Folia biologica (Kraków), vol. 55 (2007), No 1-2

Cytogenetic Characterization of the Trinidad Endemic, Arachnocoris trinitatus Bergroth: the First Data for the Tribe Arachnocorini (: Cimicomorpha: )

Valentina G. KUZNETSOVA, Snejana GROZEVA, Jo-Anne N. SEWLAL and Seppo NOKKALA

Accepted September 20, 2006

KUZNETSOVA V. G., GROZEVA S., SEWLAL J. N., NOKKALA S. 2007. Cytogenetic characterization of the Trinidad endemic, Arachnocoris trinitatus Bergroth: the first data for the tribe Arachnocorini (Heteroptera: Cimicomorpha: Nabidae). Folia biol. (Kraków) 55: 17-26.

As an extension of the ongoing cytogenetic studies of the bug family Nabidae (Heteroptera: Cimicomorpha), the first evidence for the tribe Arachnocorini (the subfamily Nabinae), with reference to the Trinidad endemic, Arachnocoris trinitatus Bergroth, is provided. This is an attempt to gain a better insight into the evolution, systematics and within-family relationships of the family Nabidae. The studies were conducted using a number of cytogenetic techniques. The male karyotype (chromosome number and size; sex chromosome system; NOR location; C-heterochromatin amount, distribution and characterization in terms of the presence of AT-rich and GC-rich DNA), and male meiosis with particular emphasis on the behavior of the sex chromosomes in metaphase II are described. Also investigated are the male and female internal reproductive organs with special reference to the number of follicles in a testis and the number of ovarioles in an ovary. A. trinitatus was found to display a number of characters differentiating it from all hitherto studied nabid species placed in the tribe Nabini of the subfamily Nabinae, and in the tribe Prostemmatini of the subfamily Prostemmatinae. Among these characters are chromosome number 2n = 12 (10 + XY), the lowest within the family, nucleolus organizer regions (NORs) situated on the autosomes rather than on the sex chromosomes as is the case in other nabid species, and testes composed of 3 follicles but not of 7 as in other nabids. All the data obtained suggest many transformations during the evolution of A. trinitatus.

Key words: Heteroptera, Nabidae, Arachnocorini, Arachnocoris trinitatus, karyotype, C-heterochromatin, NOR, CMA3, DAPI, meiosis, internal reproductive organs, evolution, karyosystematics.

Valentina G. KUZNETSOVA, Zoological Institute, Russian Academy of Sciences, Univer- sitetskaya emb., 1, 199034 St. Petersburg, Russia. E-mail: [email protected] Snejana GROZEVA, Institute of Zoology, Bulgarian Academy of Sciences, Blvd Tsar Osvobo- ditel 1, 1000 Sofia, Bulgaria. E-mail: [email protected] Jo-Anne N. SEWLAL, Department of Life Sciences, University of the West Indies St. Augustine, Trinidad and Tobago. E-mail: [email protected] Seppo NOKKALA, Laboratory of Genetics, Department of Biology, University of Turku, 20014 Turku, Finland. E-mail: [email protected]

