Bed bug cytogenetics : karyotype , sex chromosome system , FISH mapping of 18 S rDNA , and male meiosis in Cimex lectularius Linnaeus , 1758 ( Heteroptera : Cimicidae )

Bugs (Insecta: Heteroptera) are frequently used as examples of unusual cytogenetic characters, and the family Cimicidae is one of most interest in this respect. We have performed a cytogenetic study of the common bed bug Cimex lectularius Linnaeus, 1758 using both classical (Schiff-Giemsa and AgNO3-staining) and molecular cytogenetic techniques (base-specifi c DAPI/CMA3 fl uorochromes and FISH with an 18S rDNA probe). Males originated from a wild population of C. lectularius were found to have 2n = 26 + X1X2Y, holokinetic chromosomes, 18S rRNA genes located on the X1 and Y chromosomes; achiasmate male meiosis of a collochore type; MI and MII plates nonradial and radial respectively.


INTRODUCTION
The bеd bug genus Cimex Linnaeus, 1758 is a relatively small group of highly specialized hematophagous ectoparasites, with 17 species distributed primarily across the Holarctic and associated with humans, bats, and birds (Schuh, Slater, 1995;Simov et al., 2006).Till now, the chromosomal complement and different aspects of chromosome behavior during male meiosis have been studied in 14 Cimex species (Ueshima, 1963(Ueshima, , 1966;;Grozeva, Nokkala, 2002; for other references see Ueshima, 1979), including the common bed bug C. lectularius Linnaeus 1758 (Slack, 1938(Slack, , 1939a, b;, b;Darlington, 1939;Ueshima, 1966).As is the case with other Heteroptera, C. lectularius displays holokinetic chromosomes, i.e. chromosomes having, instead оf localized centromere, a kinetochore plate spread along their whole or almost whole length.Among several peculiarities of Cimex cytogenetics, multiple sex chromosome systems are the most conspicuous.Although, within Cimicidae, multiple sex chromosomes are not unique to the genus Cimex (some species in at least nine other Cimicidae genera have multiple X chromosomes; see Ueshima, 1979), the number of X is, by far, greatest in C. lectularius.In the latter, the number of autosomes is consistently 26, whereas the number of X chromosomes varies from two (X 1 X 2 Y) to 15 (X 1 X 2 Y + 13 extra Xs) in different populations and sometimes between males of one population.Autosomal bivalents show normal a sequence Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics of meiotic divisions, with homologous chromosomes segregating in the fi rst round of meiosis, and sister chromatids separating in the second.This order is however reversed in sex chromosomes, and this is true even where 15 Xs exist.Sex chromosomes always undergo post-reductional meiosis, i.e. the equational separation at anaphase I and the reductional segregation at anaphase II, the sex chromosome behavior typical of the Heteroptera.Autosomal bivalents are suggested to be chiasmate, with the single chiasma being formed in every bivalent (Ueshima, 1966(Ueshima, , 1979)).
All the published cytogenetic studies on C. lectularius have used a standard chromosome technique.In the present study, a wild North-Western Russian population of C. lectularius was analyzed using the standard Schiff-Giemsa technique, AgNO 3 -staining, fl uorochrome DAPI/CMA 3 -banding, and fl uorescent in situ hybridization (FISH) with an 18S rDNA probe.As a result, the karyotype, sex chromosome system, the distribution and nucleotide sequences of C-heterochromatin regions, chromosomal location of 18S rRNA genes, and meiosis in males are reported here.

Insects
Adults and nymphs of Cimex lectularius were collected in 2009 from a wild population (St.Petersburg).A total of 6 males were studied.Specimens were fi xed alive in 96% ethanol: glacial acetic acid (3:1) and stored in fi xative at 4 o C until further use.

Preparations
The gonads were dissected out and squashed in a drop of 45% acetic acid.The cover slip was removed by the dry ice method.Slides were dehydrated in fresh fi xative and air dried.The preparations were fi rst analyzed with a phase contrast microscope at 400x.The best 28 chromosome preparations were used for different staining techniques.

