Cytogenetic Characterization of Eight Odonata Species Originating from the Curonian Spit (the Baltic Sea, Russia) Using C-Banding and FISH with 18S rDNA and Telomeric (TTAGG)n Probes.

We studied the karyotypes of 8 dragonfly species originating from the Curonian Spit (the Baltic Sea, Russia) using C-banding and FISH with 18S rDNA and "insect" telomeric (TTAGG)n probes. Our results show that Leucorrhinia rubicunda, Libellula depressa, L. quadrimaculata, Orthetrum cancellatum, Sympetrum danae, and S. vulgatum from the family Libellulidae, as well as Cordulia aenea and Epitheca bimaculata from the family Corduliidae share 2n = 25 (24 + X) in males, with a minute pair of m-chromosomes being present in every karyotype except for that of C. aenea. Major rDNA clusters are located on one of the large pairs of autosomes in all the species. No hybridization signals were obtained by FISH with the (TTAGG)n probe in the examined species with the only exception of S. vulgatum. In this species, clear signals were detected at the ends of almost all chromosomes. This finding raises the possibility that in Odonata the canonical "insect" (TTAGG)n telomeric repeat is in fact present but in very low copy number and is consequently difficult to detect by in situ hybridization. We conclude that more work needs to be done to answer questions about the organization of telomeres in this very ancient and thus phylogenetically important insect order.

Screening cleavage of Factor XIII V34X Activation Peptides by thrombin mutants: A strategy for controlling fibrin architecture.

In blood coagulation, thrombin converts fibrinogen into fibrin monomers that polymerize into a clot network. Thrombin also activates Factor XIII by cleaving the R37-G38 peptide bond of the Activation Peptide (AP) segment. The resultant transglutaminase introduces covalent crosslinks into the fibrin clot. A strategy to modify clot architecture would be to design FXIII AP sequences that are easier or more difficult to be thrombin-cleaved thus controlling initiation of crosslinking. To aid in this design process, FXIII V34X (28-41) Activation Peptides were kinetically ranked for cleavage by wild-type thrombin and several anticoagulant mutants. Thrombin-catalyzed hydrolysis of aromatic FXIII F34, W34, and Y34 APs was compared with V34 and L34. Cardioprotective FXIII L34 remained the variant most readily cleaved by wild-type thrombin. The potent anticoagulant thrombins W215A and W215A/E217A (missing a key substrate platform for binding fibrinogen) were best able to hydrolyze FXIII F34 and W34 APs. Thrombin I174A and L99A could effectively accommodate FXIII W34 and Y34 APs yielding kinetic parameters comparable to FXIII AP L34 with wild-type thrombin. None of the aromatic FXIII V34X APs could be hydrolyzed by thrombin Y60aA. FXIII F34 and W34 are promising candidates for FXIII - anticoagulant thrombin systems that could permit FXIII-catalyzed crosslinking in the presence of reduced fibrin formation. By contrast, FXIII Y34 with thrombin (Y60aA or W215A/E217A) could help assure that both fibrin clot formation and protein crosslinking are hindered. Regulating the activation of FXIII is predicted to be a strategy for helping to control fibrin clot architecture and its neighboring environments.

Homoploid hybrid speciation and genome evolution via chromosome sorting.

Genomes of numerous diploid plant and animal species possess traces of interspecific crosses, and many researches consider them as support for homoploid hybrid speciation (HHS), a process by which a new reproductively isolated species arises through hybridization and combination of parts of the parental genomes, but without an increase in ploidy. However, convincing evidence for a creative role of hybridization in the origin of reproductive isolation between hybrid and parental forms is extremely limited. Here, through studying Agrodiaetus butterflies, we provide proof of a previously unknown mode of HHS based on the formation of post-zygotic reproductive isolation via hybridization of chromosomally divergent parental species and subsequent fixation of a novel combination of chromosome fusions/fissions in hybrid descendants. We show that meiotic segregation, operating in the hybrid lineage, resulted in the formation of a new diploid genome, drastically rearranged in terms of chromosome number. We also demonstrate that during the heterozygous stage of the hybrid species formation, recombination was limited between rearranged chromosomes of different parental origin, representing evidence that the reproductive isolation was a direct consequence of hybridization.

