The present study aimed to cytogenetically analyse five Ctenidae species Ctenus ornatus (Keyserling, 1877), Ctenus medius (Keyserling, 1891), Phoneutria nigriventer (Keyserling, 1891), Viracucha andicola (Simon, 1906), and Enoploctenus cyclothorax (Philip Bertkau, 1880), from Brazil. All species presented a 2n♂ = 28 except for V. andicola, which showed 2n♂ = 29. Analysis of segregation and behavior of sex chromosomes during male meiosis showed a sex chromosome system of the type X₁X₂0 in species with 28 chromosomes and X₁X₂X₃0 in V. andicola. C banding stained with fluorochromes CMA₃ and DAPI revealed two distributions patterns of GC-rich heterochromatin: (i) in terminal regions of most chromosomes, as presented in C. medius, P. nigriventer, E. cyclothorax and V. andicola and (ii) in interstitial regions of most chromosomes, in addition to terminal regions, as observed for C. ornatus. The population of Ubatuba (São Paulo State) of this same species displayed an additional accumulation of GC-rich heterochromatin in one bivalent. Fluorescent in situ hybridization revealed that this bivalent corresponded to the NOR-bearing chromosome pair. All analyzed species have one bivalent with 18S rDNA site, except P. nigriventer, which has three bivalents with 18S rDNA site. Karyotypes of two species, C. medius and E. cyclothorax, are described for the first time. The latter species is the first karyotyped representative of the subfamily Acantheinae. Finally, 18S rDNA probe is used for the first time in Ctenidae at the present study.

C-banding, FISH, fluorochrome, meiosis, spider cytogenetics, sex chromosomes

Ctenidae is a family of Araneae distributed throughout the tropical region of the planet (World Spider Catalog 2017). This family includes wandering and nocturnal spiders, with some species of medical interest, such as those of the genus Phoneutria Perty, 1833 (Ministério da Saúde 2017). Ctenidae is divided into five subfamilies, namely Acanthocteninae, Viridasiinae, Cteninae, Calocteninae, and Acantheinae (Silva-Dávila 2003; Polotow and Brescovit 2014). Although ctenids are of great ecological and medical importance, studies on their cytogenetics are scarce (Table 1) and cytogenetic data for the last two subfamilies are not available to date.

Three karyotypes have been observed in the family: (i) 2n♂ = 22 (20 + X₁X₂0); (ii) 2n♂ = 28 (26 + X₁X₂0); and (iii) 2n♂ = 29 (26 + X₁X₂X₃0) (Table 1). The sex chromosome systems (SCS) in spiders are considered highly diverse by many authors (Král et al. 2006; 2011; Araujo et al. 2012) ranging from simple systems, such as XY or X0, to multiple SCS as X_nY_n or X_n0 (Araujo et al. 2017). Based on findings in a specimen of Ctenus ornatus (Keyserling, 1877) Araujo et al. (2014) suggested that the X₁X₂X₃0 system in Ctenidae, might have arisen from a supernumerary chromosome and, according to literature evidence, this system arose repeatedly in the evolutionary history of Entelegynae and its conversion into the X₁X₂0 system and vice-versa is a recurring event. Bole-Gowda (1952) also suggested the involvement of a supernumerary element in the origin of the X₃ chromosome in Sparassidae species. Other hypotheses on the conversion of a X₁X₂0 into a X₁X₂X₃0 were also proposed by some authors (Pätau 1948; Postiglioni and Brum-Zorrilla 1981; Parida and Sharma 1986). The conversion of a X₁X₂X₃0 into a X₁X₂0 was firstly proposed in the spider genus Malthonica Simon, 1898 (Agelenidae) by Král (2007), suggesting that tandem fusions occurred in this process.

Chromosome banding techniques, as identification of nucleolus organizer regions (NORs) using silver nitrate impregnation, have been performed in Ctenidae. Araujo et al. (2014) found a single terminal NOR on one autosomal pair in C. ornatus and Phoneutria nigriventer (Keyserling, 1891), and on two pairs in Viracucha andicola (Simon, 1906). Kumar et al. (2016) also detected NORs on two autosomal pairs in Ctenus indicus (Gravely, 1931). However, molecular cytogenetic studies are scarce in spiders. There have been only five studies about distribution of some sequences using fluorescence in situ hybridization (FISH): location of 18S rDNA sites in Wadicosa fidelis (O. Pickard-Cambridge, 1872) (Lycosidae) (Forman et al. 2013) and Brachypelma albopilosum Valerio, 1980 (Theraphosidae) (Král et al. 2013); 5S rDNA sites in Oxyopes sertatus L. Koch, 1878 (Oxyopidae) (Suzuki and Kubota 2011); mapping of silk genes in Latrodectus hesperus Chamberlin & Ivie, 1935 and Latrodectus geometricus C. L. Koch, 1841 (Theridiidae) (Zhao et al. 2010); and ocurrence of telomeric repeats in Brachypelma albopilosa Valerio, 1980 (Vítková et al. 2005).