Standard staining
To study the number and behavior of chromosomes, the preparations were stained following the Schiff-Giemsa method developed by Grozeva, Nokkala (1996).The preparations were fi rst subjected to hydrolysis in 1 N HCl at room temperature for 20 min, then in 1 N HCl at 60°C for 8 min, and stained in Schiff's reagent for 20 min.After rinsing thoroughly in distilled water, the preparations were additionally stained in 4% Giemsa in Sorensen's buffer, pH 6.8 for 20 min, rinsed with distilled water, air-dried, and mounted in Entellan.
AgNO 3 -staining To check the localization of nucleolar organizer regions (NORs), the 1-step method with a protective colloidal developer (Howell, Black, 1980) was followed.

Fluorochrome banding
To reveal the base composition of C-heterochromatin, staining by GC-specifi c chromomycin A 3 (CMA 3 ) and AT-specifi c 4-6-diamidino-2-phenylindole (DAPI) were used according to Schweizer (1976), andDonlon, Magenis (1983) respectively, with some modifi cations.C-banding pretreatment was fi rst carried out using 0.2 N HCl at room temperature for 30 min, followed by 7-8 min treatment in saturated Ba(OH)2 at room temperature and then an incubation in 2x SSC at 60°C for 1 h.The preparations (without Giemsa) were stained fi rst with CMA 3 (2,5 μg/ ml) for 25 min and then with DAPI (0.4 μg/ ml) for 5 min.After staining, the preparations were rinsed in the McIlvaine buffer, pH 7 and mounted in an antifade medium (700 μl of glycerol, 300 μl of 10 mM McIlvaine buffer, pH 7, and 10 mg of N-propyl gallate).

Microscopy and imaging
Chromosome preparations were analyzed under a Leica DM 4000B microscope (Leica Microsystems Wetzlar GmbH, Germany) with a 100x objective.Fluorescence images were taken with a Leica DFC 350 FX camera using Leica Application Suite 2.8.1 software with an Image Overlay module.The preparations were stored partly at the Institute of Biodiversity and Ecosystem Research, BAS in Sofi a and partly at the Zoological Institute, RAS in St Petersburg.