Distribution of 18S rDNA sites and absence of the canonical TTAGG insect telomeric repeat in parasitoid Hymenoptera.

Karyotypes of six species belonging to three main clades of parasitoid Hymenoptera, the superfamilies Ichneumonoidea (Ichneumonidae: Ichneumon amphibolus), Cynipoidea (Cynipidae: Diplolepis rosae) and Chalcidoidea (Eurytomidae: Eurytoma robusta, Eu. serratulae and Eu. compressa, and Torymidae: Torymus bedeguaris) were studied using FISH with 18S rDNA and telomeric (TTAGG)n probes. Haploid karyotypes of D. rosae, Eu. robusta and Eu. serratulae carried the only 18S rDNA hybridization signal, whereas those of I. amphibolus and Eu. compressa carried three and two rDNA clusters respectively. In addition, three rDNA sites were visualized in the aneuploid female of T. bedeguaris. The number of rDNA clusters in parasitoid Hymenoptera generally correlates to the chromosome number. Apart from the overwhelming majority of the studied species of aculeate Hymenoptera, no hybridization signals were obtained from FISH with the telomeric (TTAGG)n probe in the examined parasitoid species. These data suggest absence of the canonical (TTAGG)n insect telomeric motif in the Ichneumonoidea, Cynipoidea and Chalcidoidea, and perhaps in parasitoid Hymenoptera in general.

Karyotype diversity in the genus Nysius Dallas, 1852 (Hemiptera, Heteroptera, Lygaeidae) is much greater than you might think.

We studied the karyotype and chromosomal distribution of 18S rDNA clustered in nucleolar organizer regions (NORs) in Nysiusgraminicola (Kolenati, 1845), belonging to the subfamily Orsillinae (Lygaeidae). It is shown that this species has a karyotype with 2n = 22(18+mm+XY), previously known in only one of 24 studied species of the genus Nysius Dallas, 1852, characterized by a similar karyotype, 2n = 14(12+mm+XY). In N.graminicola, 18S loci are located on sex chromosomes, which is a previously unknown trait for this genus. Our results in a compilation with previous data revealed dynamic evolution of rDNA distribution in Nysius. It is concluded that molecular chromosomal markers detected by FISH contribute to a better understanding of the structure and evolution of the taxonomically complex genus Nysius.

Expanding the Chromosomal Evolution Understanding of Lygaeioid True Bugs (Lygaeoidea, Pentatomomorpha, Heteroptera) by Classical and Molecular Cytogenetic Analysis.

The Lygaeoidea comprise about 4660 species in 790 genera and 16 families. Using standard chromosome staining and FISH with 18S rDNA and telomeric (TTAGG)n probes, we studied male karyotypes and meiosis in 10 species of Lygaeoidea belonging to eight genera of the families Blissidae, Cymidae, Heterogastridae, Lygaeidae, and Rhyparochromidae. Chromosome numbers were shown to range from 12 to 28, with 2n = 14 being predominant. All species have an XY system and all but one have a pair of m-chromosomes. The exception is Spilostethus saxatilis (Lygaeidae: Lygaeinae); in another species of Lygaeinae, Thunbergia floridulus, m-chromosomes were present, which represents the first finding for this subfamily. All species have an inverted sequence of sex chromosome divisions ("post-reduction"). The 18S rDNA loci were observed on one or both sex chromosomes in Kleidocerys resedae and Th. floridulus, respectively (Lygaeidae), while on an autosomal bivalent in all other species. The rDNA loci tended to be close to the end of the chromosome. Using (TTAGG)n-FISH, we were able to show for the first time that the Lygaeoidea lack the canonical "insect" telomere motif (TTAGG)n. We speculate that this ancestral motif is absent from the entire infraorder Pentatomomorpha being replaced by some other telomere repeat motif sequences.

Comparative Cytogenetics of Lace Bugs (Tingidae, Heteroptera): New Data and a Brief Overview.

