Research Article |
Corresponding author: Francisco Panzera ( fcopanzera@gmail.com ) Academic editor: Seppo Nokkala
© 2016 Sebastián Pita, Francisco Panzera, Pablo Mora, Jesús Vela, Teresa Palomeque, Pedro Lorite.
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:
Pita S, Panzera F, Mora P, Vela J, Palomeque T, Lorite P (2016) The presence of the ancestral insect telomeric motif in kissing bugs (Triatominae) rules out the hypothesis of its loss in evolutionarily advanced Heteroptera (Cimicomorpha). Comparative Cytogenetics 10(3): 427-437. https://doi.org/10.3897/compcytogen.v10i3.9960
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Next-generation sequencing data analysis on Triatoma infestans Klug, 1834 (Heteroptera, Cimicomorpha, Reduviidae) revealed the presence of the ancestral insect (TTAGG)n telomeric motif in its genome. Fluorescence in situ hybridization confirms that chromosomes bear this telomeric sequence in their chromosomal ends. Furthermore, motif amount estimation was about 0.03% of the total genome, so that the average telomere length in each chromosomal end is almost 18 kb long. We also detected the presence of (TTAGG)n telomeric repeat in mitotic and meiotic chromosomes in other three species of Triatominae: Triatoma dimidiata Latreille, 1811, Dipetalogaster maxima Uhler, 1894, and Rhodnius prolixus Ståhl, 1859. This is the first report of the (TTAGG)n telomeric repeat in the infraorder Cimicomorpha, contradicting the currently accepted hypothesis that evolutionarily recent heteropterans lack this ancestral insect telomeric sequence.
Cimicomorpha , kissing bugs, holocentric chromosomes, telomeres, NGS, (TTAGG)n
Telomeres, the physical ends of eukaryote chromosomes, are defined as specialized DNA-protein structures essential for chromosome replication, meiotic pairing and chromosome stability. In most organisms, telomeric DNA is composed by simple G-rich sequences repeats that extend for tens of base pairs (bp) as much as 150 kb, depending on the organism. Although telomeric repeats are diverse in their DNA sequence composition among different organisms (
Among Hemiptera, the ancestral motif is present in the suborder Sternorrhyncha (coccids and aphids with some exceptions) (
Kissing bugs (Triatominae, Reduviidae) are included within the infraorder Cimicomorpha (Heteroptera), constituting a group of medical relevance because they act as vectors of Chagas disease, also known as American trypanosomiasis. This subfamily includes 150 species, of which more than 80 have been cytogenetically studied (
Four species where analyzed, involving three different genera from the two principal tribes of the subfamily: Triatomini (Dipetalogaster maxima, Triatoma infestans, and T. dimidiata) and Rhodniini (Rhodnius prolixus). The last three species are the main vectors of Chagas disease. Origin and cytogenetic traits of each species are detailed in Table
Geographical origin and male diploid chromosome number in the four species here analyzed. A = autosomes.
Species | Geographical origin | Male diploid chromosome number (2n) |
---|---|---|
Tribe Rhodniini | ||
Rhodnius prolixus | Guatemala, Quezaltenango, Insectary CDC (USA) | 22= 20A + XY |
Tribe Triatomini | ||
Dipetalogaster maxima | Baja California, Mexico | 22= 20A + XY |
Triatoma dimidiata | Jutiapa, Guatemala | 23= 20A + X1X2Y |
Triatoma infestans | Tacuarembó, Uruguay | 22= 20A + XY |
A Triatoma infestans (non-Andean lineage) specimen collected in Tacuarembó (Uruguay) was used for sequencing. Approximately 3 µg of genomic DNA were employed in a low coverage Illumina® Hiseq™ 2000 paired-end sequencing. Graph-based clustering analysis was carried out using RepeatExplorer (
Chromosome preparations for FISH analyses were obtained from male gonads. Testes were removed from live adult insects, fixed in an ethanol–glacial acetic acid mixture (3:1) and stored at -20°C. Squashes were made in a 50% acetic acid drop, coverslips were removed after freezing in liquid nitrogen and the slides were air dried and then stored a 4°C.
Telomeric TTAGG probe generation and FISH assays were carried out following
Previously to hybridization, slides were treated with RNase A, pepsin and formaldehyde and dehydrated in 70%, 90% and 100% ethanol for 5 min each. Hybridization was performed applying 25 µl of DNA labelled solution to each slide, which was heated for 3 min at 80°C to denature the DNA, and immediately chilled on ice for 3 min. The hybridization mix consisted of (final concentrations) 50% formamide, 2xSSC, 50 mM sodium phosphate, 0.1 mg/ml sonicated salmon sperm DNA, 0.1 mg/ml yeast RNA, and 5 ng/ml labeled telomere probe. The slides were transferred to a moist chamber humidified with formamide (50%) and incubated overnight at 37°C. After incubation, the slides were washed in 50% formamide at 37°C, three times, 3 min each; followed by 2xSSC, 0.05% Tween-20, pH 7.5, three times, 5 min each. Fluorescence immunological detection was performed using the avidin-FICT/ anti-avidin-biotin system with four rounds of amplification. Slides were mounted with Vectashield (Vector). DAPI in the antifade solution was used to counterstain chromosomes.
The data obtained from the T. infestans genome sequencing were analyzed with RepeatExplorer (
Fluorescence in situ hybridization with (TTAGG)n telomeric probe (green signals) on mitotic and meiotic chromosomes (counterstained with DAPI in blue) of four Triatominae species. A Triatoma infestans (2n=22), spermatogonial prometaphase B Triatoma dimidiata (2n=23), spermatogonial prometaphase C Dipetalogaster maxima (2n=22), pachytene stage D Rhodnius prolixus (2n=22), first meiotic division showing 10 bivalents and two sex chromosomes (X and Y). Scale bar: 5 µm.
Furthermore, we tested the telomeric motif presence by FISH in other three triatomine species with (TTAGG)n probe. Hybridization signals were clearly seen on the chromosomal ends of mitotic and meiotic chromosomes (Fig.
Given our positive FISH hybridization results on R. prolixus chromosomes, we additionally conducted a BLAST search of telomeric sequences in the published genome of this species, available at https://www.vectorbase.org/. Similar as reported by
Heteroptera or true bugs are a hemipteran suborder comprising seven infraorders and 40,000 species. All phylogenetic studies agreed that the infraorders Cimicomorpha and Pentatomomorpha are the most evolutionarily derived groups, with a common ancestor and involving about 80% of heteropteran species (
This study was supported by project grants (No. 370) from the ‘‘Comisión Sectorial de Investigación Científica’’ (CSIC-Udelar-Uruguay), Programa de Desarrollo de las Ciencias Básicas (PEDECIBA Uruguay), Agencia Nacional de Investigación e Innovación (ANII, Uruguay) and by the “Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía”, sponsor of Program of Academic Mobility of AUIP (Ibero-American University Postgraduate Association) for S.P. and F.P. This work was also supported by the Spanish Junta de Andalucía (through the program “Ayudas a Grupos de Investigación”, Group BIO220). This paper is included in the Ph.D. Thesis of Sebastián Pita (Udelar-University of Jaén), partially funded by a grant from the University of Jaén through the program “Ayudas para la realización de tesis doctoral en régimen de cotutela”.