RESULTS
We analyzed meiosis in males of C. lectularius using the standard chromosome technique, AgNO 3 -staining, fl uorochrome DAPI/CMA 3 staining and 18S rDNA FISH technique.Based primarily upon the behavior of chromosomes in the consecutive stages of meiosis, the chromosome complement in males was expressed as a formula 2n = 29 (26A + X 1 X 2 Y).Although females were not studied, sex chromosome mechanism was referred to a X 1 X 2 Y system (rather than XY 1 Y 2 ) due to the Ueshima's (1966) observations of several populations of C. lectularius in which females showed 4Xs (X 1 X 2 X 3 X 4 ) while males 2Xs  9).The location of the NORs on the sex chromosomes was confi rmed by the AgNO 3 -staining (Fig. 10) The mitotic chromosomes gradually decrease in size and lack visible constrictions since they are holokinetic.Consequently, neither homologous chromosomes nor sex chromosomes are possible to identify in the routinely stained preparations (not shown).The X 1 and Y chromosomes can be however easily detected both in metaphase and prometaphase cells after FISH with the 18S DNA probe (Figs 1-3).The majority of the mitotic cells show two chromosomes, most likely X 1 and Y, with clusters of rRNA genes (Figs 1, 2); however, hybridization signals are well over two in some prometaphase cells (Fig. 3).
Among the meiotic stages, those referred to the prophase and early metaphase I (MI) are of chief interest for characterisation of male meiosis in C. lectularius.During prophase the chromosomes gradually condense from leptotene to pachytene, and no diplotene or diakinesis stages are observed.In the course of condensation, the homologues of bivalents open out, while remaining connected with each other, at least, at one site by tenacious threadlike structures (Figs 4,5).By the metaphase I (MI), bivalents are completely condensed and consist of parallel-aligned chromosomes with occasionally one or both ends diverging, and no signs of the existence of chiasmata are seen during this stage (Figs 6-9).Taken together these observations are indicative of achiasmatе meiosis, the connecting threads between homologues representing the collochores previously described in several Miridae species (Nokkala, Nokkala, 1986) and recently also in C. emarginatus Simov, Ivanova, Schunger, 2006(as Cimex sp. in Grozeva, Nokkala, 2002).
At MI, 13 autosomal bivalents and 3 sex chromosome univalents are observed (Figs 7-9).MI plates are nonradial with X 1 , X 2 , and Y chromosomes located among the bivalents.The sex chromosomes are usually recognized at this stage because they are composed of two chromatids (contrary to 4 in autosomal bivalents).The autosomal bivalents are of gradually decreasing size with neither very large nor very small entities as do the autosomes at the mitotic metaphases.The sex chromosomes are similar in size to the larger half-bivalents, the X 1 being clearly longer than the X 2 while of similar size with the Y. Sex chromosomes, X 1 and Y at least, tend to be located close to each other or even connected by ends probably due to the presence of  8).Among the 26 MI cells examined, only two showed X 1 and Y chromosomes lying separately (Fig. 9).
In two MI nuclei after DAPI and CMA 3 -staining respectively, the autosomal bivalents and one of the Xs (X 2 ) display a fairly homogenous CMA 3 /DAPI staining, however at least two bivalents (arrowed) show CMA 3 /DAPI positive, AT/GC rich heterochromatic regions on the telomeres of every homologue (Figs 11,12).The other X (X 1 ) and the Y chromosome exhibit several CMA 3 -bright bands, two-three on the X 1 and one-two on the Y, indicating they are GC-rich.FISH experiments are in general agreement with the results of CMA 3 -staining, i.e. the 18S rDNA loci are co-localized with some of the CMA 3 -bands suggesting that these CMA 3positive sites represent rDNA cistrons.X 1 and Y chromosomes each show hybridization signals indicating clusters of 18S rRNA genes are located near the end of X 1 and near the end of Y chromosomes respectively (Figs 7-9).Noteworthy is a size polymorphism for the 18S rDNA clusters on X 1 and Y, which is occasionally observed even within the same male (Figs 6-9).
The daughter metaphase II (MII) cells each contain 16 chromosomes, including 13 autosomes, 2Xs, and the Y (Figs 14-17), indicative of the reductional segregation of autosomes and equational separation of sex chromosomes at anaphase I (AI) (Fig. 13).At MII, sex chromosomes undergo a characteristic "touch-and-go" pairing, with 2Xs lying on one side of the equatorial plate and the Y on the other.In contrast to MI, metaphase II (MII) plates are clearly radial, and sex chromosomes appear here as a pseudotrivalent located inside a ring formed by autosomes.
At anaphase II (AII), autosomes divide equationally, but sex chromosomes undergo a reductional division, i.e. both X chromosomes moved to one pole and the Y to the other (the so-called sex chromosome post-reduction) as evidenced by the only 18S rDNA/FISH signal in every daughter telophase II (TII) plate (Fig. 18) as well as in every spermatid and every sperm (Figs 19,20).

DISCUSSION
The common bed bug, Cimex lectularius, is one of the most widely recognized insects all over the world.Slack (1938) was the fi rst to study chromosome cytology of C. lectularius.Shortly afterwards Darlington (1939) and then Ueshima (1966Ueshima ( , 1967Ueshima ( , 1979) ) studied and discussed the unique aspects of male meiosis in this species.Based on the observations of these authors, C. lectularius males have the standard complement of 2n = 26 + X 1 X 2 Y, the X chromosomes varying in number from two (X 1 X 2 Y) to 15 (X 1 X 2 Y + 13 extra Xs) in different populations while occasionally between males of the same population and even between cells of the same male.For example, Ueshima (1966) has investigated males and females in six populations originated from USA (Berkeley, California; Columbus, Ohio), Mexico (Monterey; La Piedad), Japan (Nagasaki), France (Durtal), Egypt (Cairo), and Czech Republic (Moravia) respectively.The number of sex chromosomes was shown to be stable within every population (2Xs in Berkeley, La Piedad, Nagasaki, and Durtal; 6Xs in Cairo and Moravia) except for the Ohio population, in which males had either 7Xs or 9Xs.Notice that the transmission of additional sex chromosomes throughout meiosis was, except in a very few cases, quite regular, and they seemed not to be important for sex determination.
Multiple (above two) X chromosomes have been described in both natural populations and laboratory stocks of C. lectularius.According . Cytogenet., 2010 4(2) Comparative Cytogenetics