The lace bug family Tingidae comprises more than 2600 described species in 318 genera that are classified into the subfamilies Tinginae (about 2500 species and 300 genera), Cantacaderinae, and Vianadinae. We provide data on karyotypes of 16 species belonging to 10 genera of the tribes Tingini and Acalyptaini (Tinginae) studied using conventional chromosome staining and FISH. The species of Tingini possess 2n = 12A + XY, whereas those of Acalyptaini have 2n = 12A + X(0). FISH for 18S rDNA revealed hybridization signals on one of the medium-sized bivalents in species of both tribes. FISH with a telomeric probe TTAGG produced no signals in any species. In addition, we provide a list of all data obtained to date on Tingidae karyotypes, which includes 60 species from 22 genera of Tinginae. The subfamily is highly conservative in relation to the number and size of autosomes, whereas it shows diversity in the number and chromosomal distribution of the rDNA arrays, which may be located either on a pair of autosomes (the predominant and supposedly ancestral pattern), on one or both sex chromosomes, or on an autosome pair and the X. The absence of the "insect" telomeric sequence TTAGG in all species implies that Tinginae have some other, yet unknown, telomere organization.

Proteolytic and nonproteolytic activation mechanisms result in conformationally and functionally different forms of coagulation factor XIII A.

Factor XIIIA (FXIIIA) is a transglutaminase that cross-links intra- and extracellular protein substrates. FXIIIA is expressed as an inactive zymogen, and during blood coagulation, it is activated by removal of an activation peptide by the protease thrombin. No such proteolytic FXIIIA activation is known to occur in other tissues or the intracellular form of FXIIIA. For those locations, FXIIIA is assumed instead to undergo activation by Ca2+ ions. Previously, we demonstrated a monomeric state for active FXIIIA. Current analytical ultracentrifugation and kinetic experiments revealed that thrombin-activated FXIIIA has a higher conformational flexibility and a stronger affinity toward glutamine substrate than does nonproteolytically activated FXIIIA. The proteolytic activation of FXIIIA was further investigated in a context of fibrin clotting. In a series of fibrin cross-linking assays and scanning electron microscopy studies of plasma clots, the activation rates of FXIIIA V34X variants were correlated with the extent of fibrin cross-linking and incorporation of nonfibrous protein into the clot. Overall, the results suggest conformational and functional differences between active FXIIIA forms, thus expanding the understanding of FXIIIA function. Those differences may serve as a basis for developing therapeutic strategies to target FXIIIA in different physiological environments. ENZYMES: Factor XIIIA ( EC 2.3.2.13).

New evidence for the presence of the telomere motif (TTAGG) n in the family Reduviidae and its absence in the families Nabidae and Miridae (Hemiptera, Cimicomorpha).

Male karyotype and meiosis in four true bug species belonging to the families Reduviidae, Nabidae, and Miridae (Cimicomorpha) were studied for the first time using Giemsa staining and FISH with 18S ribosomal DNA and telomeric (TTAGG)n probes. We found that Rhynocoris punctiventris (Herrich-Schäffer, 1846) and R. iracundus (Poda, 1761) (Reduviidae: Harpactorinae) had 2n = 28 (24 + X1X2X3Y), whereas Nabis sareptanus Dohrn, 1862 (Nabidae) and Horistus orientalis (Gmelin, 1790) (Miridae) had 2n = 34 (32 + XY) and 2n = 32 (30 + XY), respectively. FISH for 18S rDNA revealed hybridization signals on a sex chromosome, the X or the Y, in H. orientalis, on both X and Y chromosomes in N. sareptanus, and on two of the four sex chromosomes, Y and one of the Xs, in both species of Rhynocoris Hahn, 1834. The results of FISH with telomeric probes support with confidence the absence of the "insect" telomere motif (TTAGG)n in the families Nabidae and Miridae and its presence in both species of genus Rhynocoris of the Reduviidae, considered as a basal family of Cimicomorpha. Increasing evidence reinforces the hypothesis of the loss of the canonical "insect" telomere motif (TTAGG)n by at least four cimicomorphan families, Nabidae, Miridae, Tingidae, and Cimicidae, for which data are currently available.

FISH-based karyotyping of Pelmatohydraoligactis (Pallas, 1766), Hydraoxycnida Schulze, 1914, and H.magnipapillata Itô, 1947 (Cnidaria, Hydrozoa).

