according to the phylogenetic analyses of Agnarsson (2004) and Arnedo et al. (2004), only recently by Koc¸ak and Kemal (2008).
deposited in the collection of the Laborato´rio de Artro´podes, Instituto Butantan, city of Sa˜o Paulo, state of Sa˜o Paulo, Brazil (IBSP 91671, 91672, 91673, 91674, 91675). Gonads were dissected in physiologic solution for insects (7.5 g NaCl, 2.38 g Na2HPO4, 2.72 g KH2PO4, in 1 L of distilled water), colchicine solution (0.16% in physiologic solution for insects) was added to the material and left for 2 h. A volume of hypotonic solution (tap water) equal to that of colchicine solution was added. After 15 min of hypotonization, the gonads were placed in methanol:acetic acid fixative (3: 1) for 60 min. Pieces of tissue were
dissociated in a drop of 60% acetic acid on the surface of microscope slides. The chromosome preparations were dried on a metal heating plate (from 35 8C to 40 8C), stained with a 3% Giemsa solution (47 mL distilled water, 1.5 mL phosphate buffer, pH 6.8, 1.5 mL Giemsa) for 15 min, and rinsed briefly with distilled water. Approximately 50 cells of Latrodectus gr. curacaviensis were recorded using a Motic1 light microscope. Morphology of chromosomes was classified according to Levan et al. (1964). Karyotype was constructed in Corel Draw1 12 image editing software.
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Fig. 1. Male Latrodectus sp. from Rio Grande do Norte, Brazil. Scale bar = 1 mm. Fig. 3. Male metaphase I of Latrodectus sp., showing 12II + X1X2. The arrows indicate terminal chiasma. Scale bar = 10 mm.
3. Results The analysis of spermatogonial metaphases revealed 26 telo/ acrocentric chromosomes in males of Latrodectus gr. curacaviensis. In spermatogonial metaphases, the sex chromosomes did not show any different characteristics. No heteropycnotic regions were noticed in any chromosome (Fig. 2). Diplotene nuclei and metaphase I plates showed 12 autosomal bivalents. Each bivalent contained only one terminal chiasma. Two small univalents, placed always close to each other, corresponding to the sex chromosomes X1 and X2 (Fig. 3). These results indicated that male karyotype of Latrodectus gr. curacaviensis is composed of 24 autosomes and two sex chromosomes, X1 and X2. Size of sex chromosome univalents suggests that the X1 and X2 chromosomes are among the smallest ones of the complement. Metaphases II showed n = 12 or n = 14 chromosomes (Fig. 4). Difference of number reflected segregation of the X chromosomes to the same pole during anaphase I. 4. Discussion The only work on Latrodectus cytogenetics is the abstract of Martindale (1980), which is based only on female specimens and contains no data on chromosomal morphology, sex chromosome system, and meiosis (Table 1). The present work describes for the first time the chromosome morphology, course of meiosis, and SCS of the genus Latrodectus. According to a phylogenetic analysis (Garb et al., 2004), the genus Latrodectus is divided in two clades. The geometricus clade is composed only by two species, Latrodectus geometricus and Latrodectus rhodesiensis. The mactans clade contains much more species, namely Latrodectus antheratus (Badcock, 1932); Latrodec-
tus bishopi Kaston, 1938; Latrodectus corallinus Abalos, 1980; Latrodectus diaguita Carcavallo, 1960; Latrodectus hasselti Thorell, 1870; Latrodectus hesperus Chamberlin & Ivie, 1935; Latrodectus katipo Powell, 1871; Latrodectus mactans; Latrodectus menavodi Vinson, 1863; Latrodectus mirabilis (Holmberg, 1876); Latrodectus pallidus O. P.