Comp
to Darlington (1939) the average number of Xs is higher in wild populations than in laboratory cultures, however a closer look at the presently available data on this species is called for.The most common sex chromosome mechanism in the Heteroptera is XX/XY system, and the multiple sex chromosome systems are suggested to have originated from the original XY system (Ueshima, 1979).In the Cimicidae, a total of 45 species have been hitherto cytologically studied and both putative ancestral XY and a great number of derived multiple systems, among which X 1 X 2 Y clearly prevails, were found (Ueshima, 1966, 1979 andreferences therein;Manna, 1984;Grozeva, Nokkala, 2002;Poggio et al., 2009).This is also true for the genus Cimex in which as many as 14 of the 17 described species have been studied (Ueshima, 1963(Ueshima, , 1966(Ueshima, , 1967;;Grozeva, Nokkala, 2002; for other references see Ueshima, 1979).The origin of multiple systems in the Heteroptera is usually ascribed to simple transverse dissociation (fi ssion) of the original X chromosome (Schrader, 1947;Ueshima, 1966Ueshima, , 1979)), the process which is facilitated by the holokinetic nature of the bugs' chromosomes.The distinguishing features of multiple sex chromosome systems formed by dissociation are that the newly originated sex chromosomes are smaller than the original ones, and there is no accompanying change in the number of autosomes in a derived complement compared to that with XY sex chromosome system.Ueshima (1966) has discovered XY, X 1 X 2 Y, and X 1 X 2 X 3 X 4 Y systems in 7 species of the Cimex pilosellus (Horváth, 1910) complex and argued for the dissociation hypothesis on the basis that as the number of X chromosomes increases, their size decreases.However this problem clearly calls for further investigation using modern cytological techniques.It is noteworthy that the application of C-banding to study the chromosomes of several Triatominae (Reduviidae) species, led Panzera et al. (2010) to the conclusion that chromosomal rearrangements other than dissociations might have been involved in the formation of the multiple sex chromosome systems in the Heteroptera.
As is typical in the Heteroptera, chromosomes of C. lectularius are holokinetic, i.e. without localized centromere.In males studied herein meiosis follows a standard for the Heteroptera pattern as previously described in other populations of this species (Slack, 1939b;Darlington, 1939;Ueshima, 1966Ueshima, , 1979)).The sex chromosomes behave as univalents during the fi rst round of meiosis and undergo equational separation at anaphase I.At metaphase II, the Xs and Y appear associated end-to-end to form a pseudotrivalent, which is located inside the ring of autosomes.During anaphase II, Xs and Y chromosomes undergo reductional division segregating to opposite poles.This reversed order of sex chromosome behavior during meiotic divisions is characteristic of the Heteroptera, referred to as inverted meiosis or sex chromosome postreduction (Hughes-Schrader, Schrader, 1961).
At present, the cytogenetic studies of the Heteroptera are mainly focused on C-heterochromatin and nucleolus organizer regions (NORs), whereas molecular cytogenetic techniques such as immunofl uorescence ones and different modifi cations of FISH (fl uorescent in situ hybridization), including chromosome painting, BAC-FISH technique, and GISH/FISH mapping of genes, are not yet used in the Heteroptera.By now, some of these techniques have been applied to some economically important holokinetic insects (aphids Acyrthosiphon pisum (Harris, 1776); cabbage moth Mamestra brassica (Linnaeus, 1758); silkworm Bombyx mori (Linnaeus, 1758)) and have provided useful insight into Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics the understanding of their genome constitution (Mandrioli et al. 2003;Yoshido et al., 2005;Mandrioli, Borsatti, 2007;Marec et al., 2010).
