The genus Lethocerus Mayr, 1853 is a member of the family Belostomatidae (electric light bugs, toe biters), the subfamily Lethocerinae (Perez Goodwin 2006). The giant water bug Lethocerus patruelis is the largest European true bug and the largest European water insect. The adult bugs reach 80 mm in length. The information on the karyotypesof the genus Lethocerus has been recently summarized by Bardella et al. (2012). In Lethocerus species, chromosome numbers vary from 2n = 4 to 2n = ca. 30 with intermediate numbers of 2n = 8, 26 and 28. The cytogenetic mechanisms of sex determination are also diversified with XY, neo-XY and multiple X_nY encountered in different species. In several species, a pair of m-chromosomes (= microchromosomes) has been described (Ueshima 1979). As is common in Belostomatidae and in Heteroptera as a whole, all so far studied species of Lethocerus have been shown to have an inverted meiosis for the sex chromosomes in males (the so-called “sex chromosome post-reduction”) with the sex chromosomes undergoing equational separation during the first division while reductional segregation during the second division (Ueshima 1979, Papeschi and Bressa 2006, Bardella et al. 2012).

In the present work, we studied for the first time the structure of the internal reproductive organs, karyotype and meiosis in males of Lethocerus patruelis (Stål, 1854). In addition, we summarize here data on the contemporary distribution of Lethocerus patruelis in Bulgaria and in the northern Aegean Islands, and provide the first information on the reproduction of this species in Bulgaria.

The range of Lethocerus patruelis includes Balkan Peninsula, Anatolia, Israel, Syria, Saudi Arabia, Yemen, the United Arab Emirates, Kuwait, Iraq, Iran, Afghanistan, Oriental Region (Pakistan, India, Nepal, Burma), and recently this species was recorded from Italy (Polhemus 1995, Protić 1998, Perez Goodwyn 2006, Olivieri 2009, Fent et al. 2011).

In Bulgaria, only few records of Lethocerus patruelis specimens migrating from southern parts of the Balkan Peninsula, attracted to light, were published up to 2000 year (Buresch 1940; Josifov 1960, 1974, 1986, 1999) (Fig. 8). During the last ten years, many new findings of Lethocerus patruelis were made by us in Bulgaria: Kresna Gorge, Eastern Rhodopes, Maritsa River Valley (from Kapitan Andreevo to Peshtera) and southern Black Sea Coast (near Burgas). In some of these regions, this species was very abundant; more than 60 specimens per night were attracted to light (Kapitan Andreevo Checkpoint, August–September 2011).

A number of facts (records of the breeding population in Marena; the existence of similar habitats in other regions with records of Lethocerus patruelis at light; the last years’ tendency to milder winters) led us to suppose that this species would breed successfully also in other regions in southern Bulgaria (Maritsa River Valley, Burgas lakes). If such is the case, it would be a further evidence of the recent changes of European bug fauna caused by climate changes and global warming (Rabitsch 2008).

We have studied Lethocerus patruelis in respect of male reproductive organs, karyotype and meiosis. The internal reproductive system in this species appeared to be quite similar to that in Diplonychus rusticus (Fabricius, 1871) (Belostomatinae), the only belostomatid species studied so far on this point (Pendergrast 1957, as Sphaerodema rusticum). In Lethocerus patruelis, each testis consists of the only follicle which is rolled up into a ball; each vas deferens is dilated to form a large vesicula seminalis; bulbus ejaculatorius (representing usually, if present, a dilated anterior end of the ductus ejaculatorius) and accessory glands (diverticula of the ductus ejaculatorius) are absent. Pendergrast (1957) found a similar condition in Diplonychus rusticus, however he did not provide information about the number of follicles in testes.

We found that Lethocerus patruelis had 2n = 26 (22 + 2m + XY). The eight Lethocerus species studied so far with respect to karyotypes (Table 1) represent a large proportion of the 22 species currently known in this genus (Perez Goodwyn 2006). Hence, some preliminary inferences about cytological features of Lethocerus and also of the family Belostomatidae as a whole can be deduced.

Belostomatidae are composed of 3 subfamilies (Belostomatinae, Horvathiniinae, Lethocerinae) with 10 genera and approximately 150 species (Lauck and Menke 1961, Schuh and Slater 1995, Perez Goodwyn 2006, Ribeiro 2007). Up to now, there have been cytogenetically analyzed 32 species of the following 6 genera: Abedus (1 species), Belostoma (18 species) and Diplonychus (3 species) from Belostomatinae and Benacus (1 species), Kirkaldyia (1 species) and Lethocerus (8 species) from Lethocerinae (Table 1). In Belostomatidae, chromosome numbers vary from 2n = 4 in Lethocerus sp. from Michigan to 2n = ca. 30 and 2n = 30 in Lethocerus uhleri and Belostoma dilatatum, respectively. Similarly, the cytogenetic mechanisms of sex determination are variable. Among those, there is the simple XY system (inherent in each of the genera), and the derived neo-XY (in Lethocerus) and multiple X₁X₂Y or X₁X₂X₃Y (in Belostoma) systems. In several species, both from Belostomatinae (Belostoma and Diplonychus) and Lethocerinae (Lethocerus), the presence of m-chromosomes has been reported.

