The significance of cytogenetics for the study of karyotype evolution and taxonomy of water bugs (Heteroptera, Belostomatidae) native to Argentina

Abstract Male meiosis behaviour and heterochromatin characterization of three big water bug species were studied. Belostoma dentatum (Mayr, 1863), Belostoma elongatum Montandon, 1908 and Belostoma gestroi Montandon, 1903 possess 2n = 26 + X1X2Y (male). In these species, male meiosis is similar to that previously observed in Belostoma Latreille, 1807. In general, autosomal bivalents show a single chiasma terminally located and divide reductionally at anaphase I. On the other hand, sex chromosomes are achiasmatic, behave as univalents and segregate their chromatids equationally at anaphase I. The analysis of heterochromatin distribution and composition revealed a C-positive block at the terminal region of all autosomes in Belostoma dentatum, a C-positive block at the terminal region and C-positive interstitial dots on all autosomes in Belostoma elongatum, and a little C-positive band at the terminal region of autosomes in Belostoma gestroi. A C-positive band on one bivalent was DAPI negative/CMA3 positive in the three species. The CMA3-bright band, enriched in GC base pairs, was coincident with a NOR detected by FISH. The results obtained support the hypothesis that all species of Belostoma with multiple sex chromosome systems preserve NORs in autosomal bivalents. The karyotype analyses allow the cytogenetic characterization and identification of these species belonging to a difficult taxonomic group. Besides, the cytogenetic characterization will be useful in discussions about evolutionary trends of the genome organization and karyotype evolution in this genus.

tum and B. gestroi are described for the first time. These results allowed us to distinguish morphologically similar species and, also, led us to propose a scenario of karyotype evolution in the genus Belostoma.

Chromosome preparations
The captured specimens were brought alive to the laboratory and reared until their gonads were dissected out. For meiotic analysis, the adults and nymphs were fixed for 15-30 min in freshly prepared fixative (ethanol:glacial acetic acid, 3:1). Afterwards, gonads were dissected out and kept at 4º C in 70% ethanol. Slides were prepared by the squash technique in a drop of 2% iron-propionic haematoxylin following conventional procedures (Sáez 1960). For C-and fluorescent bandings, and FISH technique, gonads were dissected in a physiological saline solution for Ephestia Guenée, 1845 (Glaser 1917: cited by Lockwood 1961, swollen for 15 min in a hypotonic solution (0.075 M KCl), and fixed for 15-30 min in freshly prepared Carnoy fixative (ethanol:chloroform:glacial acetic acid, 6:3:1). Spread chromosome preparations were made in a drop of 60% acetic acid with the help of tungsten needles and spread on the slide using a heating plate at 45° C as described in Traut (1976). The preparations were dehydrated in an ethanol series (70, 80 and 96%, 30 sec each) and stored at -20º C until use.

Fluorescence in situ hybridization
Unlabelled 18S ribosomal DNA (rDNA) probes were generated by polymerase chain reaction (PCR) using universal arthropod primers: forward 5´-CCTGAGAAACG-GCTACCACATC-3´ and reverse 5´-GAGTCTCGTTCGTTATCGGA-3´ (Whiting 2002). Total genomic DNA of Dysdercus albofasciatus Berg, 1878, obtained by standard phenol-chloroform-isoamylalcohol extraction, was used as a template. PCR was done following the procedure described in Fuková et al. (2005). The PCR product showed a single band of about 1,000 bp on a 1% agarose gel. The band was recovered from the gel and purified by using a QIAquick Gel Extraction Kit (Quiagen GmbH, Hilden, Germany). The 18S rDNA fragment was re-amplified by PCR and then labeled with biotin 14-dATP by nick translation using a BioNick Labeling System (Invitrogen, Life Technologies Inc., San Diego, CA, USA). FISH with a biotinylated 18S rDNA probe was carried out following the procedure in Sahara et al. (1999) with several modifications described by Fuková et al. (2005) and Bressa et al. (2009).

C-positive heterochromatin and DNA measurements
Data of C-positive heterochromatin percentage and the haploid DNA content in B. dentatum, B. elongatum and B. gestroi are part of the results obtained by Papeschi in her Ph.D. Thesis (1992). The procedures of C-positive heterochromatin percentage and the haploid DNA content were published by Papeschi in 1991 and1988, respectively. Hence, these results were mentioned only in this paper to analyze and discuss the extent of karyotype uniformity in these three species. Briefly, the study of the C-positive heterochromatin measurements was performed on meiotic cells. For each species at least 10 C-banded cells at diakinesis and without superimposed bivalents were photographed. The percentage of C-positive heterochromatin was calculated as the C-positive area divided by the total chromosome area. The DNA content was carried out by Feulgen microdensitometry (Papeschi 1988) in individuals fixed similar time ago (1-3 months) (Papeschi 1991).