The base-specifi c fl uorochromes DAPI and CMA 3 are currently widely applied in cytogenetic studies of the Heteroptera to get information about the distribution of the AT and GC repeats along the chromosomes.In most heteropteran species the only pair of NOR-bearing chromosomes has been detected by different techniques such as fl uorochrome banding, Ag-NOR staining or occasionally FISH with an rDNA probe.The location of NORs is however variable.In different species, the NORs are revealed on autosomes or on sex chromosomes (see reviews by Papeschi and Bressa, 2006a, b) but occasionally they occur on both sex chromosomes and autosomes of a species (Morielle-Souza, Azeredo-Oliveira, 2007;Bressa et al., 2008).
In our study of C. lectularius, the two CMA 3positive GC-rich bands were discovered at the telomeres of sex chromosomes (X 1 and Y).NORs are known to be largely GC-rich in the Heteroptera (Grozeva et al., 2004;Papeschi, Bressa, 2006a, b;Kuznetsova et al., 2007), and CMA 3 + /DAPI --regions of C. lectularius were therefore interpreted as the sites of NORs, and FISH with the 18S rDNA probe confi rmed this assessment.As expected, FISH/rDNA signals were observed in every spermatid, confi rming thus the location of NORs on one of the X chromosomes (X 1 ) and on the Y chromosome, which underwent a reductional division at anaphase II.Ueshima (1966Ueshima ( , 1979) ) has argued for chiasmate meiosis in C. lectularius with one chiasma being formed in every bivalent.However, our study did not support this generalization suggesting the occurrence of achiasmate meiosis in C. lectularius, and this pattern seems to be characteristic of the family Cimicidae as a whole (Grozeva, Nokkala, 2002;Poggio et al., 2009).Strong support for this suggestion is known to come from the absence of diplotene and diakinesis stages (Nokkala, Nokkala, 1986) and such is the case in C. lectularius.According to our observations, males of C. lectularius follow achiasmate meiosis of the specifi c collochore type.In this meiosis there are no chiasmata; after synapsis, the opening-out of bivalents takes place, and tenacious threads, the so-called collochores, are formed to hold homologous chromosomes together in the absence of chiasmata.The collochore meiosis was fi rst described in Drosophila melanogaster Meigen, 1830 (Cooper, 1964), thereafter in four Miridae species (Nokkala, Nokkala, 1986) and quite recently in Cimex emarginatus (as Cimex sp. in Grozeva, Nokkala, 2002) and some other Cimicidae species (Poggio et al., 2009).One further type of achiasmate meiosis known in the Heteroptera is the alignment type.In this meiosis, homologous chromosomes are held together along all their length during prophase, this alignment remaining unchanged till the beginning of anaphase I (Nokkala, Nokkala, 1983, 1984;Nokkala, Grozeva, 2000;Grozeva et al., 2008).Furthermore, a pattern intermediate between collochore and alignment meioses has been quite recently described in Arachnocoris trinitatus Bergroth, 1916 from the Nabidae (Kuznetsova et al., 2007;Kuznetsova, Grozeva, 2008), the cimicomorphan family with primarily meiosis of the alignment type (Nokkala, Nokkala, 1984;Kuznetsova, Maryańska-Nadachowska, 2000;Kuznetsova et al., 2004).
The form of metaphase I and metaphase II plates in C. lectularius is noteworthy, and this is the pattern which seems to be species-specifi c in the Heteroptera (Ueshima, 1979).In this species, the metaphase I plates were found to be nonradial but metaphase II plates radial.

CONCLUSIONS
The results of the present study support the following assertions: (1) Cimex lectularius displays holokinetic chromosomes; (2) male karyotype includes 2n = 26 + X 1 X 2 Y; (3) 18S rRNA genes are located on the X 1 and Y chromosomes; (4) males have achiasmatic meiosis of the collochore type; (5) sex chromosomes undergo post-reductional meiosis, i.e. the equational separation at AI and the reductional segregation at AII; (6) MI plates are radial and MII plates are nonradial.