Nabidae are one of the primitive families in the tribe, Nabini. The subfamily Prostemmatinae is di- large infraorder Cimicomorpha (Heteroptera). vided into the tribes Phorticini and Prostemmatini, The family shows Holarctic distribution and in- each with two genera only. Prostemmatini consid- cludes approximately 400 species belonging to 20 ered to be more primitive (KERZHNER 1981, genera and 4 subfamilies: the very small subfami- 1996). Data on chromosome numbers, sex chro- lies Velocipedinae and Medocostinae, which are mosome systems and patterns of male meiosis (re- sometimes considered separate families, and the viewed by KUZNETSOVA et al. 2004) are presently sister subfamilies Nabinae (includes more than 2/3 available only for the tribes Nabini (25 species of the species of the family) and Prostemmatinae. karyotyped from the genera Nabis, Lasiomerus, The systematics of Nabinae is not sufficiently ad- Himacerus, and Hoplistoscelis) and Prostem- vanced. The subfamily is classified into 4 tribes: matini (2 species karyotyped from the genera Pa- Arachnocorini, Carthasini, Gorpini and the largest gasa and Prostemma). The tribes are distinguished 18 V. G. KUZNETSOVA et al. by chromosome numbers and behaviour of the sex Arena Forest Reserve (10o34N 61o14W), Trini- chromosomes in male meiosis. Among common dad, W.I. (Table 1). The bugs were found to inhabit characters are holokinetic chromosomes, XY sex the webs of the Mesabolivar aurantiacus chromosome system, the nucleolus organizers (Mello-Leitao, 1930) (Araneae: ), where (NORs) positioned on the sex chromosomes, and they are suggested to catch prey and find mates achiasmatic male meiosis with the post-reduction (SEWLAL 2005) The freshly picked specimens of sex chromosomes (UESHIMA 1979; NOKKALA were fixed in the field in a 3:1 ethanol-glacial ace- &NOKKALA 1984; KUZNETSOVA &MARYAÑSKA-NA- tic acid solution. DACHOWSKA 2000; GROZEVA &NOKKALA 2003; KUZNETSOVA et al. 2004; GROZEVA et al. 2004). In addition, the tribes are characterized by similar Table 1 structure of male testes and female ovaries, consist- ing of 7 follicles and 7 ovarioles respectively in all Arachnocoris trinitatus: the material the species studied (CARAYON 1950, 1951; MIYA- examined, with place and date of col- MOTO 1957, 1959; KERZHNER 1981; KUZNETSOVA lection et al. 2004). Collection Samples Although the above-mentioned data have al- Collection place date examined lowed a number of preliminary taxonomical con- % & clusions (KUZNETSOVA et al. 2004), it is necessary 08.04.05 5 ,3 to examine more representatives of Nabidae be- 19.05.05 2% Arena Forest Reserve, fore an adequate appraisal of the value of the char- Trinidad W.I., 24.06.04 3% 10o34’N 61o14’W acters studied in the classification and phylogeny % & of the family can be made. 13.07.05 3 ,3 08.08.05 4%,4&,2% larvae In the present paper the first data on the tribe Arachnocorini (Nabinae), with reference to the en- demic of Trinidad, Arachnocoris trinitatus Ber- For examining the internal reproductive organs, groth, are provided. Arachnocorini occur in the the abdomen of a male or a female was open on a New World and represent a morphologically and slide in a drop of 45% acetic acid. In males, gonads biologically highly specialized group, with only were removed dorsally with fine-tipped needles two genera. Of these, the genus Arachnocoris through an incision between the fourth and ninth Scott, 1881 includes about dozen species, all of abdominal segments and spread on the slide to al- which inhabit spider’s web nests, whereas the low tracing all the parts and calculating number of taxonomic position of the monotypic genus Parar- testicular follicles in every testis. In females, struc- achnocoris Reuter, 1908 is not conclusively estab- ture of gonads was not studied in detail, except for lished (KERZHNER 1981). the number of ovarioles in every ovary. The studies of A. trinitatus were conducted using For cytogenetic studies, testicular follicles were a number of cytogenetic techniques. We have ana- squashed on the slide under the cover slip in a drop lyzed the male karyotype (with reference to chro- of 45% acetic acid. The preparations were exam- mosome number and size; sex chromosome ined initially by a phase contrast. The cover slips system; NOR location; C-heterochromatin amount, were removed by a dry ice-technique. The prepa- distribution, and molecular characterization in rations were then dehydrated in freshly prepared terms of the presence of AT-rich and GC-rich 3:1 fixative for 20 min, and air-dried. Several DNA) and male meiosis with particular emphasis methods were applied to stain the preparations: on the behavior of sex chromosomes in MII. Also the male and female internal reproductive organs (1)Standard staining.To have been investigated with special reference to study the standard karyotype and course of meiosis the number of follicles in a testis and the number a Feulgen-Giemsa – method (GROZEVA &NOKKALA of ovarioles in an ovary. 1996) was applied. The preparations were sub- jected to hydrolysis in1NHClfirst at room tem- This study aims to gain a better insight into the perature for 20 min and then at 60oC for 8 min, and evolution, systematics and within-family relation- stained in Schiff's reagent for 20 min. After rinsing ships of Nabidae. thoroughly in distilled water, the slides were addi- tionally stained by 4% Giemsa in Sörensen's buffer pH 6.8 for 20 min. The slides were rinsed Material and Methods briefly in distilled water, air-dried and made per- manent with Entellan. Specimens of A. trinitatus have been repeatedly (2)C-banding.Todetecttheamount and collected by J. N. SEWLAL in 2004 and 2005 in distribution of constitutive heterochromatin Cytogenetic Characterization of A. trinitatus (Nabidae) 19

