New Evidence of the Monophyletic Relationship of the Genus Psammolestes Bergroth, 1911 (Hemiptera, Reduviidae, Triatominae).

The genus Psammolestes within the subfamily Triatominae and tribe Rhodniini comprises the species Psammolestes arthuri, Psammolestes coreodes, and Psammolestes tertius, all potential vectors of Chagas disease. A feature of Psammolestes is their close association with birds, which makes them an interesting model for evolutionary studies. We analyzed cytogenetically Psammolestes spp., with the aim of contributing to the genetic and evolutionary knowledge of these vectors. All species of the Psammolestes showed the same chromosomal characteristics: chromocenter formed only by sex chromosomes X and Y, karyotype 2n = 22 and constitutive heterochromatin, and AT base pairs restricted to the sex chromosome Y. These results corroborate the monophyly of the genus and lead to the hypothesis that during the derivation of P. tertius, P. coreodes, and P. arthuri from their common ancestor, there was no reorganization in the number or structure of chromosomes.

Sub-kb Hi-C in D. melanogaster reveals conserved characteristics of TADs between insect and mammalian cells.

Topologically associating domains (TADs) are fundamental elements of the eukaryotic genomic structure. However, recent studies suggest that the insulating complexes, CTCF/cohesin, present at TAD borders in mammals are absent from those in Drosophila melanogaster, raising the possibility that border elements are not conserved among metazoans. Using in situ Hi-C with sub-kb resolution, here we show that the D. melanogaster genome is almost completely partitioned into >4000 TADs, nearly sevenfold more than previously identified. The overwhelming majority of these TADs are demarcated by the insulator complexes, BEAF-32/CP190, or BEAF-32/Chromator, indicating that these proteins may play an analogous role in flies as that of CTCF/cohesin in mammals. Moreover, extended regions previously thought to be unstructured are shown to consist of small contiguous TADs, a property also observed in mammals upon re-examination. Altogether, our work demonstrates that fundamental features associated with the higher-order folding of the genome are conserved from insects to mammals.

High-resolution TADs reveal DNA sequences underlying genome organization in flies.

Despite an abundance of new studies about topologically associating domains (TADs), the role of genetic information in TAD formation is still not fully understood. Here we use our software, HiCExplorer (hicexplorer.readthedocs.io) to annotate >2800 high-resolution (570 bp) TAD boundaries in Drosophila melanogaster. We identify eight DNA motifs enriched at boundaries, including a motif bound by the M1BP protein, and two new boundary motifs. In contrast to mammals, the CTCF motif is only enriched on a small fraction of boundaries flanking inactive chromatin while most active boundaries contain the motifs bound by the M1BP or Beaf-32 proteins. We demonstrate that boundaries can be accurately predicted using only the motif sequences at open chromatin sites. We propose that DNA sequence guides the genome architecture by allocation of boundary proteins in the genome. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogenome.ie-freiburg.mpg.de .

Thiazole Orange as an Alternative to Antibody Binding for Detecting Triple-helical DNA in Heterochromatin of Drosophila and Rhynchosciara.

The standard method for detecting triple-stranded DNA over the last 1.5 decades has been immune detection using antibodies raised against non-canonical nucleic acid structures. Many fluorescent dyes bind differentially to nucleic acids and often exhibit distinctive staining patterns along metaphase chromosomes dependent upon features, including binding to the major and minor DNA grooves, level of chromatin compaction, nucleotide specificity, and level of dye stacking. Relatively recently, the fluorochrome Thiazole Orange (TO) was shown to preferentially bind to triplex DNA in gels. Here, we demonstrate that TO also detects triplex DNA in salivary gland chromosomes of Drosophila melanogaster and Rhynchosciara americana identical in location and specificity to observations using antibodies. This finding may enable triple-stranded DNA investigations to be carried out on a much broader and reproducible scale than hitherto possible using antibodies, where a frequently encountered problem is the difference in detection specificity and sensitivity between one antibody and another.