-Cambridge, 1872; Latrodectus renivulvatus Dahl, 1902; Latrodectus revivensis Shulov, 1948; Latrodectus tredecimguttatus (Rossi, 1790); Latrodectus variegatus Nicolet, 1849; Latrodectus variolus Walckenaer, 1837; Latrodectus sp. from Brazil (Bahia); Latrodectus sp. from Chile. Considering the diploid numbers, two cytotaxonomic groups of Latrodectus can be distinguished: (1) a group in which the diploid number remains the same or increases in relation to the other latrodectines (Crustulina and Steatoda, 2n< = 22), composed by L. mactans (2n, = 26 or 2n, = 24), Latrodectus indistinctus (2n, = 28) (Martindale, 1980) and L. gr. curacaviensis (2n< = 26) (present work) and (2) a group in which the diploid number decreases in relation to the other latrodectines, composed by L. geometricus (2n, = 18) and L. rhodesiensis (2n, = 16) (Martindale, 1980). Unfortunately, Latrodectus curacaviensis (Mu¨ller, 1776) was not included in the work of Garb et al. (2004), but McCrone and Levi (1964) proposed that this species, based on the genital structures, could form a group with L. variolus and L. bishopi, species belonging to the mactans clade in the hypothesis of Garb et al. (2004). This grouping is reinforced by our cytogenetic analysis, revealing Latrodectus gr. curacaviensis as a probable member of the mactans clade and not as part of a distinctive group as some researchers proposed in the past (McCrone and Levi, 1964; Pinter, 1967; Meier and White, 1995), whereas the two species of the geometricus clade presented lower diploid numbers (2n = 18 and 2n = 16), reinforcing
Fig. 2. Male karyotype of Latrodectus sp. with 2n = 26 telo/acrocrocentric chromosomes. Scale bar = 10 mm.
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Fig. 4. Male metaphase II cells of Latrodectus sp. with n = 12 (A) and n = 14 = 12 + X1X2 (B). Scale bar = 10 mm.
the taxonomical and evolutionary proximity recovered by Lotz (1994) and Garb et al. (2004), respectively. It is interesting to note that the other genera belonging to the subfamily Latrodectinae (Agnarsson, 2004; Arnedo et al., 2004), namely Crustulina Menge, 1868 and Steatoda, presented 2n< = 22, i.e. the same diploid number showed by the majority of the theridiids. Thus, in spite of the three genera of latrodectines being closely related according to morphological (Agnarsson, 2004) and molecular data (Arnedo et al., 2004), only the genus Latrodectus revealed a high interspecific variation of the diploid number. It is important to emphasize that, in contrast to Theridiidae, in which 2n< = 22 prevails (Table 1), all other families of Araneoidea karyotyped to date (Araneidae, Linyphiidae, Nephilidae, Nesticidae and Tetragnathidae), possess predominantly species with 2n< = 24. Considering the theridiid subfamilies recognized by Agnarsson (2004) and Arnedo et al. (2004), only Hadrotarsinae and Spintharinae were not chromosomally analyzed until now, and further cytogenetic analyses on species belonging to these clades are a priority to verify or not the occurrence of the 2n< = 22 in all theridiid subfamilies. Acknowledgements The authors are grateful for the critical reading of two anonymous reviewers and especially to Jirˇı´ Kra´l (Charles University, Prague), and to Cristina A. Rheims for reviewing the English language. Financial support was provided by Conselho Nacional de Desenvolvimento Cientı´fico e tecnolo´gico (CNPq), and Fundac¸a˜o de Apoio a` Pesquisa do Estado do Rio Grande do Norte (FAPERN). References Agnarsson, I., 2004. Morphological phylogeny of cobweb spiders and their relatives (Araneae, Araneoidea, Theridiidae). Zool. J. Linn. Soc. 141, 447–626. Arnedo, M.A., Coddington, J., Agnarsson, I., Gillespie, R.G., 2004. From a comb to a tree: phylogenetic relationships of the comb-footed spiders (Araneae, Theridiidae) inferred from nuclear and mitochondrial genes. Mol. Phylogenet. Evol. 31, 225–245. Avile´s, L., Maddison, W., 1991. When is the sex ratio biased in social spiders? Chromosome studies of embryos and male meiosis in Anelosimus species (Araneae, Theridiidae). J. Arachnol. 19, 126–135. Avile´s, L., McCormack, J., Cutter, A., Bukowski, T., 2000. Precise, highly female-biased sex ratios in a social spider. Proc. R. Soc. Lond. B: Biol. Sci. 267, 1445–1449. Chen, S.H., 1999. Cytological studies on six species of spiders from Taiwan (Araneae: Theridiidae, Psechridae, Uloboridae, Oxyopidae, and Ctenidae). Zool. Stud. 38, 423–434.