(C-bands), a procedure described in an earlier pa- DS-U1. The fluorochrome-labeled preparations per of this series (GROZEVA &NOKKALA, 2003) were analysed using a fluorescence microscope was used. Dialux 22 at 1000 H, and the photomicrographs (3)AgNOR-banding (Sil- were taken using Camera Nikon DS-U1. ver nitrate staining). For In A. trinitatus, a total of 19 males and 10 fe- visualization of nucleolar organizer regions males, and about 150 nuclei at different stages of (NORs), the 1-step method with colloidal devel- male meiosis were examined. oper (HOWELL &BLACK 1980) was followed. (4) To reveal the presence of AT-rich and GC- rich DNA of C-heterochromatin, the preparations Results were treated with fluorochromes CMA3 and DAPI, which are known to show the affinity for DNA re- Internal reproductive organs gions enriched in GC and AT respectively (SCHWEIZER 1976; DONLON &MAGENIS 1983 The internal reproductive organs in males in- with modifications by KUZNETSOVA et al. 2001) clude paired testes arranged longitudinally in the as described below. The preparations were im- abdomen. Each testis consists of 3 elongated col- mersed into 45% acetic acid for 30 min, rinsed in ourless follicles (testis tubes) and a tubular vas distilled water and incubated in saturated solution deferens with a well developed vesicula seminalis. of Ba(OH)2 at room temperature for 6 min. To stop Two sister vasa deferentia enter the common duc- the action of Ba(OH)2, the preparations were tus ejaculatorius. There are two pairs of highly dipped in 0.2 N HCl and rinsed in distilled water. branched accessory glands. The upper glands (me- The preparations were then sequentially treated in sadenia) open into vasa deferentia where they en- o 2xSSC at 60 C for 40 min, rinsed in distilled water, ter the ductus ejaculatorius. The lower glands and air-dried. Then they were immersed in Methyl (ectodenia) empty into the bulbus ejaculatorius, Green solution (28 mg Methyl Green in 75ml which represents a strongly expanded part of duc- McIlvaine buffer, pH 7.0) for 5 min, and rinsed in tus ejaculatorius. In females, each ovary includes 7 McIlvaine buffer at pH 7.0. After this, the prepara- elongated colourless ovarioles. tions were stained first with CMA3 at a final con- centration of 5 Fg/ml (in 10 mM McIlvaine buffer, Male karyotype and spermatogenesis pH 7.0; CMA3 solution contained additionally 2.5 mM MgCl in 10 mM McIlvaine buffer) for 25 2 Standard staining.Meiotic min, rinsed in the same buffer, and then stained stages are found in males, collected in different with DAPI at a final concentration of 0.4 Fg/ml (in months, April through August, whereas no sper- 10 mM McIlvaine buffer, pH 7.0) for 5 min. After matogonial mitoses are found in adult males and staining, the preparations were rinsed in the buffer only few spermatogonial mitoses in male larvae, and mounted in an anti-fade medium (700 Flof probably indicating an early maturation of the go- glycerol, 300 Fl of 10 mM McIlvaine buffer at pH nads. The spermatocyte condensation stage and 7.0, and 10 mg of N-propyl gallate). MI display 5 autosomal bivalents and univalent X The measuring of chromosome areas in the and Y chromosomes (Figs 1a, 2), thus the meiofor- well-spread metaphases I (MI) was performed mula of male A. trinitatus isn=5A+X+Ysug- with the aid of a Videotest Image Analyzer (Ista- gesting 2n = 12 (10 + XY). The bivalents form a Videotest, St. Petersburg, Russia: STEIN et al. gradual size series, except for the first bivalent, 1998) with a digital camera attached. True and which stands out because of its very large size. The relative areas of the chromosomes (since auto- X is the second largest chromosome, whereas the somes are represented by bivalents in MI, the Y is the smallest chromosome of the complement. measured area of every bivalent was divided by 2) The true and relative areas of chromosomes are were calculated. Relative chromosome area is the presented in Table 2, the middle-sized bivalents area of each chromosome expressed as a percent- (2-4) being placed in one group, as they could not age of the total area of the complement in the nu- be satisfactory distinguished from one another in cleus. In the aggregate, 32 MI plates, 27 from one the majority of nuclei examined. male and 5 from another male, were measured. The chromosomes show no localized centro- Likewise, the chromosomes from 7 MI plates of meres, thus they are holokinetic. In meiotic pro- Nabis (Aspilaspis) viridulus Spinola, 1837 were phase I, diplotene and diakinesis stages are absent. measured for comparison. In prophase and MI, bivalents consist of parallel- The preparations were analysed by means of an aligned chromosomes without any traces of chias- Olympus BX 51 light microscope at 1000x, and mata between them. However, at condensation the photomicrographs taken using Camera Nikon stage, which precedes MI, the homologous chro- 20 V. G. KUZNETSOVA et al.

