Chromosomal distribution of interstitial telomeric sequences as signs of evolution through chromosome fusion in six species of the giant water bugs (Hemiptera, Belostoma)

Abstract Tandem arrays of TTAGG repeats show a highly conserved location at the telomeres across the phylogenetic tree of arthropods. In giant water bugs Belostoma, the chromosome number changed during speciation by fragmentation of the single ancestral X chromosome, resulting in a multiple sex chromosome system. Several autosome–autosome fusions and a fusion between the sex chromosome pair and an autosome pair resulted in the reduced number in several species. We mapped the distribution of telomeric sequences and interstitial telomeric sequences (ITSs) in Belostoma candidulum (2n = 12 + XY/XX; male/female), B. dentatum (2n = 26 + X1X2Y/X1X1X2X2), B. elegans (2n = 26 + X1X2Y/X1X1X2X2), B. elongatum (2n = 26 + X1X2Y/X1X1X2X2), B. micantulum (2n = 14 + XY/XX), and B. oxyurum (2n = 6 + XY/XX) by FISH with the (TTAGG)n probes. Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS. From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere–telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.

Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS.
From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere-telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.

K E Y W O R D S
chromosomal fusion, interstitial telomeric repeats, karyotype evolution, telomere FISH
Besides keeping chromosome integrity, telomeres are involved in chromosome pairing during meiosis and telomere-telomere sister chromatid cohesion during mitotic anaphase as found in very different organisms (Antoniacci & Skibbens, 2006;Carlton & Cande, 2002;Danjinou et al., 1999;Lee, Conrad, & Dresser, 2012;Rockmill & Roeder, 1998). Studies in several vertebrate species also suggest a potential role of telomeric repeats in karyotype evolution through additional intrachromosomal sites, the so-called interstitial telomeric sequences (ITSs) (Bruschi, Rivera, Lima, Zúñiga, & Recco-Pimentel, 2014;Meyne et al., 1990). In some species, the occurrence of ITS can be correlated with the evolutionary changes of karyotypes due to telomere-telomere fusions of the chromosomes, intrachromosomal rearrangements (inversions), unequal crossing over, or the insertion of telomeric DNA into unstable sites during the repair of double-strand breaks (Bolzán & Bianchi, 2006;Lin & Yan, 2008;Meyne et al., 1990). In insects, ITSs consisting of the (TTAGG) n motif were so far identified only in a species with holokinetic chromosomes, the vapourer moth Orgiya antiqua (Linnaeus) (Rego & Marec, 2003). This species has a reduced chromosome number, and the observed ITSs most probably reflect remnants of multiple chromosome fusions of ancestral chromosomes.
The giant water bugs Belostomatidae play an important role as biological agents in freshwater ecosystems because they are intermediate-stage predators in the food chain of their communities and are useful in the control of the most efficient vector species for malaria and dengue transmission, Aedes and Anopheles, given that they feed effectively on their larvae and pupae (Kweka et al., 2011;Saha, Aditya, Bal, & Saha, 2007;Schaefer & Panizzi, 2000;Sivagnaname, 2009). In the genus Belostoma (Heteroptera, Belostomatidae), previous cytogenetic studies showed that 17 species differ from one another in chromosome number, sex chromosome system, and several other chromosomal characters (Bozini Gallo et al., 2017;Chirino & Bressa, 2014;Chirino, Papeschi, & Bressa, 2013;Papeschi & Bidau, 1985;Papeschi & Bressa, 2006). This genus is the most diverse by including 61 species mainly distributed from Colombia and Brazil to Argentina and Chile (Heckman, 2011;Polhemus & Polhemus, 2008;Ribeiro & Estévez, 2009;Schnack, 1976). However, species delimitation is difficult due to they are very similar in coloration and appearance, only males or rarely only females can be identified, and there is no efficient key ( Figure 1). Besides, it was also found out that Argentinean and Brazilian allopatric populations of both B. candidulum Montandon and B. cummingsi De Carlo, which are geographically separated by long distances and are restricted to small geographic areas (Ribeiro, 2007;Ribeiro & Estévez, 2009), should be considered as chromosomal races or cryptic species by having different chromosome complements (Bozini Gallo et al., 2017;Chirino & Bressa, 2014;Papeschi & Bidau, 1985). In Belostoma, it has been proposed that the ancestral chromosome number of 2n = 26 + XY/XX (male/ female) changed during speciation by fragmentation of the X chromosome, resulting in a multiple sex chromosome system and the male karyotype of 2n = 26 + X 1 X 2 Y while preserving the ancestral pair of NOR-autosomes. Alternatively, several autosome-autosome fusions and a fusion between the ancestral sex chromosome pair and the pair of NOR-autosomes led to reduced chromosome numbers (2n = 14 + XY, 2n = 12 + XY, 2n = 6 + XY) and the increase in chromosome size. The fusion of sex chromosomes with both NOR-autosomes has been demonstrated by the presence of major ribosomal DNA (rDNA) clusters in both X and Y chromosomes (Chirino & Bressa, 2014;Chirino et al., 2013;Papeschi & Bressa, 2006). In male meiosis of all Belostoma species studied, at least one chiasma per bivalent is found, which is thought to be necessary for the regular segregation of homologs to opposite poles during meiosis I. The terminal/ subterminal end-to-end connections between homologs facilitate their recombination and help to align them at metaphase plate, and thus ensure the pole-to-pole orientation of homologous chromosomes (Chirino & Bressa, 2014;Chirino et al., 2013;Papeschi, 1988;Papeschi & Bidau, 1985;Papeschi & Bressa, 2006). Hence, one could F I G U R E 1 Belostoma giant water bugs from Argentina cytogenetically analyzed. Bar = 1 cm argue that the telomeres are hot spots of pairing and recombination as they are restricted to chromosome ends.