Statistical analysis
The total chromosome length measurements (TCL) were performed with Micro Measure for Windows, version 3.3. The TCL of all bivalents and sex chromosomes were performed in metaphase I. Differences in TCL among species were compared by using one-way analysis of variance (ANOVA), with Fisher adjusted a posterior contrast. Statistical analyses were done using Statview software (SAS Institute Inc., 1992-1998.

Microscopy, photographs and image processing
Preparations were observed in epifluorescence microscopes: Zeiss Laborlux (Carl Zeiss, Germany) equipped with an analogue camera and Leica DMLB equipped with a Leica DFC350 FX CCD camera and Leica IM50 software, version 4.0 (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). Photomicrographs from meiotic chromosome preparations were taken using Kodak colour Supra print film 400 ASA. Blackand-white images of chromosomes from C-and fluorescent bandings and FISH technique were recorded separately for each fluorescent dye with the CCD camera. Images were pseudo-coloured (light blue for DAPI, green for CMA 3 , and red for Cy3), and processed with an appropriate software.

Male chromosome complement and meiosis
Male meiotic karyotypes based on metaphase I autosomal bivalents (II) and sex univalents of B. dentatum, B. elongatum and B. gestroi show a male diploid chromosome number 2n = 13II + X 1 X 2 Y (Fig. 1). In the three species, the autosomes decrease gradually in size, both X chromosomes differ slightly in size and the Y chromosome is the smallest of the complement. The chromosome complement and male meiotic behaviour of B. dentatum have already been described (Papeschi and Bidau 1985). The three species of Belostoma show statistical differences in total chromosome length (TCL) (F 2, 93 = 8.484; P = 0.0004), which is higher in B. dentatum (39.43 ± 3.72 µm), intermediate in B. elongatum (37.03 ± 2.96 µm) and lower in B. gestroi (33.31 ± 3.64 µm).
Analysis of spermatogonial prometaphase of B. elongatum and B. gestroi revealed a diploid number of 29 chromosomes; both karyotypes were as described by Papeschi (1992) (Fig. 2a). Male meiotic behaviour in B. elongatum and B. gestroi was similar and followed the same pattern as previously described for other Belostoma species. Thus, we showed a single and combined Figure 2 with meiotic stages from both species. At synizesis, the first meiotic identifiable stage of meiosis, chromatin condenses eccentrically in the nucleus (Fig. 2b). At pachytene, an autosomal bivalent is associated with the nucleolus and the 13 autosomal bivalents are joined through their positive heteropycnotic terminal regions. The condensed sex chromosomes, close to each other, may be distinguished (Fig. 2c). In this cell, the two X chromosomes have a secondary constriction, but these constrictions are observed in only one specimen of B. elongatum. During the diffuse stage, all bivalents decondense completely, except for some chromocentres (Fig. 2d). In B. gestroi, at early diakinesis, both X chromosomes are negative heteropycnotic, and the Y chromosome is positive heteropycnotic (Fig. 2e, f). At late diakinesis, the three sex univalents and the 13 autosomal bivalents becomes isopycnotic in both species (Fig. 2g,  h). Each bivalent has a single chiasma in either subterminal or terminal positions ( Fig. 2e-h). At metaphase I, autosomal bivalents arrange in a ring, but the Xs and Y univalents do not show a defined position (Fig. 2i). During anaphase I, the bi-  valents divide reductionally, whereas the sex chromosomes do so equationally (Fig.  2j). All telophase I nuclei exhibit 16 chromosomes in each pole (13 + X 1 X 2 Y). The second meiotic division follows without an interkinesis stage. At metaphase II, the 13 autosomes dispose forming a ring and in the centre of it, the sex chromosomes are associated in a pseudo-trivalent. The Y chromosome is negatively heteropycnotic and is oriented towards the opposite spindle pole to that of X 1 and X 2 (Fig. 2k). At anaphase II, 14 chromosomes migrate to one pole (13 + Y) and 15 to the opposite one (13 + X 1 X 2 ) (Fig. 2l).