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation.

The fluorescent dye 4'-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.

The three-dimensional genome organization of Drosophila melanogaster through data integration.

BACKGROUND: Genome structures are dynamic and non-randomly organized in the nucleus of higher eukaryotes. To maximize the accuracy and coverage of three-dimensional genome structural models, it is important to integrate all available sources of experimental information about a genome's organization. It remains a major challenge to integrate such data from various complementary experimental methods. Here, we present an approach for data integration to determine a population of complete three-dimensional genome structures that are statistically consistent with data from both genome-wide chromosome conformation capture (Hi-C) and lamina-DamID experiments. RESULTS: Our structures resolve the genome at the resolution of topological domains, and reproduce simultaneously both sets of experimental data. Importantly, this data deconvolution framework allows for structural heterogeneity between cells, and hence accounts for the expected plasticity of genome structures. As a case study we choose Drosophila melanogaster embryonic cells, for which both data types are available. Our three-dimensional genome structures have strong predictive power for structural features not directly visible in the initial data sets, and reproduce experimental hallmarks of the D. melanogaster genome organization from independent and our own imaging experiments. Also they reveal a number of new insights about genome organization and its functional relevance, including the preferred locations of heterochromatic satellites of different chromosomes, and observations about homologous pairing that cannot be directly observed in the original Hi-C or lamina-DamID data. CONCLUSIONS: Our approach allows systematic integration of Hi-C and lamina-DamID data for complete three-dimensional genome structure calculation, while also explicitly considering genome structural variability.

Differentiation between Triatoma arthurneivai and Triatoma wygodzinskyi (Hemiptera: Reduviidae: Triatominae) using cytotaxonomy.

Using classic morphometric techniques to examine the head and thorax of Triatoma specimens, researchers identified a possible taxonomic problem involving T. arthurneivai (Lent & Martins) and T. wygodzinskyi (Lent). A recent geometric morphometric study indicated that the insects captured outside the Serra do Cipó region, State of Minas Gerais, Brazil, were T. wygodzinskyi. The misidentification of T. arthurneivai as T. wygodzinskyi could result in several problems associated with entoepidemiological lifting, the biological characterization of the species, and phylogenetic reconstruction. For the first time, we describe the use of cytogenetic analysis as a tool for differentiation between T. arthurneivai and T. wygodzinskyi. The results indicated that both species had the same number of chromosomes 2n = 22 (20A + XY). However, analyses of spermatocytes during early prophase indicated that it was possible to differentiate T. arthurneivai and T. wygodzinskyi, because only T. arthurneivai exhibited heteropycnotic blocks distributed in the chromatin. Therefore, we highlight the analysis of spermatocytes as a taxonomic tool for the characterization of T. arthurneivai and T. wygodzinskyi, and suggest that the technique can be used for entoepidemiological lifting in vector control programs. Thus, the results presented here, in conjunction with morphometric analyses, are of utmost taxonomic and epidemiological importance for the identification of T. arthurneivai and T. wygodzinskyi specimens.

New record and cytogenetic analysis of Psammolestes tertius Lent & Jurberg, 1965 (Hemiptera, Reduviidae, Triatominae) from Bahia State, Brazil.

This paper reports on the first occurrence of Psammolestes tertius in the Chapada Diamantina region, located in the city of Seabra, Bahia State, in northeastern Brazil. Following an active search, 24 P. tertius specimens were collected from Phacellodomus rufifrons (rufous-fronted thornbird) nests. The insects did not present any symptoms of infection by Trypanosoma cruzi. P. tertius males were cytogenetically analyzed, and the results were compared with those of other specimens from the Brazilian State of Ceará. Triatomines from both locations presented the same cytogenetic characteristics: 22 chromosomes, little variation in the size of the autosomes, Y chromosomes that were larger than the X chromosomes, a chromocenter formed only by the sex chromosomes during prophase, and autosomes lacking constitutive heterochromatin. However, it is important to note that this species shows intraspecific chromosomal variation. In light of the results obtained, it is recommended that more studies be performed to characterize P. tertius. These studies will be particularly helpful in understanding this species in ecological, biological, biogeographical, and phylogenetic terms.