| Chromosome preparations
Specimens were brought to the laboratory alive and their gonads dissected out in a physiological solution, swollen in a hypotonic solution, and fixed (Chirino et al., 2013). Gonads were transferred into a drop of 60% acetic acid, and their cells were dissociated with the help of tungsten needles and spread on the slide using a heating plate at 45°C (Traut, 1976). The preparations were dehydrated in an ethanol series (70%, 80%, and 96%, 30 s each) and stored at −20°C until use.

| Fluorescence in situ hybridization (FISH)
Chromosome preparations were removed from freezer, dehydrated in an ethanol series, and air-dried. The preparations were treated with 10 mmol/L HCl for 10 min at 37°C in shaking water bath to remove cytoplasm, washed three times in 2× SSC for 5 min each at RT, digested with 100 μg/ml RNase A (Sigma-Aldrich, St. Louis, MO, USA) in 2× SSC for 60 min at 37°C in a humid chamber, and incubated in 5× Denhardt's solution (50× Denhardt is 1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin) for 30 min at 37°C (Sahara et al., 1999). Then, they were denatured in 70% deionized formamide for 3 min 30 s at 68°C, dehydrated in a cold ethanol series, and air-dried.
For each slide, 10 μl of hybridization mixture containing 50 ng of the biotin-labeled telomere probe, 10 μg of salmon sperm DNA (Sigma-Aldrich), 70% deionized formamide, and 20% dextran sulfate in 2× SSC was used. The mixture was denatured for 5 min at 90°C and immediately chilled on ice for at least 3 min. After denaturation, 10 μl of the mixture was spotted on each slide, and the slides were incubated overnight at 37°C in a humid chamber. Posthybridization washes, de-tection of hybridization probe signals using Cy3-conjugated streptavidin (Jackson ImmunoRes. Labs. Inc., West Grove, PA, USA), and one round of amplification with biotinylated antistreptavidin (Vector Labs. Inc., Burlingame, CA, USA) and Cy3-conjugated streptavidin were performed (Sahara et al., 1999). The preparations were coun- BioChemika, Sigma-Aldrich Production GmbH, Buchs, Switzerland) and mounted in antifade based on DABCO (Sigma-Aldrich Production GmbH, Buchs, Switzerland).

| Microscopy and image processing
Preparations were observed in a Leica DMLB epifluorescence microscope equipped with a Leica DFC350 FX CCD camera and Leica IM50 software, version 4.0 (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). Black-and-white images were recorded separately for each fluorescent dye. Images were pseudo-colored (light blue for DAPI and red for Cy3) and processed with Adobe Photoshop CS6 version 6.1 (1999-2012) software (Adobe Systems Inc.). for contrasts between treatments, as the data were not normally distributed and were not homoscedastic (Daniel, 1990). Statistical analyses were performed using Statview software (SAS Institute, 1992.