Chromosome bandings
C-banding reveals differences in the amount and location of heterochromatin among the three species analysed. In B. elongatum, very large C-positive blocks can be detected terminally on all bivalents from prophase I to metaphase I, and interstitial dots are also observed (Fig. 3a-c). In B. gestroi, in contrast, C-positive bands are very small and are always located terminally (Fig. 3d, e). The results observed in B. dentatum matched data previously described by Papeschi (1991) with C-positive bands terminally located in all bivalents (Fig. 3f, g). Furthermore, the two X chromosomes in the three species show terminally located bands, whereas the Y chromosome is C-negative (Fig. 3a, c-g).
All chromosomes stain homogenously with both fluorochromes on mitotic and meiotic metaphase cells in the three species, except for one of the medium-sized autosomal bivalents in B. dentatum (Fig. 4a-c) and B. elongatum (Fig. 4d-f), and one of the large-sized in B. gestroi (Fig. 4g-i), which show a DAPI negative/CMA 3 positive band at one terminal position.

Location of rDNA
In chromosome preparations of B. dentatum, FISH experiments with the 18S rDNA probe show a cluster of rDNA genes located at one end of two homologous chromosomes each (Fig. 5a). A single cluster of signals is observed in an autosomal bivalent at pachytene (Fig. 5b). During diffuse stage, hybridization signals are observed in the decondensed mass of autosomal chromatin, whereas the sex chromosomes remain condensed forming a conspicuous DAPI bright chromatin body without any signals (Fig.  5c). At diakinesis-metaphase I, one medium-sized autosomal bivalent show hybridization signals at both ends (Fig. 5d). In concordance with the results of B. dentatum, in mitotic metaphases of B. elongatum and B. gestroi, hybridization signals are detected in two homologous autosomes (Fig. 5e, g). At diakinesis-metaphase I, a single cluster of rRNA genes is located at both ends of a medium-sized autosomal bivalent of B. elongatum (Fig. 5e-f ) and of a one large-sized of B. gestroi (Fig. 5h).   (g-i). a Oogonial metaphase (2n = 30 = 26 + X 1 X 1 X 2 X 2 ) b Diakinesis c Metaphase II d Spermatogonial metaphase (2n = 29 = 26 + X 1 X 2 Y) e Diakinesis f Metaphase II g Spermatogonial metaphase (2n = 29 = 26 + X 1 X 2 Y) h Diakinesis i Metaphase II. Arrows indicate DAPI negative/CMA 3 positive bands. Arrowheads show sex chromosomes (d, g). X, Y = sex chromosomes. pIII = pseudo-trivalent. Bar = 10 µm.