Considering the great importance of ctenids and the scarcity of cytogenetic studies in the group, our study analyzed the mitotic and meiotic chromosomes of five species of this family. To understand better the karyotype structure in this group of spiders, we evaluated the behavior of sex chromosomes, heterochromatin composition/distribution pattern, and the location of 18S rDNA sites.

Ctenus ornatus, Ctenus medius Keyserling, 1891, Phoneutria nigriventer, and Enoploctenus cyclothorax (Bertkau, 1880) exhibited 2n♂ = 28, as observed in mitotic metaphases (Fig. 1A, E, I, M), whereas Viracucha andicola presented 2n♂ = 29 (Fig. 1Q). All chromosomes were identified in metaphases II as acrocentric (Fig. 1D, H, L), except for E. cyclothorax and V. andicola, in which it was difficult to determine accurately the morphology of all chromosomes (Fig. 1P, T).

At male diakinesis 13 bivalents in all species were found and two univalent X in parallel association in the species with 28 chromosomes (Fig. 1C, G, K, O) and three univalent X in the species with 29 chromosomes (Fig. 1S). Three sex chromosomes in V. andicola showed parallel association (Fig. 1S-box). In some plates at pachytene and diplotene X are not associated in species with the two X chromosomes (Fig. 1C, G, K, O-boxes). Species with 2n♂ = 28 showed metaphases II with 13 and 15 chromosomes (Fig. 1D, H, L, P), and species with 2n♂ = 29 showed cells with 13 and 16 chromosomes (Fig. 1T), that confirm sex chromosome systems of the types X₁X₂0 and X₁X₂X₃0, respectively. In species with 28 chromosomes, two positively heteropycnotic bodies were observed in pachytene stage (Fig. 1B, F, J, N) and V. andicola exhibited three positive heteropycnotic bodies (Fig. 1R), identified as the sex chromosomes.

Ctenus ornatus presented interstitial and terminal CMA₃⁺ bands (Fig. 2A). Nevertheless, the population of Ubatuba (São Paulo state) presented an additional large terminal CMA₃⁺ block in a bivalent (Fig. 2C). In C. medius (Fig. 2E), P. nigriventer (Fig. 2G), V. andicola (Fig. 2I), and E. cyclothorax (Fig. 2K), all populations showed only CMA₃⁺ terminal blocks. Karyotypes contained no DAPI⁺ blocks (Fig. 2B, D, F, H, J, L).

The FISH revealed one bivalent with 18S rDNA site in C. ornatus (Fig. 3A), C. medius (Fig. 3B), V. andicola (Fig. 3D), and E. cyclothorax (Fig. 3E). C. ornatus presented size polymorphism of the 18S rDNA site (Fig. 3A-box). P. nigriventer showed three bivalents exhibiting 18S rDNA site; however, one of these bivalents presented site only in one chromosome (Fig. 3C).

Metaphase II of C. ornatus from the Ubatuba population submitted to FISH and subsequently to CMA3/DAPI also revealed that CMA⁺ sites with higher accumulation of GC-rich heterochromatin are co-localized to the sites carrying 18S rDNA (Fig. 4).

Figure 1.

Male mitotic and meiotic cells of Ctenidae species stained with Giemsa. Boxes – X chromosomes without association (C, G, K, O), and with association (S). C. medius (A–D), C. ornatus (E–H), P. nigriventer (I–L), E. cyclothorax (M–P), V. andicola (Q–T). The arrowheads show sex chromosomes. Mitotic metaphases with 2n = 28 (A, E, I, M) and 2n =29 (Q). Pachytene cells (B, F, J, N, R) with positively heteropycnotic sex chromosomes. Diakinesis cells (C, G, K, O, S), note parallel association of two X chromosomes (C, G, K, O) or three X chromosomes without association (S). Metaphase II cells with n = 13 and n = 13 + X₁X₂(D, H, L, P) and n = 13 and n = 13 + X₁X₂X₃(T). Bar = 10 µm.

Figure 2.

Ctenidae male mitotic and meiotic cells, C-banding and staining with base-specific fluorochromes CMA₃ (A, C, E, G, I, K) and DAPI (B, D, F, H, J, L). Arrowhead - X chromosomes. A, B mitotic metaphase of Ctenus ornatus, 28 chromosomes, arrow – interstitial CMA₃⁺ region C, D diakinesis of C. ornatus, Ubatuba population, arrow – bivalent with large CMA₃⁺ block E, F diakinesis of C. medius G, H mitotic metaphase of Phoneutria nigriventer, 2n=28 I, J diakinesis of Viracucha andicola K, L diakinesis of Enoploctenus cyclothorax. Bar = 10 µm.

Figure 3.

