Comparative cytogenetics of Insecta

The structure of the polytene chromosomes in the salivary gland of two hard for distinguishing species Cricotopus silvestris Fabricius and Cricotopus ornatus Meigen was studied. For the both salivary chromosomes mapes are worked out. Using the karyologicalanalysis is established the hybrid origin of the species Cricotopus silvestris. The comparative-karyologic analysis of the taxons showed some common features in their karyotypes. There is some resemblance between II chromosomes of the species Cr. silvestris and III chromosomes of the species Cr. ornatus and between I chromosomes of Cr. silvestris and II chromosomes of Cr. ornatus. At the same time these species are well distinguished karyologically according to the number of chromosomes (the species Cr. silvestris has 2” = 4, but the species Cr. ornatus has 2n = 6) and the chromosomal polymorphism- Cr silvestris is more polymorphic. These date indicate the philogenetic jouth of the species Cr. silvestris.

A monoclonal antibody (Mab 2a8) has been generated against a DNA tight binding protein of eukaryotic cells. Its distribution was investigated in salivary gland polytene chromosomes of two Chironomid species, Endochironomus tendens and Glyptotendipes gripekoveni, by immunofluorescence. The results indicate: 1. Mab 2a8 is distributed in some distinct condensed bands along the entire lenght of chromosomes; 2. The banding pattern obtained with fluorescent antibody does almost strictly correspond to some Hoechst positive bands and to constitutive heterochromatin sites established by «C» banding method. Other condensed (Hoechst positive) bands on chromosomes were Mab 2a8 immunonegative. 3. No immunofluorescent was observed in genetically active regions of polytene chromosomes (puffs, Balbiani rings and nuclear organizer). The mode of distribution in defined bands of polytene chromosomes suggests the residence of this protein in polytene chromosomes, whose presence appears to be well in correlation with the centromere and intercalary heterochromatin regions. This protein exibits preferential binding to specific chromosome sites containing DNA sequences preferably involved in a specific interaction with target protein. This protein is probably conserved during the evolution being represented in species distant from a phylogenetical point of view (mammalian cells, polytene chromosomes of Chironomid).

Telomeres are terminal regions of chromosomes, which protect them from fusion with other chromosomes and stabilize their structure. Telomeres usually contain specific DNA repeats (motifs), which are maintained by telomerase, a kind of reverse transcriptase. In this review, we survey the current state of knowledge of telomere motifs in insects. Among Hexapoda, data on telomere composition are available for more than 350 species from 108 families and 25 orders. The telomere motif (TTAGG)n is considered ancestral for the class Insecta. However, certain insects have different and often unknown telomeric sequences. This apparently happens because telomerase‐dependent mechanisms usually coexist with various means of alternative lengthening of telomeres as backup mechanisms of telomere maintenance. This coexistence can explain losses and reappearances of the TTAGG repeat and telomerase‐dependent telomere replication in insect evolution. For example, a few higher taxa, such as Heteroptera (Hemiptera) and Hymenoptera, show presence of the (TTAGG)n motif in their basal clades as well as a subsequent loss and, at least in the Hymenoptera, independent reappearance of this repeat in some advanced groups. Analogously, most members of Coleoptera also retain the TTAGG repeat, although it is changed to TCAGG in certain families. Furthermore, the (TTAGG)n motif seems to have been irreversibly lost in the order Diptera. In this group, telomeric sequences are represented either by long terminal repeats or by retrotransposons. Retrotransposons are also interspersed with other telomeric sequences in many groups of insects. The accumulating data demonstrate that the class Insecta is substantially diverse in terms of its telomere structure. Telomeres are terminal regions of chromosomes, which protect and stabilize their structure. Telomeres usually contain specific DNA repeats (motifs) that are maintained by telomerase. In this review, we survey the current state of knowledge of telomere motifs in insects. The telomeric repeat TTAGG is considered ancestral for the class Insecta. However, certain insects have different and often unknown telomeric sequences. This happens because telomerase‐dependent mechanisms coexist with various means of alternative lengthening of telomeres as backup mechanisms of telomere maintenance.