Discussion
The Belostoma species analyzed here shared apparently similar karyotypes, since they possess the same chromosome complement (2n = 29 = 26 + X 1 X 2 Y, male), with chromosomes progressively decreasing in size. In Belostomatidae, this 2n is the modal diploid chromosome number and is present in 10 species of Belostoma Bressa 2006, Chirino and. On the other hand, in B. elongatum and B. gestroi the male meiotic behaviour followed a similar pattern as previously described for other species of this genus (Papeschi and Bidau 1985, Papeschi 1996, Bardella et al. 2012). Both B. elongatum and B. gestroi, as well as B. dentatum, showed the following cytogenetic characteristics: a) synizesis observed, b) a multiple sex chromosome system (X 1 X 2 Y, male), c) Y chromosome negatively heteropycnotic at metaphase II, d) variation in the TCL and in the C-banding pattern, and e) a single pair of NOR-autosomes. Within Heteroptera, the synizesis stage was described in a few species of Belostoma Bidau 1985, Papeschi 1992) and Dysdercus Guérin-Méneville, 1831 (Bressa 2003. In this stage the chromatin condenses eccentrically in the nucleus and chromosome pairing begins.
Heterochromatin characterization in the three species revealed differences in the amount, distribution and location of the constitutive heterochromatin in autosomes and both X chromosomes: i) terminal C-positive bands in B. dentatum, ii) conspicuous C-positive bands at terminal and interstitial positions in B. elongatum, and iii) very scarce C-positive bands terminally located in B. gestroi. This variation in the constitutive heterochromatin of these three species could imply changes in the DNA content in the karyotype evolution in the genus, which could modify the size of the chromosome complement. In accordance with this suggestion, the analysis of TCL showed a significant variation among the three species, which means that certain chromosomal changes, must have taken place during their evolution. Papeschi (1992) found a great interspecific variation in DNA content as well as differences in C-positive heterochromatin percentage among B. dentatum (1.93 pg, 58.54 %), B. elongatum (1.75 pg, 59.47 %) and B. gestroi (1.13 pg, 37.24 %). Taking into account the data previously described by Papeschi (1992), together with the results obtained from the analysis of the TCL in these three species, we propose the existence of positive relationships between TCL and DNA content and between TCL and C-positive heterochromatin percentage (Fig. 6). Thus, it is apparent that differences in the TCL in the three species might represent changes or variations in DNA content since the accumulation/addition of satellite DNA in all chromosomes of the complement. The comparison between the DNA content and the C-positive heterochromatin percentage of the three species shows a general trend, i.e. an increase in the DNA content is accompanied by an increase in the amount of C-positive heterochromatin. However, B. dentatum and B. elongatum have a very different DNA content and a similar percentage of C-positive heterochromatin. On the other hand, B. gestroi shows the lowest DNA content and the lowest C-positive heterochromatin percentage. In accordance with the earlier reports on six other species of Belostoma (Papeschi and Bidau 1985, Papeschi 1988, 1991, 1992, the genome size differences between B. dentatum and B. elongatum could be due to a proportionate variation of both C-positive heterochromatin and C-negative chromatin occurred during evolution. The chromosomes of B. gestroi could have gained low amount of C-positive heterochromatin, or else during evolution some C-positive bands became lost.
In Heteroptera, the classical distribution pattern of C-positive heterochromatin is terminal in some or all chromosomes. Interstitial C-positive bands are described in a few species and some of them correspond to secondary constrictions and NORs. In concordance with these cytogenetic features, the C-banding pattern observed in B. elongatum with respect to both terminal and interstitial C-positive regions agrees with most previous reports within Belostoma (Papeschi 1995) and Heteroptera (Camacho et al. 1985, Panzera et al. 1995, Grozeva and Nokkala 2001, Angus et al. 2004, Ituarte and Papeschi 2004, Bressa et al. 2005, Franco et al. 2006.
The results with fluorescent banding indicate that all C-positive bands in the species analysed were not enriched with AT or CG base pairs, as all chromosomes were stained homogeneously with both DAPI and CMA 3 fluorochromes, except for the C-positive band observed in the medium-sized autosomal bivalent of B. dentatum and B. elongatum and in one of the large-sized of B. gestroi, which was DAPI negative/ CMA 3 positive. Therefore, the CMA 3 bright band is enriched in GC base pairs and could represent an NOR (see below). The presence of a CMA 3 bright band was also reported not only in other species of Belostoma  but also in other heteropteran species, at interstitial or terminal position, either on autosomes or sex chromosomes, and they are generally associated to NORs (González-García et al. 1996, Papeschi et al. 2001, 2003, Rebagliati et al. 2003, Cattani et al. 2004, Grozeva et al. 2004, Poggio et al. 2011. In Belostomatidae, the location of NORs was previously analysed by FISH with 18S rDNA probe in B. oxyurum (Dufour, 1863) (2n = 6 + XY, NOR in sex chromosomes), B. micantulum (Stål, 1860) (2n = 14 + XY, NOR in sex chromosomes), B. elegans (Mayr, 1871) (2n = 26 + X 1 X 2 Y, NOR in a pair of autosomes) , and Lethocerus patruelis (Stål, 1854) (2n = 22 + 2m + XY, NOR in sex chromosomes) (Kuznetsova et al. 2012). The present paper presents information about the number and chromosomal