An account is given of the karyotypes of Hydramagnipapillata Itô, 1947, H.oxycnida Schulze, 1914, and Pelmatohydraoligactis (Pallas, 1766) (Cnidaria, Hydrozoa, Hydridae). A number of different techniques were used: conventional karyotype characterization by standard staining, DAPI-banding and C-banding was complemented by the physical mapping of the ribosomal RNA (18S rDNA probe) and H3 histone genes, and the telomeric (TTAGGG) n sequence by fluorescence in situ hybridization (FISH). We found that the species studied had 2n = 30; constitutive heterochromatin was present in the centromeric regions of the chromosomes; the "vertebrate" telomeric (TTAGGG) n motif was located on both ends of each chromosome and no interstitial sites were detected; 18S rDNA was mapped on the largest chromosome pair in H.magnipapillata and on one of the largest chromosome pairs in H.oxycnida and P.oligactis; in H.magnipapillata, the major rRNA and H3 histone multigene families were located on the largest pair of chromosomes, on their long arms and in the centromeric areas respectively. This is the first chromosomal mapping of H3 in hydras.

Activation of factor XIII is accompanied by a change in oligomerization state.

Factor XIII A (FXIIIA) is a member of the transglutaminase enzyme family that cross-links both intra- and extracellular protein substrates. To prevent undesired cross-linking, FXIIIA is expressed as an inactive zymogen and exists intracellularly as an A2 homodimer. In plasma, FXIII A2 is complexed with two protective factor XIII B subunits (A2 B2 ) that dissociate upon activation of the zymogen. Based on limited experimental data, activated FXIII was considered a dimer of two catalytically active A subunits. However, accumulating but indirect evidence has suggested activation may lead to a monomeric state instead. In the present study, we employed analytical ultracentrifugation (AUC) to directly explore the oligomerization state of zymogen as well as active FXIIIA in solution. We first confirmed that the zymogen was a FXIIIA2 dimer. When we activated FXIIIA nonproteolytically (by high mm Ca2+ ), the protein dissociated to monomers. More importantly, FXIIIA incubation with its physiological partner, the protease thrombin, led to a monomeric state as well. AUC studies of partially cleaved FXIIIA further suggested that thrombin cleavage of a single activation peptide in a zymogen dimer is sufficient to weaken intersubunit interactions, initiating the transition to monomer. The enzymatic activity of the thrombin-cleaved species was higher than nonproteolytically activated enzyme, suggesting that displacement of the activation peptide renders the FXIIIA more accessible to substrates. Thus, results provide evidence that FXIII undergoes a change in oligomerization state as part of the activation process, and emphasize the role of the activation peptide in preventing FXIIIA catalytic activity. ENZYMES: Factor XIIIA (EC2.3.2.13).

Cytogenetic study on antlions (Neuroptera, Myrmeleontidae): first data on telomere structure and rDNA location.

Myrmeleontidae, commonly known as "antlions", are the most diverse family of the insect order Neuroptera, with over 1700 described species (in 191 genera) of which 37 species (in 21 genera) have so far been studied in respect to standard karyotypes. In the present paper we provide first data on the occurrence of the "insect-type" telomeric repeat (TTAGG) n and location of 18S rDNA clusters in the antlion karyotypes studied using fluorescence in situ hybridization (FISH). We show that males of Palpares libelluloides (Linnaeus, 1764) (Palparinae), Acanthaclisis occitanica (Villers, 1789) (Acanthaclisinae) and Distoleon tetragrammicus (Fabricius, 1798) (Nemoleontinae) have rDNA clusters on a large bivalent, two last species having an additional rDNA cluster on one of the sex chromosomes, most probably the X. (TTAGG) n - containing telomeres are clearly characteristic of Palpares libelluloides and Acanthaclisis occitanica; the presence of this telomeric motif in Distoleon tetragrammicus is questionable. In addition, we detected the presence of the (TTAGG) n telomeric repeat in Libelloides macaronius (Scopoli, 1763) from the family Ascalaphidae (owlflies), a sister group to the Myrmeleontidae. We presume that the "insect" motif (TTAGG) n was present in a common ancestor of the families Ascalaphidae and Myrmeleontidae within the neuropteran suborder Myrmeleontiformia.