parts (Fig. 1a). At diffuse stage sex chromosomes Table 2 could be seen both separated or associated with True and relative chromosome areas each other (not shown). At condensation stage and of Arachnocoris trinitatus karyotype in MI, X and Y chromosomes lie separately in all the cells examined. The first division is reduc- Relative areas tional for the autosomal bivalents, but the sex True areas (% of total Number of 2 chromosomes undergo equational separation in Chromo- (Fm ): area of males/cells/ anaphase I (AI) and segregation in anaphase II somes means and chromosomes): measure- (AII), that is the sex chromosomes undergo post- limits means and ments reduction. Each MII cell therefore contains both X limits and Y chromosomes (Figs 3, 4). When viewed 13.29 17.09 from the pole, MII plates are “radial” with the sex A1 2/32/32 3.79-20.65 13.46-19.93 chromosomes lying in the center of a ring formed 5.52 7.56 by autosomes (Fig. 3). Viewed from the side, sex A2-A4 2/32/96 1.50-8.25 5.67-10.05 chromosomes show a bipolar co-orientation and 4.21 5.38 are located near the opposite poles, showing the A5 2/32/32 1.09-6.75 3.76-6.70 so-called “distance pairing” (Fig. 4). 6.99 8.95 X 2/32/32 1.62-11.3 6.45-11.41 AgNOR-banding.At silver-stained 2.43 3.26 Y 2/32/32 0.68-3.88 1.7-6.59 condensation stage, a nucleolus organizer (NOR) is visible associated with the largest bivalent, and the site of NOR divides the bivalent into two une- mosomes are physically associated in several sites qual parts (Fig. 5) as also does the above- via some kind of connection (Figs 1a, b). The larg- mentioned gap (Fig. 1a). The NORs are not notice- est bivalent displays, besides these connections, a able in the highly condensed chromosomes in ma- gap, which divides the bivalent into two unequal ture MI.

Figs 1-9. Meiotic chromosomes of male Arachnocoris trinitatus Bergroth (Heteroptera, Nabidae), 2n = 10 + XY. Fig. 1. Standard staining of condensation stage. (a) The male meioformula is n=5+X+Y.Arrow indicates the gap on the largest bivalent; (b) The largest bivalent with “collochores”. Arrowheads point to the collochore regions. Fig. 2. Standard staining of MI showing 5 bivalents and univalent X and Y chromosomes. Figs 3-4. Standard staining of the two sister MII nuclei, each with n = 5A+X+Y.(3)View from the pole; (4) View from the side; X and Y chromosomes show “distance-pairing”. Fig. 5. AgNOR-staining of early condensation stage. A nucleolus organizer (NOR) is associated to the largest bivalent, N – nucleolus. Fig. 6. C-banding of condensation stage. The only interstital C-band in the largest bivalent is situated at the NOR site. Arrow points to NOR-connected C-band. Figs 7-9. DAPI-staining (a) and CMA3-staining (b) of condensation stage (Figs 7a, b), prometaphase I (Figs 8a, b), and MI (Figs 9a, b). Arrow points to CMA3-positive signal in the NOR- site in the largest bivalent. Bar = 10 Fm for Figs 1-9. Cytogenetic Characterization of A. trinitatus (Nabidae) 21

Arachnocoris trinitatus 10 Bergroth Table 3 15 True and relative chromosome areas of 10 Nabis (Aspilaspis) viridulus karyotype 5 Relative 0 hoooesize chromosome A1A2-A4 A5 X Y True chromosome Number of chromosome areas males/cells/ Chromo-areas (Fm2): (% of total measure- somes means, area of ments 11 Nabis (Aspilaspis) viridulus Spinola limits chromosomes): examined means, limits 20 3.46 4.33 15 A1-A16 1/7/112 1.52-5.6 2.04-6.46 10 18.1 22.69 5 X 1/7/7 15.5-18.8 22.62-24.64 hoooesize chromosome 0 A1-A16 XY 6.35 7.9 Y 1/7/7 5.72-6.89 6.45-8.94 Figs 10-11. Idiograms of diploid male karyotypes: 10 – Arachnocoris trinitatus;11–Nabis (Aspilaspis) viridulus. unequal parts. The smallest bivalent and one of the middle-sized bivalents have only terminal bands, whereas two other middle-sized bivalents display each three bands, two terminal and one sub- terminal. X chromosome shows C-bands on both telomeres, whereas Y chromosome bears a major portion of heterochromatin throughout its exten- sion. In highly condensed chromosomes of MI no prominent C-bands are distinctive.

