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 (Heteroptera: Cimicomorpha: Nabidae) 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 spider Mesabolivar aurantiacus chromosome system, the nucleolus organizers (Mello-Leitao, 1930) (Araneae: Pholcidae), 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.