Datta, S.N., Chatterjee, K., 1983. Chromosome number and sex-determining system in fifty-two species of spiders from North-East India. Chrom. Inf. Serv. 35, 6–8. Diaz, J.H., 2004. The global epidemiology, syndromic classification, management, and prevention of spider bites. Am. J. Trop. Med. Hyg. 71, 239–250. Diaz, M.O., Saez, F.A., 1966. Karyotypes of South-American Araneida. Mem. Inst. Butantan 33, 153–154. Garb, J.E., Gonza´lez, A., Gillespie, R.G., 2004. The black widow spider genus Latrodectus (Araneae: Theridiidae): phylogeny, biogeography, and invasion history. Mol. Phylogenet. Evol. 31, 1127–1142. Gorlov, I.P., Gorlova, O.Y.U., Logunov, D.V., 1995. Cytogenetic studies on Siberian spiders. Hereditas 122, 211–220. Hackman, W., 1948. Chromosomenstudien an Araneen mit besonderer Neru¨cksichtigung der Geschlechtschromosomen. Acta Zool. Fenn. 54, 1–101. Igarashi, H., Kondo, A., 1977. The chromosome observation technique for spiders. Acta Arachnol. 27, 157–166. Kageyama, A., Seto, T., 1979. Chromosomes of seven species of Japanese theridiid spiders. Chrom. Inf. Serv. 27, 10–12. ¨ ., Kemal, M., 2008. New synonyms and replacement names in the genus Koc¸ak, A.O group taxa of Araneida. Cent. Ent. Stud., Misc. Pap. 139–140, 1–4. Levan, A., Fredga, K., Sandberg, A.A., 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52, 201–220. Lotz, L.N., 1994. Revision of the genus Latrodectus (Araneae: Theridiidae) in Africa. Navorsing van die Nasionale Museum, Bloemfontein 10, 1–60. Maretic, Z., Levi, H.W., Levi, L.R., 1964. The theridiid spider Steatoda paykulliana, poisonous to mammals. Toxicon 2, 149–154. Martindale, C., 1980. Studies on Latrodectus. In: Proceedings of the Entomological Congress of the Entomological Society of Southern Africa, vol. 3. pp. 45–46. McCrone, J.D., Levi, H.W., 1964. North American spiders of the Latrodectus curacaviensis group. Psyche 71, 12–27. Meier, J., White, J., 1995. Handbook of Clinical Toxicology of Animal Venoms and Poisons. CRC Press, Florida. Montgomery, T.H., 1907. On the maturation mitoses and fertilization of the egg of Theridium. Zool. Jahrb. Abt. Anat. Ontogenie Tiere 25, 237–250. Pinter, L.J., 1967. Species of widow spiders in Northern Argentina (Latrodectus: Theridiidae). Psyche 74, 290–298. Platnick, N.I., 2009. The World Spider Catalog, Version 10.0. American Museum of Natural History (online at http://research.amnh.org/entomology/spiders/ catalog/index.html). Saaristo, M.I., 2006. Theridiid or cobweb spiders of the granitic Seychelles islands (Araneae, Theridiidae). Phelsuma 14, 49–89. Sokolov, I.I., 1960. Studies on nuclear structures in spiders (Araneina). I. Karyological peculiarites in spermatogenesis. Vopr. Tsitol. Protistol. 160–186. Srivastava, M.D.L., Shukla, S., 1986. Chromosome number and sex-determining mechanism in forty-seven species of Indian spiders. Chrom. Inf. Serv. 41, 23–26. Suzuki, S., 1950. Sex determination and karyotypes in spiders. Zool. Mag. 59, 31–32. Suzuki, S., 1954. Cytological studies in spiders. III. Studies on the chromosomes of fifty-seven species of spiders belonging to seventeen families, with general considerations on chromosomal evolution. J. Sci. Hiroshima Univ., Ser. B, Div. 1. Zool. 15, 23–136. Tugmon, C.R., Brown, J.D., Horner, N.V., 1990. Karyotypes of seventeen USA spiders species (Araneae, Araneidae, Gnaphosidae, Loxoscelidae, Lycosidae, Oxyopidae, Philodromidae, Salticidae and Theridiidae). J. Arachnol. 18, 41–48. Yoshida, H., 2008. A revision of the genus Achaearanea (Araneae: Theridiidae). Acta Arachnol. 57, 37–40. Youju, W., Xiuzhen, W., Sujuan, C., Zhenling, Y., 1995. On chromosomes of the Achaearanea tepidariorum. Acta Arachnol. Sinica 4, 37–40.