DAPI-and CMA3-banding. At DAPI-stained condensation stage, the distribu- tion of signals is similar to that of C-bands except for the DAPI-negative interstitial C-band in the largest bivalent (Fig. 7a). When stained by CMA3, the same chromosome plate reveals signals in none of the chromosomes except for the largest bivalent showing a bright interstitial signal (Fig. 7b). In a pro- metaphase, successively labeled with DAPI and CMA3, the chromosomes show very small, mainly telomeric DAPI-positive signals and a single bright CMA3-positive signal in the largest bivalent (Figs 8a, b). The Figures 9a and 9b demonstrate a MI plate after DAPI and CMA3 respectively. No signals are evident in the former, whereas the only interstitial signal in the largest bivalent is seen in the latter. Fig. 12. Distribution of chromosome numbers, male meiotic patterns, number of testicular follicles, and the relationships between some genera of the tribes Nabini, Arachnocorini (the Nabinae), and Prostemmatini (the Prostemmatinae). Discussion In this paper the results of the study of karyotype, C-banding.Atcondensation stage, C- male meiosis, and some features of male and fe- bands are revealed in every bivalent and in both male internal reproductive organs of A. trinitatus sex chromosomes (Fig. 6). The bivalents and X are presented. This is the first studied representa- and Y sex chromosomes each show a particular tive of the highly specialized tribe Arachnocorini C-banding pattern. The largest bivalent display no (of the subfamily Nabinae), which occurs in the bands except for an interstitial C-band situated at New World only and includes about a dozen spe- the NOR site and dividing the bivalent into two cies, all of which inhabit the ’ web nests. 22 V. G. KUZNETSOVA et al.