location of ribosomal clusters in B. dentatum, B. elongatum and B. gestroi, which have a single cluster located in an autosomal pair. In these three species the NOR is associated with a CMA 3 -bright band. The results of the fluorescent banding and FISH in these species agree with those described for B. oxyurum, B. micantulum and B. elegans, in which the NOR regions are colocalized with a CMA 3 -positive band and, therefore, the repeating unit of rDNA is GC-rich . Taking into account the data on the number and location of rDNA clusters along with the type of sex chromosome systems in Belostomatidae, we can observe two different patterns of rDNA distribution. The NOR is located at terminal position on both sex chromosomes in species that have a simple sex chromosome system (XY), or, in contrast, the NOR is placed at terminal position on an autosomal pair in the species with a multiple sex chromosome system (X 1 X 2 Y). Hence, our present results led us to propose that in Belostomatidae the location of rDNA genes could be associated with variants of the sex chromosome systems. Moreover, this relationship between the number and location of the NOR and the sex chromosome system has only been observed in this family of Heteroptera.
Previous cytogenetic data on worldwide Belostomatidae species allowed Papeschi and Bressa (2006) to propose an ancestral male karyotype 2n = 28 = 26 + XY, from  Papeschi (1991Papeschi ( , 1992. which the karyotypes with multiple sex chromosome systems (2n = 26 + X 1 X 2 Y) and those with a low 2n (6 + XY, 14 + XY, 13 + XY, 22 + XY) would have originated by fragmentation of the ancestral X chromosome and chromosomal fusions, respectively. It is generally accepted that multiple sex chromosome systems in Heteroptera are the result of fragmentation(s) of the X and/or Y chromosome(s) of an ancestral simple sex chromosome system (Heizer 1950, Hughes-Schrader and Schrader 1961, Ueshima 1979, Manna 1984, Papeschi 1996. The holokinetic nature of heteropteran chromosomes and the achiasmatic male meiosis of sex chromosomes are the main facts that support this hypothesis and may account for the variability (Ueshima 1979, Manna 1984, Thomas 1987. In most cases of multiple sex chromosomes, the increase in the number of sex chromosomes is not accompanied by a reduction in the number of autosomes. The analysis of different populations of B. orbiculatum Estévez and Polhemus, 2001(Papeschi 1996), B. plebejum (Stål, 1858 (Papeschi 1994), B. dilatatum (Dufour, 1863) (Bardella et. al 2012), Oechalia pacifica (Stål, 1859) (Heizer 1950) and Banasa zeteki Sailer, 1959 (Pentatomidae) (Schrader and Hughes-Schrader 1958), polymorphic for the sex chromosome systems, lend support to the hypothesis of a fragmentation origin of a multiple sex chromosome systems. In all these examples one chromosome of the simple system (XY) was replaced by two chromosomes of smaller size in the mutant individuals. Therefore, these interspecific polymorphisms of sex chromosomes represent a direct evidence of the origin of multiple sex chromosome system through fragmentation in Belostoma. On the other hand, the species of this genus with reduced chromosome numbers are characterized by a larger chromosome size, a low DNA content and very scarce C-positive heterochromatin . These karyotypes probably originated from the ancestral chromosome complement through chromosome fusions. The possibility of their occurrence is supported by the fact that the autosomal fusions have been found in heterozygous condition in natural populations of B. plebejum (Papeschi 1994), Triatoma infestans (Klug, 1834) (Poggio et al. 2013) and Mepraia gajardoi Frías, Henry andGonzález, 1998 (Pérez et al. 2004) (Reduviidae).
Published data on karyotype evolution in species of this genus  along with the present results of rDNA-FISH support the hypothesis that in the ancestral male karyotype (2n = 28 = 26 + XY) the NOR would have been located in a pair of autosomes (Fig. 7). A fragmentation of the single X chromosome in the ancestral karyotype resulted in multiple X chromosomes and led to a karyotype with 2n = 29 = 26 + X 1 X 2 Y, but keeping the ancestral NOR-autosome pair, as represented by B. dentatum, B. elegans, B. elongatum and B. gestroi (Fig. 7). On the other hand, autosomal fusions and the fusion of the ancestral sex chromosome pair (XY) with the autosomes carrying the NOR would result in the reduction in the diploid chromosome (2n = 8, 16), increasing the chromosomes size and led to the presence of rDNA genes in both X and Y chromosomes. Alternatively, the rDNA gene cluster could have been translocated from an autosomal location to the X and Y chromosomes, as it has been proposed in Dysdercus albofasciatus Berg, 1878 (Bressa et al. 2009). Furthermore, future studies within Belostoma about this regular pattern will shed light on the karyotype evolution within the genus and also support the mechanisms involved in the karyotype evolution.
Conventional taxonomy of water bugs has focused almost entirely on adult specimens. There are relatively few publications on interspecific differences among the larvae, and fewer still concern South American species. The literature of the genus Belostoma includes much confusion because, in many cases, the species are very similar in coloration and appearance and only males or rarely only females can be identified (Heckman 2011). In B. dentatum, B. elongatum and B. gestroi, the karyotype analyses allow us to get a detailed characterization and a better knowledge of their chromosomal structure. Hence, we conclude that the cytogenetic studies provide valuable features that can be used to solve problems on taxonomic identification, at least for this genus.