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Short communication
The first cytogenetic characterization of the poisonous black widow spider Latrodectus gr. curacaviensis from Brazil, with chromosomal review of the family Theridiidae (Arachnida, Araneae) Douglas Araujo a,*, Ulysses Madureira Maia b, Antonio Domingos Brescovit c a Universidade Estadual de Mato Grosso do Sul – UEMS, Unidade Universita´ria de Mundo Novo, BR 163, Km 20.2, Bairro Universita´rio, CEP 79980-000, Mundo Novo, Mato Grosso do Sul, Brazil b Universidade do Estado do Rio Grande do Norte – UERN, Faculdade de Cieˆncias Exatas e Naturais – FANAT, Departamento de Cieˆncias Biolo´gicas, DECB, Campus Universita´rio Central, BR 110, Km 48, Rua Professor Antoˆnio Campos, s/n, Bairro Costa e Silva, CEP 59610-090, Mossoro´, Rio Grande do Norte, Brazil c Instituto Butantan, Laborato´rio de Artro´podes, Av. Vital Brazil, 1500, CEP 05503-900, Sa˜o Paulo, Sa˜o Paulo, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 July 2009 Received in revised form 3 October 2009 Accepted 3 October 2009
In this paper we present, for the first time, cytogenetical data on Latrodectus gr. curacaviensis (Theridiidae) from Brazil, as well as the first data on meiosis and sex chromosome system of this genus. Testes were submitted to colchicine, hypotonic, and fixation treatment, and chromosomal preparations were stained with Giemsa solution. The analysis showed 2n = 26 telo/acrocentric chromosomes in spermatogonial metaphases. Metaphase I exhibited 12 autosomal bivalents and two sex chromosome univalents (12II + X1X2). All bivalents revealed one terminal chiasma. Metaphases II confirmed the sex chromosome system, showing 12 autosomes or 12 autosomes plus two X chromosomes, respectively. Male karyotype prevailing in theridiids is formed by 2n = 22 chromosomes, including sex chromosome system X1X2 in all species. The Latrodectus species of the geometricus clade analyzed until now showed smaller diploid number (2n, = 16 and 2n, = 18) than the species of the mactans clade (2n, = 24 and 2n, = 26). Thus, according to the chromosome number, the examined Latrodectus species seems to be related to the mactans clade. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Latrodectinae Chromosome Meiosis Sex chromosome system Mitosis
1. Introduction Theridiidae (comb-footed spiders) is the fifth most species-rich family of spiders, including 2295 species and 109 genera (Platnick, 2009). From a cytogenetical point of view, only 29 species, belonging to 13 genera, were studied to date, revealing a high homogeneity of the karyotype. In 23 species, male karyotype was formed by 22 telo/acrocentric chromosomes, including an X1X2 sex chromosome system (SCS) (Table 1). Among the theridiids there are the widow spiders of the genus Latrodectus Walckenaer, 1805 (Latrodectinae), composed of 30 species (Platnick, 2009), with a worldwide distribution and notorious importance in medicine because of their neurotoxic venom (Garb et al., 2004). Interestingly, other theridiids potently venomous to vertebrates are those of the genus Steatoda Sundevall, 1833, another member of the subfamily Latrodectinae (Maretic et al., 1964; Diaz, 2004). Latrodectus is an exception to the