Ctenidae male meiotic cells, FISH with rDNA 18S probe. Arrowhead - sex chromosomes. A diakinesis of Ctenus ornatus: in the box the bivalent with size heteromorphism of 18S rDNA sites B diakinesis of Ctenus medius C diakinesis of Phoneutria nigriventer: arrow - bivalent with 18S rDNA sites in only one of the chromosomes D diplotene of Viracucha andicola E diplotene of Enoploctenus cyclothorax. Bar = 10 µm.

Figure 4.

Chromosomes of Ctenus ornatus, Ubatuba/São Paulo state. A Metaphase II, FISH with rDNA 18S probe B sequential staining with DAPI/CMA₃ in the same metaphase II, showing association between sites of GC-rich heterochromatin and rDNA 18S regions. Note the presence of more than one metaphase II. Bar = 10 µm.

The conventional analysis showed diploid number, chromosomal morphology, sex chromosome system and meiotic behavior of five Ctenidae species. The present study presents the first data for Acantheinae, increasing to four the number of ctenid subfamilies with cytogenetic data (Table 1), and the first cytogenetic study in C. medius and E. cyclothorax. In Ctenidae, the diploid number variation occurs basically due to the differences in SCS: species with 2n♂ = 28 exhibit a SCS of the type X₁X₂0, whereas species with 2n♂ = 29 have the type X₁X₂X₃0. Only A. borellii (Viridasiinae) presents 2n♂ = 22, with SCS of the type X₁X₂0 (Chen 1999, Araujo et al. 2014, Kumar et al. 2016).

The parallel association between sex chromosomes during male meiosis is a common pattern observed in Entelegynae (Král et al. 2011; Araujo et al. 2012), and also found in Ctenidae (Chen 1999; Araujo et al. 2014; Kumar et al. 2016). Forman et al. (2013) observed absence of sex chromosome pairing in some plates of Wadicosa fidelis. They proposed that it might be due to chromosome preparation. A similar situation may have occurred in species analyzed in this study.

We observed two distinct distribution patterns of the GC-rich heterochromatin: (i) bands distributed in terminal regions of most chromosomes, as presented in C. medius, P. nigriventer, E. cyclothorax and V. andicola; and (ii) bands present in interstitial regions of most chromosomes, in addition to the terminal regions, as observed for C. ornatus. The first pattern could arise by dispersion of heterochromatin due to contact of chromosomes during their polarization of Rabl in mitosis or during bouquet orientation at the early prophase I as described by Schweizer and Loidl (1987). The second pattern could arise by occurrence of chromosomal rearrangements (Schweizer and Loidl 1987) or by spreading of the heterochromatin by transposable elements, as proposed for grasshopper (Rocha et al. 2015). Furthermore, despite the few species studied, GC-rich blocks seem to be common in entelegyne spiders (Araujo et al. 2005; Ramalho et al. 2008, Chemisquy et al. 2008). They were also found in Ctenidae species in the present study. The heterochromatin distribution also allowed to distinguish C. ornatus from Ubatuba population of other C. ornatus populations here analyzed.

The present study revealed a massive accumulation of GC-rich heterochromatin associated with 18S rDNA site in C. ornatus from Ubatuba. Association of GC-rich heterochromatin with NORs is common in many animal groups, for example in fishes (Ferro et al. 2001) and amphibians (Schmid 1980). In spiders, this association has been reported in Nephilingys cruentata (Araneidae) (Araujo et al. 2005).

Another characteristic observed in C. ornatus was the size heteromorphism of 18S rDNA sites. This can be explained by unequal crossing, which causes a greater accumulation of rDNA cistrons in one of the homologous chromosomes, as described by Ferro et al. (2001) and Teribele et al. (2008) in fish species. A similar situation may have occurred in P. nigriventer, very small 18S rDNA sites could exhibit low fluorescence, making detection difficult.

In Ctenidae, NOR in one bivalent seems to be the most commonly observed pattern. Only P. nigriventer presented more rDNA sites. This finding differs from Araujo et al. (2014), who observed only one chromosome pair carrying NOR in the same species using the silver nitrate impregnation that identifies only transcriptionally active sites. Specimens of V. andicola showed a single NOR as revealed by the FISH analysis. By contrast, the data exhibited by Araujo et al. (2014) showed NORs in two chromosome pairs, which could indicate an interpopulation variation, however the authors analyzed only one specimen, which hinders a more accurate study.

The present study brings new cytogenetic information and first FISH data for Ctenidae providing valuable contribution to the knowledge on karyotypes in this family.

The authors thank Dra. Renata da Rosa (UEL), reviewers and the sub-editor of Comparative Cytogenetics for their considerations and time in reviewing this article, and to Dra. Juceli Gonzalez Gouveia (UEMS) for his assistance related to molecular cytogenetic techniques. Authors also thank to Rafael Campos de Barros, Mailson Gabriel da Fonseca, and Robson Rockembacher (UEL) for their assistance with sample collection. This work was supported by the Capes and CNPq (ADB grant PQ 301776/2014-0; ALD grant 312529/2014-7).