All the species possess a number of distinctive common mechanisms of karyotype evolution op- morphological characters (e.g. the absence of ovi- erating in groups with holokinetic chromosomes positor in females), which are due to their way of and also with the data on the related to Nabidae living (KERZHNER 1981). This species was found families Miridae, Anthocoridae and Cimicidae, to differ from all the hitherto studied representatives which are characterized with high chromosome of the family Nabidae also in a number of cytoge- numbers, close or equal to 34. To decide between netic characters, such as chromosome number, these hypotheses further studies are needed. In karyotype structure, location of NOR, and, in addi- particular, comparison studies of chromosome tion, testis structure. A. trinitatus males have 2n = size in species with different chromosome num- 12 (10 + XY), the lowest chromosome number so bers can make possible testing the hypotheses, and far known in Nabidae. As was mentioned in the In- a start has been made in the present work. troduction, a total of 27 species belonging to the The true and relative chromosome areas for A. tribes Prostemmatini (2 species) of the subfamily trinitatus are presented in Table 2. Since the Prostemmatinae and Nabini (25 species) of the middle-sized bivalents (2-4) could not be satisfac- subfamily Nabinae have been karyotyped until the tory distinguished from one another in the major- present time (reviewed by KUZNETSOVA et al. ity of cells examined, they are placed in one size 2004). In Prostemmatini, two studied genera, Pa- group. The largest autosome AI is double the size gasa and Prostemma,share2n=28(26+XY).In of A2-A4. Of the sex chromosomes, the X is fairly Nabini, 3 values of chromosome number were large however approximately two times shorter found: 2n = 18 (16 + XY); 2n = 34 (32 + XY); 2n = than A1, whereas the Y is about a third as large as 38 (36 + XY). The karyotype 2n = 18 (16 + XY) the X and represents the smallest element of the predominates and occurs in the genera Lasiomerus complement (Fig. 10). A. trinitatus differs in karyo- (1 species studied), Hoplistoscelis (1 species stud- type structure from the nabid species with other ied), and in 16 representatives of the genus Nabis. chromosome numbers, for example, from Nabis The most common chromosome number of a group (Aspilaspis) viridulus having 2n = 34 (32 + XY). is usually referred to as the modal number, there- The true and relative chromosome areas of this fore at present the karyotype 2n = 18 (16 + XY) can species are given in Table 3, and the ideogram of be taken as the modal karyotype in Nabinae, at its karyotype is presented in Fig. 11. In N. viridu- least in the genus Nabis. It is noteworthy that in the lus, the great majority of bivalents could not be sat- same genus the subgenera Aspilaspis (3 species isfactory distinguished from one another, then to studied) and Halonabis (1 species studied) display avoid errors, they are placed in one size group, 2n = 34 (32 + XY), and the main peculiarity of this 1-16. In this species, in contrast to A. trinitatus, the karyotype is that it shows a precise doubling of the conventional largest autosome A1 is similar in size autosome number compared with the modal 2n = to A2, whereas the X is the largest chromosome of 18 (16 + XY). These subgenera were previously the set and three times as large as A1. Due to our treated as separate genera (KERZHNER 1981), and preliminary estimations (unpublished), differences the validity of this opinion is supported by their in chromosome size are also characteristic of the karyotype different from that of other subgenera of nabid species with the same chromosome number Nabis (KUZNETSOVA et al. 2004). It is of interest that is an evidence for many karyotype transforma- that the karyotype 2n = 34 (32 + XY) also occurs in tions in the evolution of the family. Himacerus mirmicoides (O. Costa), whereas one further studied representative of this genus, H. ma- If the chromosome formula 2n = 32 + XY is racandicus (Reuter), shows a higher number, 2n = primitive in the Nabidae, and all other karyotypes 38 (36 + XY). (except for 2n = 36 + XY) have originated from this initial state via autosome fusions (KUZNETSOVA The rearrangements resulting in chromosome et al. 2004), then the change to the 2n = 10 + XY number differences in Nabidae are open to ques- shown by A. trinitatus is most likely the result of tion. One hypothesis is that the karyotype 2n = 16 + fusion of 11 pairs of autosomes. The fact that the XY is ancestral for the family, at least for the sub- largest chromosome pair is considerably longer family Nabinae, and that 2n = 32 + XY is a result of than the remaining chromosomes of the karyotype polyploidy (LESTON 1957; THOMAS 1996) or auto- provides strong evidence for fusions. In A1 there is somal polyploidy (KUZNETSOVA &MARYAÑSKA-NA- an unstained gap, or constriction having a sub- DACHOWSKA 2000). The polyploid hypothesis has median location, and the silver impregnation tech- been recently questioned by JACOBS (2002). An- nique confirms that it is the site where the nucleo- other view holds that the ancestral nabid karyotype lar organizer region (NOR) is situated. An was 2n = 32 + XY, and all other karyotypes had important point is that in all other previously stud- originated from it by a series of autosome fusions ied nabid species the NORs are associated to sex and fission (KUZNETSOVA et al. 2004). This hy- chromosomes, both to X and Y (NOKKALA & pothesis seems to be in better agreement with the NOKKALA 1984; GROZEVA et al. 2004). It allows Cytogenetic Characterization of A. trinitatus (Nabidae) 23 suggestion that in Nabidae the NOR is originally pattern has been reported in many heteropteran located in sex chromosomes and that there has species (PAPESCHI et al. 1995; PÉREZ et al. 1997; been a translocation of the NOR material since A. GROZEVA &NOKKALA 2001), other representa- trinitatus speciation. tives of Nabidae included (GROZEVA &NOKKALA It is generally agreed that holokinetic chromo- 2003; GROZEVA et al. 2004). Constitutive hetero- somes contain only a small amount of C-hetero- chromatin is known to be gene-impoverished chromatin, the distribution of which is mainly con- (PARDUE &HENNIG 1990). It is very tempting to fined to the telomeres (BLACKMAN 1985; BRESSA assume that the Y in the Heteroptera contains no et al. 2005). It is apparent however, that neither major genes, at least those required for sex deter- structural nor behavioral features of holokinetic mination or male fertility. This provides an insight chromosomes in fact set limits on the heterochro- into why in the evolution of Heteroptera, having matin accumulation. In support of this assertion the XY sex chromosome system as an ancestral one can argue that a great deal of heterochromatin condition, the Y chromosome is not infrequently occurs in some species (PAPESCHI 1991; KUZNE- lost bringing into existence the co-generic species TSOVA et al. 1997; ANGUS et al. 2004; BRESSA et with both XY and X0 systems (NOKKALA & al. 2005) and in some specialized chromosomes NOKKALA 1984; BLACKMAN 1995). This clearly like sex chromosomes and B chromosomes, which suggests that the heteropteran Y chromosome is are sometimes completely or almost completely transcriptionally inert, but probably fulfils the role heterochromatic (BLACKMAN 1990; PAPESCHI 1995; of a pairing partner for the X chromosome, thereby MARYAÑSKA-NADACHOWSKA 2004; NECHAYEVA providing high accuracy in segregation. et al. 2004; ANGUS et al. 2004; GROZEVA et al. In common with other hitherto studied nabid 2004). The wide prevalence of telomeric localiza- species, male meiosis in A. trinitatus is achias- tion of heterochromatin in holokinetic chromo- matic, as exemplified by prophase I, where diplo- somes allows suggestion that this pattern is not tene and diakinesis are absent due to the lack of occasional but is evolutionarily fixed and caused chiasmata, as well as by M I, where in the bivalents by the biological advisability. the homologues are aligned in parallel to each The Nabidae bugs characteristically display other with no traces of chiasmata between them. small C-bands in their karyotypes, the bands There is no evidence as to whether chiasmata are showing both telomeric and interstitial localiza- formed in females, however they are most likely tion and variously distributing in different species formed. It is well known, that the abolition of chi- (GROZEVA &NOKKALA 2003; GROZEVA et al. asmata, when it occurs, is confined to one sex of a 2004). This is true even for closely related species, species, more often to the male (WHITE 1973), and such as Nabis (Aspilaspis) indicus (Stal) and N. its genetic control may be related to heterogamety (A.) viridulus Spinola, and suggests some chromo- (NOKKALA &NOKKALA 1983). However at con- some rearrangement events during speciation. These densation stage of A. trinitatus the homologous species share the same chromosome number, 2n = chromosomes are physically associated in several 34 (32 + XY), however demonstrate respectively sites via a kind of connection, a phenomenon, telomeric and interstitial C-bands (GROZEVA et al. which has never been found before in Nabidae. 