* Corresponding author. Tel.: +55 6739233187; fax: +55 6739233181. E-mail addresses: [email protected], [email protected] (D. Araujo), [email protected] (U.M. Maia), [email protected] (A.D. Brescovit). 0968-4328/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2009.10.002
chromosomal constancy observed among theridiids, with diploid number ranging from 2n = 16 to 2n = 28 in females of the four species analyzed so far (Table 1). According to Garb et al. (2004), there is great difficulty to distinguish between species of the genus Latrodectus based on morphological characters. Due to the high heterogeneity of diploid numbers within the genus, cytotaxonomy seems to be a promising approach to distinguish between Latrodectus species. In the present paper, a Latrodectus species from Brazil is chromosomally characterized, providing the first data on meiosis and SCS in the genus Latrodectus. In addition, a comparison between the cytogenetical and the cladistic data for the Latrodectus genus will be presented. 2. Materials and methods Five adult males and three adult females of Latrodectus gr. curacaviensis (Fig. 1) were collected manually, next to the roots of a carnauba wax palm, Copernicia prunifera, in the city of Ac¸u (58320 2300 S/368570 4500 W), state of Rio Grande do Norte, Brazil, in October of 2007. In contrast to females, gonads of males presented dividing cells suitable for analysis. Dissected specimens were
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Table 1 List of species of the family Theridiidae studied cytogenetically so far. Valid names extracted from Platnick (2009); cited as = species name used in the original paper; 2n = male diploid number (without parentheses), or female diploid number (between parentheses); SCS = sex chromosome system. S = supernumerary chromosome; T = telocentric; A = acrocentric. Valid name
Cited as
2n
SCS
Chromosome morphology
Collection site
Bibliography
Argyrodinae Argyrodes cyrtophorae Tikader, 1963 Argyrodes gazedes Tikader, 1970 Argyrodes gazingensis Tikader, 1970 Argyrodes sp. Argyrodes sp. Ariamnes cylindrogaster Simon, 1889 A. cylindrogaster Simon, 1889
Argyrodes cyrtophore – – – – – –
22 22 24 22 22 22 22
X1X2 X1X2 X1X2 X1X2 X1X2 X1X2 X1X2
– – – – – – 20A + X1X2A
India India India India India – Japan
Datta and Chatterjee Datta and Chatterjee Datta and Chatterjee Datta and Chatterjee Datta and Chatterjee Suzuki (1950) Suzuki (1954)
– – –
22 (18) (28)
X1X2 – –
20A + X1X2A – –
Finland South Africa South Africa
Suzuki (1954) Martindale (1980) Martindale (1980)
– – –– – – –
(24) (26) (16) 22 22 22
– – – X1X2 X1X2 X1X2
– – – 20A + X1X2A 20A + X1X2A –
Australia USA South Africa Finland Russia USA
Martindale (1980) Martindale (1980) Martindale (1980) Hackman (1948) Gorlov et al. (1995) Tugmon et al. (1990)
Enoplognatha japonica Theridium ovatum
22 22
X1X2 X1X2
20A/T + X1X2A/T –
Japan –
Kageyama and Seto (1979) Sokolov (1960)
– Chrysso venusta Theridion japonicum
22 24 22
X1X2 X1X2 X1X2