Eight species belonging to five true bug families were analyzed using DAPI/CMA3-staining and fluorescence in situ hybridization (FISH) with telomeric (TTAGG)n and 18S rDNA probes. Standard chromosomal complements are reported for the first time for Deraeocoris rutilus (Herrich-Schäffer, 1838) (2n=30+2m+XY) and Deraeocoris ruber(Linnaeus, 1758) (2n=30+2m+XY) from the family Miridae. Using FISH, the location of a 18S rDNA cluster was detected in these species and in five more species: Megaloceroea recticornis (Geoffroy, 1785) (2n=30+XY) from the Miridae; Oxycarenus lavaterae (Fabricius, 1787) (2n=14+2m+XY) from the Lygaeidae s.l.; Pyrrhocoris apterus (Linnaeus, 1758) (2n=22+X) from the Pyrrhocoridae; Eurydema oleracea (Linnaeus, 1758) (2n=12+XY) and Graphosoma lineatum (Linnaeus, 1758) (2n=12+XY) from the Pentatomidae. The species were found to differ with respect to location of a 18S rRNA gene cluster which resides on autosomes in Oxycarenus lavaterae and Pyrrhocoris apterus, whereas it locates on sex chromosomes in other five species. The 18S rDNA location provides the first physical landmark of the genomes of the species studied. The insect consensus telomeric pentanucleotide (TTAGG)n was demonstrated to be absent in all the species studied in this respect, Deraeocoris rutilus, Megaloceroea recticornis, Cimex lectularius Linnaeus, 1758 (Cimicidae), Eurydema oleracea, and Graphosoma lineatum, supporting the hypothesis that this motif was lost in early evolution of the Heteroptera and secondarily replaced with another motif (yet unknown) or the alternative telomerase-independent mechanisms of telomere maintenance. Dot-blot hybridization analysis of the genomic DNA from Cimex lectularius, Nabis sp. and Oxycarenus lavaterae with (TTAGG)n and six other telomeric probes likewise provided a negative result.

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 AgNO 3-staining) and molecular cytogenetic techniques (base-specific DAPI/CMA 3 fluorochromes and FISH with an 18S rDNA probe). Males originated from a wild population of C. lectularius were found to have 2n = 26 + X 1X 2Y, holokinetic chromosomes, 18S rRNA genes located on the X 3 and Y chromosomes; achiasmate male meiosis of a collochore type; MI and MII plates nonradial and radial respectively.

The karyotype and meiosis in males of giant water bug Lethocerus patruelis (Heteroptera: Belostomatidae: Lethocerinae) were studied using standard and fluorochrome (CMA 3 and DAPI) staining of chromosomes. The species was shown to have 2n = 22A + 2m + XY where 2m are a pair of microchromosomes. NORs are located in X and Y chromosomes. Within Belostomatidae, L. patruelis is unique in showing sex chromosome pre-reduction in male meiosis, with the sex chromosomes undergoing reductional division at anaphase I and equational division at anaphase II. Cytogenetic data on the family Belostomatidae are summarized and compared. In addition, the structure of the male internal reproductive organs of L. patruelis is presented, the contemporary distribution of L. patruelis in Bulgaria and in the Northern Ae-gean Islands is discussed, and the first information about the breeding and nymphal development of this species in Bulgaria is provided.

A cytogenetic investigation was performed in eight species of the spittlebug genus philaenus using silver-NOR (agNOR)-banding and fluorescence in situ hybridization (FISH) with 18s rDNA and (TTAGG)n telomeric probes. this is the first application of FISH technique in the auchenorrhyncha, a suborder of the hemiptera. FISH along with the rDNA probe revealed differences between species in the number and chromosomal location of major ribosomal rna gene sites, the so-called nucleolar organizer regions (NORs). however, we found a lack of perfect correlation between the results of agnor-staining and rDNA-FISH in the detection of NORs. FISH with the telomeric probe confirmed that the chromosome ends of the philaenus species are composed of the (TTAGG)n nucleotide sequence, which is a common motif of insect telomeres.