Karyotype stability in the family Issidae (Hemiptera, Auchenorrhyncha) revealed by chromosome techniques and FISH with telomeric (TTAGG)n and 18S rDNA probes.

We report several chromosomal traits in 11 species from 8 genera of the planthopper family Issidae, the tribes Issini, Parahiraciini and Hemisphaeriini. All species present a 2n = 27, X(0) chromosome complement known to be ancestral for the family. The karyotype is conserved in structure and consists of a pair of very large autosomes; the remaining chromosomes gradually decrease in size and the X chromosome is one of the smallest in the complement. For selected species, analyses based on C-, AgNOR- and CMA3-banding techniques were also carried out. By fluorescence in situ hybridization, the (TTAGG) n probe identified telomeres in all species, and the major rDNA loci were detected on the largest pair of autosomes. In most species, ribosomal loci were found in an interstitial position while in two species they were located in telomeric regions suggesting that chromosomal rearrangements involving the rDNA segments occurred in the evolution of the family Issidae. Furthermore, for 8 species the number of testicular follicles is provided for the first time.

Ribosomal DNA clusters and telomeric (TTAGG)n repeats in blue butterflies (Lepidoptera, Lycaenidae) with low and high chromosome numbers.

Ribosomal DNA clusters and telomeric repeats are important parts of eukaryotic genome. However, little is known about their organization and localization in karyotypes of organisms with holocentric chromosomes. Here we present first cytogenetic study of these molecular structures in seven blue butterflies of the genus Polyommatus Latreille, 1804 with low and high chromosome numbers (from n=10 to n=ca.108) using fluorescence in situ hybridization (FISH) with 18S rDNA and (TTAGG) n telomeric probes. FISH with the 18S rDNA probe showed the presence of two different variants of the location of major rDNA clusters in Polyommatus species: with one or two rDNA-carrying chromosomes in haploid karyotype. We discuss evolutionary trends and possible mechanisms of changes in the number of ribosomal clusters. We also demonstrate that Polyommatus species have the classical insect (TTAGG) n telomere organization. This chromosome end protection mechanism probably originated de novo in small chromosomes that evolved via fragmentations.

First evidence for (TTAGG)n telomeric sequence and sex chromosome post-reduction in Coleorrhyncha (Insecta, Hemiptera).

Telomeric repeats are general and significant structures of eukaryotic chromosomes. However, nothing is known about the molecular structure of telomeres in the enigmatic hemipteran suborder Coleorrhyncha (moss bugs) commonly considered as the sister group to the suborder Heteroptera (true bugs). The true bugs are known to differ from the rest of the Hemiptera in that they display an inverted sequence of sex chromosome divisions in male meiosis, the so-called sex chromosome post-reduction. To date, there has been no information about meiosis in Coleorrhyncha. Here we report a cytogenetic observation of Peloridium pomponorum, a representative of the single extant coleorrhynchan family Peloridiidae, using the standard chromosome staining and fluorescence in situ hybridization (FISH) with a (TTAGG) n telomeric probe. We show that Peloridium pomponorum displays 2n = 31 (30A + X) in males, the classical insect (TTAGG) n telomere organization and sex chromosome post-reduction during spermatocyte meiosis. The plesiomorphic insect-type (TTAGG) n telomeric sequence is suggested to be preserved in Coleorrhyncha and in a basal heteropteran infraorder Nepomorpha, but absent (lost) in the advanced heteropteran lineages Cimicomorpha and Pentatomomorpha. The telomere structure in other true bug infraorders is currently unknown. We consider here the inverted sequence of sex chromosome divisions as a synapomorphy of the group Coleorrhyncha + Heteroptera.

The first finding of (TTAGG)n telomeric repeat in chromosomes of true bugs (Heteroptera, Belostomatidae).

Using the fluorescence in situ hybridization (FISH), the presence of (TTAGG)n telomeric sequence was detected in the chromosomes of Lethocerus patruelis (Stål, 1854) belonging to the family Belostomatidae (Heteroptera: Nepomorpha). This sequence was exclusively present at the ends of chromosomes in this species. This is the first evidence of the insect-type TTAGG telomeric repeats in Heteroptera.