– 22A/T + X1X2A/T 20A/T + X1X2A/T
India Japan Japan
Datta and Chatterjee (1983) Kageyama and Seto (1979) Kageyama and Seto (1979)
Theridium tepidariorum
24
–
–
USA
Montgomery (1907)
Theridium tepidariorum Theridium tepidariorum Theridium tepidariorum Theridion tepidariorum Achaearanea tepidariorum Achaearanea tepidariorum Achaearanea tepidariorum Theridium sterninotatum
22 22 22 22 22 22 22 22
X1X2 X1X2 X1X2 X1X2 X1X2 X1X2 X1X2 X1X2
20A + X1X2A 20A + X1X2A 20A + X1X2A 20A + X1X2A 20A/T + X1X2A/T 20A/T + X1X2A/T – 20A + X1X2A
Finland Japan – – Japan China Taiwan Japan
Hackman (1948) Suzuki (1954) Diaz and Saez (1966) Igarashi and Kondo (1977) Kageyama and Seto (1979) Youju et al. (1995) Chen (1999) Suzuki (1954)
Theridion chikunii Theridion latifolium –
22 22 22
X1X2 X1X2 X1X2
20A/T + X1X2A/T 20A/T + X1X2A/T –
Japan Japan India
Kageyama and Seto (1979) Kageyama and Seto (1979) Srivastava and Shukla (1986)
Enoplognatha foliicola
22
X1X2
20A + X1X2A
Japan
Suzuki (1954)
– – – – – – –
22 22 22 22 22 22 22 + 1S
X1X2 X1X2 X1X2 X1X2 X1X2 X1X2 X1X2
20A/T + X1X2A/T 20A + X1X2A – 20A + X1X2A 20A + X1X2A 20A + X1X2A 20A + X1X2A
Japan Ecuador Ecuador Ecuador Ecuador Ecuador Ecuador
Kageyama and Seto (1979) Avile´s and Maddison (1991) Avile´s et al. (2000) Avile´s and Maddison (1991) Avile´s and Maddison (1991) Avile´s and Maddison (1991) Avile´s and Maddison (1991)
Latrodectinae Crustulina guttata (Wider, 1834) Latrodectus geometricus C.L. Koch, 1841 Latrodectus indistinctus O.P.-Cambridge, 1904 Latrodectus mactans (Fabricius, 1775) Latrodectus mactans (Fabricius, 1775) Latrodectus rhodesiensis Mackay, 1972 Steatoda bipunctata (Linnaeus, 1758) Steatoda grossa (C.L. Koch, 1838) Steatoda triangulosa (Walckenaer, 1802) Pholcommatinae Enoplognatha caricis (Fickert, 1876) Enoplognatha ovata (Clerck, 1757) Theridiinae Achaearanea budana Tikader, 1970 Chrysso scintillans (Thorell, 1895) Parasteatoda japonica (Bo¨senberg & Strand, 1906)a Parasteatoda tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a P. tepidariorum (C.L. Koch, 1841)a Platnickina sterninotata (Bo¨senberg & Strand, 1906)b Takayus chikunii (Yaginuma, 1960) Takayus latifolius (Yaginuma, 1960) Theridion sp. Remaining theridiids Anelosimus crassipes (Bo¨senberg & Strand, 1906) A. crassipes (Bo¨senberg & Strand, 1906) Anelosimus domingo Levi, 1963 A. domingo Levi, 1963 Anelosimus eximius (Keyserling, 1884) Anelosimus jucundus (O.P.-Cambridge, 1896) Anelosimus studiosus (Hentz, 1850) A. studiosus (Hentz, 1850)
(1983) (1983) (1983) (1983) (1983)
a The species Parasteatoda japonica and Parasteatoda tepidariorum were included in Theridiinae because they were transferred from Achaearanea Strand, 1929, a theridiine genus according the phylogenetic analyses of Agnarsson (2004) and Arnedo et al. (2004), only recently by Yoshida (2008) and Saaristo (2006), respectively. b The genus Platnickina was included in Theridiinae because it was transferred from Keijia Yoshida, 2001, a theridiine genus according to the phylogenetic analyses of Agnarsson (2004) and Arnedo et al. (2004), only recently by Koc¸ak and Kemal (2008).