Euproctis kogistana Lukhtanov et al., 1995, sp. nova, pest moth, is described from Tajikistan. The new species differs from the similar E. chrysorrhoea by the haploid chromosome number (n=22 in E. kogistana and n=14 in E. chrysorrhoea) and by numerous small morphological peculiarities.

The current state of the comparative insect karyology and the main applications of chromosomal analysis of insects are reviewed. The most important characteristics of insect karyotypes in mitosis and meiosis are considered. Karyological studies provide important information on the genetic structure, life cycles and ecological characteristics, evolution, taxonomy, and phylogeny of insects. Insects have two principal mechanisms of sex determination: more common mechanism is based on the presence of sex chromosomes, and another, on haplodiploidy. Karyotypic analysis allows a number of linkage groups as well as the primary sex ratio to be determined. Chromosomal rearrangement can be used for the ecological monitoring of various insect populations and for pest control. Although a full-scale use of karyotypic details for constructing phylogenies of large insect taxa (except for Diptera which have polygene chromosomes) is possible only in combination with other features, chromosomal characters are still very important for phylogenetic purposes because their evolution is more or less independent of the environment. Chromosomal analysis can be used to reveal and identify sibling species, as well as to identify immature phases of insects. Studies of insect chromosomes may reveal cases of hybridization between forms with different karyotypes. At present, squashed and air-dried preparations are used for studying insect chromosomes. Together with morphometric analysis, differential chromosome staining methods, such as C-and AgNOR-banding, fluorochrome staining, in situ hybridization (including chromosome painting), restriction banding, etc. are being used to detect and document karyotypic differences. An outline of trends and prospects of the comparative insect karyology is given.

We have investigated for the first time the chromosomes of Karoophasma biedouwense, a species belonging to the Manto-phasmatodea, a recently discovered order of carnivorous insects. Our study has revealed that males of this species display testes with numerous seminal tubes (follicles), as in other Polyneoptera, and short tubular seminal vesicles embedded in a utricular gland. The karyotype consists of 2n = 12A + X monocentric and biarmed, meta/submetacentric chromosomes (fundamental number of arms: FN = 26) with blocks of heterochromatin around centromeres. The autosomes are classified into two size groups, one represented by a single, very large pair of autosomes, the other by five smaller pairs which constitute a continuous series gradually decreasing in size. Among "monocentric" orders of Polyneoptera, K. biedouwense shares its low chromosome number, 2n = 13, as also found with some Orthoptera (Acridoidea, Grylloidea, Gryllacridoidea). Male meiosis is of the classical pre-reductional type and the X(0) sex determination system is probably an ancestral state. Use of FISH along with an 18S rDNA probe revealed multiple ribosomal clusters, which most likely represent an apomorphic condition. We identified the ancestral insect telomeric sequence (TTAGG)(n) in the terminal areas of the chromosomes. Currently available data on the polyneopteran orders putatively related to Mantophasmatodea showed a wide variability of cytogenetic characteristics within and between them. The only character allowing some tentative inference to be made on the ancestry of K. biedouwense is its low chromosome number, the karyotypic pattern so far unreported for the Polyneoptera except in certain Orthoptera.

The karyotype and male meiosis, with a particular focus on the presence or absence of chiasmata between the homologs, were studied in the water boatman species Cymatia rogenhoferi (Fieber) and C. coleoptrata (Fabricius) (Corixidae, Cymatiainae). It is shown that the species have 2n = 33 (28A+2m+X1X2Y) and 2n = 24 (20A+2m+XY) respectively, post-reduction of sex chromosomes, and achiasmate meiosis of an alignment type in males. Cytogenetic and some morphological diagnostic characters separating Cymatia Flor from the rest of Corixidae are discussed.