The genus Lethocerus shows a fairly wide range of chromosome numbers, with both extreme for Belostomatidae 2n = 4 and 2n = ca. 30, and three intermediate ones of 2n = 8, 26 and 28 (Table 1). The species studied share the conventional cytological features of Heteroptera, such as holokinetic chromosomes (lacking centromeres, that facilitates karyotype evolution via occasional fusion/fission events; Kuznetsova et al. 2011), an XY sex chromosome system (with derivative neo-XY and multiple X_nY systems presumed to be inherent in three species), and m-chromosomes (detected to date in Lethocerus patruelis and suggestedin Lethocerus uhleri, see Table 1). Within the genus, Lethocerus patruelis seems to be similar to Lethocerus indicus in chromosome complement. This resemblance is confined not only to chromosome number and the presence of m-chromosomes but also to that the sex chromosomes in Lethocerus patruelis (present paper) and Lethocerus indicus (Bagga 1959, Jande 1959) are indistinguishable from autosomes at meiotic metaphases if a routine staining is used. In Lethocerus patruelis, it was due to the size resemblance of X and Y chromosomes causing the almost homomorphic form of the XY-pseudobivalent at MI. Noteworthy however that Lethocerus indicus was speculated to have the sex chromosome system of a neo-XY type originated as a result of the evolutionary translocation of both sex chromosomes to one pair of autosomes in the ancestral karyotype (Jande 1959). Another example of a neo-XY system seems to be Lethocerus sp. from Michigan. For this species, Chickering and Bacorn (1933) reported 2n = 4 with no distinguishable sex chromosomes. These authors suggested that this karyotype might has originated via a translocation of X and Y chromosomes to one pair of autosomes with a subsequent fusion between two more pairs of autosomes.

Ueshima (1979) considered the karyotype of 2n = 24 + 2m + XY as the modal (the commonest) in the genus Lethocerus and the ancestral (i.e., plesiomorphic) one in its evolution. All other karyotypes were suggested to have originated from this ancestral one through autosome fusions and fragmentations, translocations of sex chromosomes to autosomes and loss of m-chromosomes (see Fig. 12 in Ueshima 1979). However, here it should be noted that the most common karyotype needs not to be plesiomorphic in a group (White 1973). In addition, the data available at that time for Lethocerus (see Table 4 in Ueshima 1979) were in fact not indicative of the modality of 2n = 24 + 2m + XY in the genus, and some data presented in Ueshima’s scheme were not universally correct (Fig. 9). For example, in the karyotype formulae of some of the species (for instance Lethocerus sp. from New Orleans) Ueshima included m-chromosomes which however have not been mentioned in the original paper (Table 1).

On the other hand, the ancestrality of a XY sex determination in Lethocerus is beyond question, since neo-XY and X_nY systems occurring in Belostomatidae (both), including Lethocerus (at least neo-XY), are clearly derivative being originated by X-autosome fusions or X-chromosome fissions, respectively. It cannot be doubted also that low chromosome numbers such as 2n = 8 in Lethocerus americanus and 2n = 4 in Lethocerus sp. from Michigan, are the derived characters brought about a series of autosome fusions during the course of evolution in this genus.

It seems likely that the ancestral karyotype in Lethocerus includes 26 autosomes and XY mechanism as found in many representatives of this genus and Belostomatidae as a whole (Table 1). It is not possible even to suggest whether this karyotype includes a pair of m-chromosomes as was speculated by Ueshima (1979). It is evident that these minute chromosomes easily escape detection by bug cytogeneticists, and hence many species recorded as lacking m-chromosomes in fact have them in their karyotypes.

CMA₃ staining of Lethocerus patruelis C-banded chromosomes revealed GC-rich sites corresponding to NORs in the X and Y chromosomes. This is the first case of NOR detection in Lethocerus. On the other hand, ribosomal genes have been already located in Belostoma chromosomes using various techniques such as fluorochrome staining, silver staining and FISH (Table 1). In Belostoma, five species were shown to have NORs also in sex chromosomes while three other species in a pair of autosomes. Noteworthy, the species with the same chromosome complement sometimes differ in rDNA location (for example, in sex chromosomes in Belostoma cummingsi while in autosomes in Belostoma dentatum, both with 2n = 26 + X₁X₂Y).

In the greatest majority of living organisms, during the first division of meiosis all chromosomes reduce in number (reductional division), whereas during the second division the chromatids separate (equational division), and this pattern is named “pre-reduction” (White 1973). However true bugs characteristically have an inverted sequence of sex chromosome divisions in male meiosis, the so-called sex chromosome “post-reduction” when sex chromosomes undergo equational division at anaphase I and reductional division at anaphase II (Ueshima 1979, Kuznetsova et al. 2011), and this is also true for Belostomatidae (Table 1). Interestingly, Lethocerus patruelis appeared unique in showing no inverted sequence of sex chromosome divisions in male meiosis. In this species, X and Y chromosomes form a pseudobivalent at prophase and segregate to opposite poles at anaphase I, and the first division of meiosis is thus reductional both for autosomes and sex chromosomes. As a result of sex chromosome pre-reduction, second spermatocytes carry a single sex chromosome, either X or Y. The second division is then equational for all the chromosomes. Although pre-reduction of sex chromosomes is not usual in Heteroptera, it does occur in some groups (for example, all so far studied species of the family Tingidae have shown pre-reduction; Ueshima 1979, Grozeva and Nokkala 2001). Moreover, closely related species occasionally differ in this pattern (Ueshima 1979, Grozeva et al. 2006, 2007) as is also true of Lethocerus species.

Male meiosis in Heteroptera can further be characterized by radial configuration of one or sometimes both MI and MII plates. In this case, autosomal bivalents at MI and autosomes at MII form a ring on the periphery of the spindle, while the sex chromosomes are located in the center of this ring (Ueshima 1979). However in Lethocerus patruelis, both MI and MII plates are non-radial with random distribution of all the chromosomes on the spindle.