2004). It is likely that the inversions have played a Till now, two basic types of achiasmatic meiosis role in the redistributing C-blocks along the chro- have been reported in Insecta, including Heterop- mosomes of N. viridulus. The analysis of constitu- tera. By the description of NOKKALA and NOKKALA tive heterochromatin content and distribution in (1986a), in the most widespread type, the align- A. trinitatus has revealed the presence of conspicuous ment of homologous chromosomes attained early C-positive bands of the terminal or sub-terminal in meiosis remained externally unchanged until location in all the bivalents except for A1, where a the beginning of anaphase, whereas in the other single interstitial C-band is connected to NOR. All type, after synapsis, the so-called “collochores” the bands are DAPI-positive except for the NOR- are formed, and homologous chromosomes are related band, which is CMA3-bright indicating an connected with each other at specific positions via excess of GC base pairs. The correspondence of these tenacious structures. Within Heteroptera, the CMA-bright bands with NORs has also been re- “alignment” type occurs in the families Saldidae, ported in other heteropteran species (GONZÁLEZ- Anthocoridae, Microphysidae, and Nabidae -GARCÍA et al. 1996; REBAGLIATI et al. 2003), in- (NOKKALA &NOKKALA 1983, 1984, 1986, b; cluding Nabidae (GROZEVA et al. 2004). In A. trini- KUZNETSOVA &MARYAÑSKA-NADACHOWSKA tatus, heterochromatin is available in both sex 2000; NOKKALA &GROZEVA 2000; KUZNETSOVA chromosomes, the X showing C-bands on both te- et al.2004), whereas the “collochore” type is en- lomeres, whilst the Y looking as if it is totally het- countered in the families Miridae and Cimicidae erochromatic or at least contains a very great (NOKKALA &NOKKALA 1986a; GROZEVA & amount of heterochromatin. With the Y, the same NOKKALA 2002). The “alignment” type is consid- 24 V. G. KUZNETSOVA et al. ered to be more primitive as compared with the NOKKALA 1984; KUZNETSOVA &MARYAÑ- “collochore” one, and the occurrence of these SKA-NADACHOWSKA 2000; KUZNETSOVA et al. types was used for the determination of phyloge- 2004), and the same pattern is also found in A. netic affinities within Cimicomoprha (NOKKALA trinitatus. This provides evidence in support of a &NOKKALA 1986a). However the data are pres- recent assumption (KUZNETSOVA et al. 2004) that ently available for solely separate representatives this meiotic pattern is an autapomorphy of the sub- of all the above-mentioned families, so the conclu- family Nabinae within the family Nabidae. sions can be accepted just as preliminary. For ex- On the basis of the morphology of the male and ample, NOKKALA &NOKKALA (1984) reported female internal reproductive apparatus in a number achiasmatic meiosis of the “alignment” type for of heteropteran families, PENDERGRAST (1957) Anthocoridae based on a study of Anthocoris ne- has identified several characters, which could pro- morum (L.), however, according to the assertion of vide phylogenetic indications at various taxo- WANG et al. (2003), while not provided by any nomic levels. He refers, in particular, to the number proofs, “two kinds of achiasmatic meiosis exist in of seminal follicles per testis in males and the A. montanus Zheng”. Because of connections ob- number of ovarioles per ovary in females. In many served between homologous chromosomes, the families male testes and female ovaries were shown achiasmatic meiosis of A. trinitatus is reminiscent to consist of 7 follicles and 7 ovarioles respectively of the “collochore” type. It is however significant, (WOODWARD 1950; PENDERGRAST 1957), and this that these connections are visible during meiotic number is taken as the generalized or plesiomor- prophase only, whereas in MI homologous chro- phic condition in Heteroptera (AKINGBOHUNGBE mosomes show parallel alignment along all their 1983). Other numbers, within a range from 1 to 8, length. This evidence suggests that “collochore” also occur, and in most species for which the num- and “alignment” types are derivable from each bers are known they are the same in both sexes other, and hence, these terms can be considered de- (WOODWARD 1950). However, in the case when scriptive. The “alignment” and “collochore” types the numbers are different, the number of follicles seem to differ in such a way that numerous col- in males is generally lower as compared to that of lochores occur between the pairing chromosomes ovarioles in females (see Table 1 in GROZEVA & in the first case, whereas single (one or two) col- KUZNETSOVA 1992). In Nabidae, male testes and lochores occur in the second case. Thus, achias- female ovaries (concerning the females, the data matic meiosis encountered in A. trinitatus can be are however fewer in number) are invariably com- considered as an intermediate one between “col- posed of 7 lobes (CARAYON 1950, 1951; MIYAMOTO lochore” and “alignment” types. At present, little 1957, 1959; KERZHNER 1981; KUZNETSOVA et al. is known about the nature of collochores, primar- 2004), except for A. trinitatus, in which males dis- ily if they are the remnants of synaptinemal com- play only 3 follicles in a testis, even if females re- plexes or represent some independent structures tain the plesiomorphic state of ovaries, each with 7 destined for holding the homologous chromo- ovarioles. somes together in meiotic bivalents when chias- mata are absent (COOPER 1964; NOKKALA 1986). Thus, A. trinitatus displays a number of charac- Undeniably, a closer look at the nature of col- ters differentiating it from all hitherto studied lochores and at the problem of achiasmatic meiosis nabid species. Among these characters are chro- as a whole is called for. mosome number, 2n = 12 (10 + XY), the lowest within the family; NORs situated on the autosomes In A. trinitatus, as in the overwhelming majority rather than on the sex chromosomes; testes com- of Heteroptera (UESHIMA 1979), sex chromo- posed of 3 seminal follicles but not of 7. These dis- somes in male meiosis are subjected to post- tinctive characters indicate many transformations reduction so that they separate equationally in AI during evolution of A. trinitatus and probably rep- while segregate reductionally in AII. Likewise, resent, at least the reduced follicle number, a result typical for Heteroptera is a so-called “touch-and- of extreme specialization shown by this species if go pairing” of sex chromosomes, that is they asso- not by the entire tribe Arachnocorini, all represen- ciate transiently as a pseudo-bivalent in MII and tatives of which are known to be morphologically segregate (poleward) in AII (UESHIMA 1979). In and biologically highly specialized. Clearly fur- Nabidae this, most likely ancestral, pattern is re- ther studies of this group could be useful. tained in the primitive tribe Prostemmatini (KUZNETSOVA et al. 2004). However, the tribe Nabini is characterized by another pattern which is Acknowledgements known as “distance pairing” of sex chromosomes, where they do not associate but are located near the The authors wish to thank N. KHABAZOVA for opposite poles, forming a “distance bivalent” in MII technical assistance and C. K. STARR for assistance followed by their segregation in AII (NOKKALA & in the field collection and transfer of the material. Cytogenetic Characterization of A. trinitatus (Nabidae) 25

