Research Article |
Corresponding author: Matheus Pires Rincão ( rincaom@gmail.com ) Corresponding author: Ana Lúcia Dias ( anadias@uel.br ) Academic editor: Jiří Král
© 2017 Matheus Pires Rincão, João Lucas Chavari, Antonio Domingos Brescovit, Ana Lúcia Dias.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Rincão MP, Chavari JL, Brescovit AD, Dias AL (2017) Cytogenetic analysis of five Ctenidae species (Araneae): detection of heterochromatin and 18S rDNA sites. Comparative Cytogenetics 11(4): 627-639. https://doi.org/10.3897/CompCytogen.v11i4.10620
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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 (
Cytogenetic data of Ctenidae species, updated from
Species | Karyotype (♂) | NORs | Reference | |
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Silver Nitrate | detection of 18S rDNA | |||
Acantheinae | ||||
Enoploctenus cyclothorax (Bertkau, 1880) | 28, X1X20 | 2 | Present study | |
Acanthocteninae | ||||
Nothroctenus sp. | 29, X1X2X30 |
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Viracucha andicola (Simon, 1906) | 29, X1X2X30 | 4 |
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2 | Present study | |||
Cteninae | ||||
Anahita fauna Karsch, 1879 | 29, X1X2X30 | Chen, 1999 | ||
Ctenus indicus (Gravely, 1931) | 28, X1X20 | 4 |
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Ctenus medius Keyserling, 1891 | 28, X1X20 | 2 | Present study | |
Ctenus ornatus (Keyserling, 1877) | 28, X1X20 | 2 |
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Ctenus sp. | 28, X1X20 |
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Parabatina brevipes (Keyserling, 1891) | 28, X1X20 |
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Phoneutria nigriventer (Keyserling, 1891) | 28, X1X20 | 2 |
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6 | Present study | |||
Viridasiinae | ||||
Asthenoctenus borelli Simon, 1897 | 22, X1X20 |
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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
Chromosome banding techniques, as identification of nucleolus organizer regions (NORs) using silver nitrate impregnation, have been performed in Ctenidae.
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.
Adults and juveniles of five ctenid species from different collection sites in Brazil were analyzed, as listed in Table
List of collected species, with the number of the individuals, collection sites, and voucher numbers. PR = Paraná State. SP = São Paulo State.
Species | Individuals (♂) | Collection Site | Voucher Number |
Ctenus medius | 5 | Londrina (23°19'37.5"S, 51°12'13.4"W), PR | 166439, 167462, 167463, 167466, 167490 |
Ctenus ornatus | 11 | Londrina (23°19'37.5"S, 51°12'13.4"W), PR | 166426–166430, 166440–166442, 166449, 166458–166459 |
9 | Céu Azul (25°09'15.8"S, 53°50'42.1"W), PR | 166399–166401, 167467–167470, 167476–167477 | |
2 | Foz do Iguaçu (25°37'41.2"S 54°27'47.2"W), PR | 166416, 167465 | |
4 | Ubatuba (23°24'14.3"S 45°03'54.0"W), SP | 166453-166454, 167402, 167406 | |
Enoploctenus cyclothorax | 3 | Céu Azul (25°09'15.8"S, 53°50'42.1"W), PR | 166397, 166398, 166407 |
Phoneutria nigriventer | 5 | Londrina (23°19'37.5"S, 51°12'13.4"W), PR | 166441, 167407, 167489, 167494, 167495 |
1 | Céu Azul (25°09'15.8"S, 53°50'42.1"W), PR | 166412 | |
1 | Foz do Iguaçu (25°37'41.2"S 54°27'47.2"W), PR | 167405 | |
Viracucha andicola | 6 | Londrina (23°19'37.5"S, 51°12'13.4"W), PR | 166434, 166445, 166447, 167398–167400 |
2 | Céu Azul (25°09'15.8"S, 53°50'42.1"W), PR | 166411, 166413 |
Chromosomal preparations were obtained according to
Genomic DNA of C. ornatus was extracted using a standard phenol/chloroform procedure (
The sequence was analyzed using the free software BioEdit, version 7.2.5 (
The 18S rDNA sites were identified using the FISH technique according to
Ctenus ornatus, Ctenus medius Keyserling, 1891, Phoneutria nigriventer, and Enoploctenus cyclothorax (Bertkau, 1880) exhibited 2n♂ = 28, as observed in mitotic metaphases (Fig.
At male diakinesis 13 bivalents in all species were found and two univalent X in parallel association in the species with 28 chromosomes (Fig.
Ctenus ornatus presented interstitial and terminal CMA3+ bands (Fig.
The FISH revealed one bivalent with 18S rDNA site in C. ornatus (Fig.
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.
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.
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.
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.
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
The parallel association between sex chromosomes during male meiosis is a common pattern observed in Entelegynae (
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
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 (
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
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
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).