deposited in the collection of the Laborato´rio de Artro´podes, Instituto Butantan, city of Sa˜o Paulo, state of Sa˜o Paulo, Brazil (IBSP 91671, 91672, 91673, 91674, 91675). Gonads were dissected in physiologic solution for insects (7.5 g NaCl, 2.38 g Na2HPO4, 2.72 g KH2PO4, in 1 L of distilled water), colchicine solution (0.16% in physiologic solution for insects) was added to the material and left for 2 h. A volume of hypotonic solution (tap water) equal to that of colchicine solution was added. After 15 min of hypotonization, the gonads were placed in methanol:acetic acid fixative (3: 1) for 60 min. Pieces of tissue were
dissociated in a drop of 60% acetic acid on the surface of microscope slides. The chromosome preparations were dried on a metal heating plate (from 35 8C to 40 8C), stained with a 3% Giemsa solution (47 mL distilled water, 1.5 mL phosphate buffer, pH 6.8, 1.5 mL Giemsa) for 15 min, and rinsed briefly with distilled water. Approximately 50 cells of Latrodectus gr. curacaviensis were recorded using a Motic1 light microscope. Morphology of chromosomes was classified according to Levan et al. (1964). Karyotype was constructed in Corel Draw1 12 image editing software.
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Fig. 1. Male Latrodectus sp. from Rio Grande do Norte, Brazil. Scale bar = 1 mm. Fig. 3. Male metaphase I of Latrodectus sp., showing 12II + X1X2. The arrows indicate terminal chiasma. Scale bar = 10 mm.
3. Results The analysis of spermatogonial metaphases revealed 26 telo/ acrocentric chromosomes in males of Latrodectus gr. curacaviensis. In spermatogonial metaphases, the sex chromosomes did not show any different characteristics. No heteropycnotic regions were noticed in any chromosome (Fig. 2). Diplotene nuclei and metaphase I plates showed 12 autosomal bivalents. Each bivalent contained only one terminal chiasma. Two small univalents, placed always close to each other, corresponding to the sex chromosomes X1 and X2 (Fig. 3). These results indicated that male karyotype of Latrodectus gr. curacaviensis is composed of 24 autosomes and two sex chromosomes, X1 and X2. Size of sex chromosome univalents suggests that the X1 and X2 chromosomes are among the smallest ones of the complement. Metaphases II showed n = 12 or n = 14 chromosomes (Fig. 4). Difference of number reflected segregation of the X chromosomes to the same pole during anaphase I. 4. Discussion The only work on Latrodectus cytogenetics is the abstract of Martindale (1980), which is based only on female specimens and contains no data on chromosomal morphology, sex chromosome system, and meiosis (Table 1). The present work describes for the first time the chromosome morphology, course of meiosis, and SCS of the genus Latrodectus. According to a phylogenetic analysis (Garb et al., 2004), the genus Latrodectus is divided in two clades. The geometricus clade is composed only by two species, Latrodectus geometricus and Latrodectus rhodesiensis. The mactans clade contains much more species, namely Latrodectus antheratus (Badcock, 1932); Latrodec-
tus bishopi Kaston, 1938; Latrodectus corallinus Abalos, 1980; Latrodectus diaguita Carcavallo, 1960; Latrodectus hasselti Thorell, 1870; Latrodectus hesperus Chamberlin & Ivie, 1935; Latrodectus katipo Powell, 1871; Latrodectus mactans; Latrodectus menavodi Vinson, 1863; Latrodectus mirabilis (Holmberg, 1876); Latrodectus pallidus O. P.-Cambridge, 1872; Latrodectus renivulvatus Dahl, 1902; Latrodectus revivensis Shulov, 1948; Latrodectus tredecimguttatus (Rossi, 1790); Latrodectus variegatus Nicolet, 1849; Latrodectus variolus Walckenaer, 1837; Latrodectus sp. from Brazil (Bahia); Latrodectus sp. from Chile. Considering the diploid numbers, two cytotaxonomic groups of Latrodectus can be distinguished: (1) a group in which the diploid number remains the same or increases in relation to the other latrodectines (Crustulina and Steatoda, 2n< = 22), composed by L. mactans (2n, = 26 or 2n, = 24), Latrodectus indistinctus (2n, = 28) (Martindale, 1980) and L. gr. curacaviensis (2n< = 26) (present work) and (2) a group in which the diploid number decreases in relation to the other latrodectines, composed by L. geometricus (2n, = 18) and L. rhodesiensis (2n, = 16) (Martindale, 1980). Unfortunately, Latrodectus curacaviensis (Mu¨ller, 1776) was not included in the work of Garb et al. (2004), but McCrone and Levi (1964) proposed that this species, based on the genital structures, could form a group with L. variolus and L. bishopi, species belonging to the mactans clade in the hypothesis of Garb et al. (2004). This grouping is reinforced by our cytogenetic analysis, revealing Latrodectus gr. curacaviensis as a probable member of the mactans clade and not as part of a distinctive group as some researchers proposed in the past (McCrone and Levi, 1964; Pinter, 1967; Meier and White, 1995), whereas the two species of the geometricus clade presented lower diploid numbers (2n = 18 and 2n = 16), reinforcing
Fig. 2. Male karyotype of Latrodectus sp. with 2n = 26 telo/acrocrocentric chromosomes. Scale bar = 10 mm.