This work was supported (for V. G. Kuznetsova) GROZEVA S., NOKKALA S. 2002. Achiasmatic male meiosis by the Russian Foundation for Basic Research, in Cimex sp. (Heteroptera, Cimicidae). Caryologia 55: 189-192. Grant No. 05-04-48387, the program of the Presid- GROZEVA S., NOKKALA S. 2003: C-heterochromatin and ex- tra (B) chromosome distribution in six species of the Nabis ium of the Russian Academy of Sciences “Dynam- (Heteroptera, Nabidae) with the modal male karyotype 2n = ics of Gene Pools in , Plants and Man”, the 16 + XY. Folia biol. (Kraków) 51: 13-21. program of the St. Petersburg Scientific Centre of HOWELL W. M., BLACK D. A. 1980. Controlled silver stain- the Russian Academy of Sciences “The Diver- ing of nucleolus organizer region with a protective colloidal developer: a 1-step method. Experientia 36: 1014-1015. gence of Genetic Material in the Evolution of Phy- JACOBS D. H. 2002. Cytogenetics and karyotype evolution of logenetic Branches of Eukaryotes”, and (for S. the genus Miteronotus Jacobs (Heteroptera: Aradidae: Car- Grozeva) by the National Science Fund, Bulgarian ventinae) with assessment of the possible role of pseudo- polyploidy in their karyotype evolution. African Entomol. Ministry of Education and Science, Grant B-1304. 10: 171-184. The study was also supported by the Russian KERZHNER I. M. 1981. Nasekomye khobotnye (Insecta: Academy of Sciences, the Bulgarian Academy of Rhynchota). (In: Fauna SSSR, vol. 13). Nauka, Leningrad): Sciences and the Academy of Finland. 1-326. (In Russian). KERZHNER I. M. 1996. Family Nabidae A. Costa 1983 – dam- sel bugs. (In: Catalogue of the Heteroptera of the Palaearctic Region, vol. 2. Aukema B. & Rieger C. eds, Netherlands En- tomol. Soc. Amsterdam): 84-107. References KUZNETSOVA V. G., GROZEVA S., NOKKALA S. 2004. New cytogenetic data on Nabidae (Heteroptera: Cimicomorpha), ANGUS R. B., KEMENY C. K., WOOD E. 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