| RESULTS
FISH experiments with (TTAGG) n probe showed twin hybridization signals at terminal and/or subterminal positions of the autosomes and sex chromosomes in all six Belostoma species (Figure 2). The telomeric signals were observed at different stages of mitosis in both sexes as well as in male meiosis. However, in some chromosome complements, not all chromosome ends showed hybridization signals.
In species with a high chromosome number (2n = 26 + X 1 X 2 Y/ X 1 X 1 X 2 X 2 , male/female), namely B. dentatum (Figure 2a) Figure 2i). However, more interstitial signals were observed at mitotic prophase and pachytene stages than at metaphase I (Figure 2f, i). These discrepancies likely suggest that ITSs are present in a low copy number to be detected by standard FISH when the chromosomes are highly condensed. We also observed differences in telomere signal intensities. Both the telomeric and ITS signals were not always balanced between homologous chromosomes within the same cell and in the same chromosome. This variation could be partly due to the structure and condensation of chromatin and partly reflect the length of telomeric sequences.
In addition, we observed differences in TCL between the six spe- between the presence and/or absence of interstitial signals and chromosome number (Figure 2).
Nevertheless, the presence of (TTAGG) n motif was recently confirmed in the telomeres of many phylogenetically distant Auchenorrhyncha  previously published cytogenetic data (Chirino & Bressa, 2014;Chirino et al., 2013;Papeschi, 1988Papeschi, , 1994Papeschi, , 1996Papeschi & Bidau, 1985;Papeschi & Bressa, 2006) support a hypothesis that the karyotype evolution in Belostoma species proceeded through fragmentation of the ancestral X chromosome and several autosome and/or autosome-sex chromosome fusions. In species with the modal diploid number of autosomes (26) and the multiple sex chromosome system (X 1 X 2 Y/X 1 X 1 X 2 X 2 ), only true telomeric signals were found at the ends of chromosomes. However, in species with reduced autosome numbers (14, 12, 6) and the XY/XX sex chromosome system, several sites with ITS were identified in addition to the terminal telomeric sequences.
The presence of ITS in B. micantulum, B. candidulum, and B. oxyurum chromosomes as well as differences in the number of hybridization signals detected by FISH suggest the origin of their karyotypes by means of several telomere-telomere fusions of chromosomes of the ancestral karyotype (2n = 26 + XY/XX) (Figure 3). Thus, the B. micantulum karyotype (2n = 14 + XY/XX) probably originated by six telomere-telomere fusions of the ancestral chromosomes, five of them between autosomal pairs, and one between an autosomal pair and the sex chromosome pair. Correspondingly, six pairs of chromosomes showed ITSs and two pairs were without ITS (Figures 2e and 3c). A similar mechanism of telomere-telomere fusions could be involved in the origin of B. candidulum from Brazil (2n = 16) (Figure 3d; revised in Chirino & Bressa, 2014). In accordance with this hypothesis, the B. candidulum karyotype from Argentina (2n = 12 + XY/XX) can be explained by an extra fusion between two pairs of autosomes, resulting in a pair of very large autosomes which bear two ITS sites each (Figures 2d   and 3d). Finally, the karyotype of B. oxyurum (2n = 6 + XY/XX) could originate by a total of ten fusions, resulting in the largest pair of autosomes with three ITS sites, the second pair with three ITS sites, the third pair with only one ITS site, and the sex chromosome pair with one ITS (Figures 2f and 3e). Therefore, the most parsimonious mechanism could be the independent fusion in tandem of chromosomes of ancestral species leading to the observed karyotypes in the analyzed species of Belostoma.
As telomeres are required for maintaining chromosome stability and integrity (Blackburn, 1991;Fajkus et al., 2005;de Lange, 2004;Louis & Vershinin, 2005), a prerequisite for the formation of telomere-telomere fusions should be either elimination or inactivation of telomeres. Therefore, terminal chromosomal fusions imply that the interstitial telomere sequences became dysfunctional. Epigenetic modification of DNA is likely the factor that confers the stability of ITS in Belostoma because these repetitive sequences could be hypermethylated, leading to the protection of the chromosomal integrity by gene disruption, repressing recombination, and silencing of neighboring gene replication (Benetti, García-Cao, & Blasco, 2007;Gonzalo et al., 2006). However, we cannot exclude other options, such as amplification that would lead to different patterns of ITS between homologous chromosomes, deletion that would lead to the absence of ITS in some homologs, and substitution that would produce several hundred base pairs of tandem repeats with many degenerate units (Bolzán & Bianchi, 2006;Danjinou et al., 1999;Fajkus et al., 2005;Lin & Yan, 2008).
In summary, Belostoma constitutes a very interesting group from a cytogenetic point of view, as it exhibits a great variety of chromosome complements with simple and multiple sex chromosome systems. The procedures applied here provide very valuable cytogenetic markers to compare karyotypes of phylogenetically related species. They may also contribute to a better understanding of taxonomic relationships and elucidate the high plasticity of nuclear genomes at the chromosomal level and the potential for genome modification in the course of the speciation processes.