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Fig. 4. Male metaphase II cells of Latrodectus sp. with n = 12 (A) and n = 14 = 12 + X1X2 (B). Scale bar = 10 mm.
the taxonomical and evolutionary proximity recovered by Lotz (1994) and Garb et al. (2004), respectively. It is interesting to note that the other genera belonging to the subfamily Latrodectinae (Agnarsson, 2004; Arnedo et al., 2004), namely Crustulina Menge, 1868 and Steatoda, presented 2n< = 22, i.e. the same diploid number showed by the majority of the theridiids. Thus, in spite of the three genera of latrodectines being closely related according to morphological (Agnarsson, 2004) and molecular data (Arnedo et al., 2004), only the genus Latrodectus revealed a high interspecific variation of the diploid number. It is important to emphasize that, in contrast to Theridiidae, in which 2n< = 22 prevails (Table 1), all other families of Araneoidea karyotyped to date (Araneidae, Linyphiidae, Nephilidae, Nesticidae and Tetragnathidae), possess predominantly species with 2n< = 24. Considering the theridiid subfamilies recognized by Agnarsson (2004) and Arnedo et al. (2004), only Hadrotarsinae and Spintharinae were not chromosomally analyzed until now, and further cytogenetic analyses on species belonging to these clades are a priority to verify or not the occurrence of the 2n< = 22 in all theridiid subfamilies. Acknowledgements The authors are grateful for the critical reading of two anonymous reviewers and especially to Jirˇı´ Kra´l (Charles University, Prague), and to Cristina A. Rheims for reviewing the English language. Financial support was provided by Conselho Nacional de Desenvolvimento Cientı´fico e tecnolo´gico (CNPq), and Fundac¸a˜o de Apoio a` Pesquisa do Estado do Rio Grande do Norte (FAPERN). References Agnarsson, I., 2004. Morphological phylogeny of cobweb spiders and their relatives (Araneae, Araneoidea, Theridiidae). Zool. J. Linn. Soc. 141, 447–626. Arnedo, M.A., Coddington, J., Agnarsson, I., Gillespie, R.G., 2004. From a comb to a tree: phylogenetic relationships of the comb-footed spiders (Araneae, Theridiidae) inferred from nuclear and mitochondrial genes. Mol. Phylogenet. Evol. 31, 225–245. Avile´s, L., Maddison, W., 1991. When is the sex ratio biased in social spiders? Chromosome studies of embryos and male meiosis in Anelosimus species (Araneae, Theridiidae). J. Arachnol. 19, 126–135. Avile´s, L., McCormack, J., Cutter, A., Bukowski, T., 2000. Precise, highly female-biased sex ratios in a social spider. Proc. R. Soc. Lond. B: Biol. Sci. 267, 1445–1449. Chen, S.H., 1999. Cytological studies on six species of spiders from Taiwan (Araneae: Theridiidae, Psechridae, Uloboridae, Oxyopidae, and Ctenidae). Zool. Stud. 38, 423–434.
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