Cytogenetic data on the threatened leafcutter ant Atta robusta Borgmeier, 1939 (Formicidae: Myrmicinae: Attini).

The karyotype of the threatened ant species Atta robusta is described so as to establish the evolutionary relationships of this taxon with other leafcutter ants. Standard Giemsa staining, C-banding, NOR banding, fluorochromes CMA3/DAPI, Hsc-FA technique and Fluorescence in situ Hybridization (FISH) using 18S rDNA probe were conducted on a population from Aracruz, state of Espírito Santo, Brazil, allowing for comparisons with data available on Atta and other fungus-growing ant species. The diploid chromosome number observed for A. robusta was 2n=22, and the karyotypic formula was 18m+2sm+2st. Heterochromatic blocks were observed in the centromeric region of most chromosomes, where one pair of metacentric chromosomes is characterized by a GC-rich heterochromatic band in the interstitial region of its long arm. The detection of 18S rDNA using FISH confirmed the presence of single NOR for A. robusta. This is the first report of rDNA 18S detection using FISH for leafcutter ants. The cytogenetic results of this study confirm the information available for Atta and allow us to confirm the conserved chromosome number, morphology and banding pattern within the genus for the taxa studied to date, which included species from three out of the four groups of Atta indicated by molecular data. The accumulation of cytogenetic data on fungus-growing ants enhances the understanding of the genomic evolutionary patterns of Atta, since it belongs to a group of recent origin between the most well studied ants. Cytogenetic data does not indicate restrictions in relocation or reintroduction in areas where populations were extinct due to the conserved karyotype. This study allows for cytogenetic comparison of A. robusta with other ants of Atta, emphasizing the importance of chromosomal information for species conservation.

Aurora A Kinase Contributes to a Pole-Based Error Correction Pathway.

Chromosome biorientation, where sister kinetochores attach to microtubules (MTs) from opposing spindle poles, is the configuration that best ensures equal partitioning of the genome during cell division. Erroneous kinetochore-MT attachments are commonplace but are often corrected prior to anaphase. Error correction, thought to be mediated primarily by the centromere-enriched Aurora B kinase (ABK), typically occurs near spindle poles; however, the relevance of this locale is unclear. Furthermore, polar ejection forces (PEFs), highest near poles, can stabilize improper attachments by pushing mal-oriented chromosome arms away from spindle poles. Hence, there is a conundrum: erroneous kinetochore-MT attachments are weakened where PEFs are most likely to strengthen them. Here, we report that Aurora A kinase (AAK) opposes the stabilizing effect of PEFs. AAK activity contributes to phosphorylation of kinetochore substrates near poles and its inhibition results in chromosome misalignment and an increased incidence of erroneous kinetochore-MT attachments. Furthermore, AAK directly phosphorylates a site in the N-terminal tail of Ndc80/Hec1 that has been implicated in reducing the affinity of the Ndc80 complex for MTs when phosphorylated. We propose that an AAK activity gradient contributes to correcting mal-oriented kinetochore-MT attachments in the vicinity of spindle poles.

Adaptive Role of Inversion Polymorphism of Drosophila subobscura in Lead Stressed Environment.

Local adaptation to environmental stress at different levels of genetic polymorphism in various plants and animals has been documented through evolution of heavy metal tolerance. We used samples of Drosophila subobscura populations from two differently polluted environments to analyze the change of chromosomal inversion polymorphism as genetic marker during laboratory exposure to lead. Exposure to environmental contamination can affect the genetic content within a particular inversion and produce targets for selection in populations from different environments. The aims were to discover whether the inversion polymorphism is shaped by the local natural environments, and if lead as a selection pressure would cause adaptive divergence of two populations during the multigenerational laboratory experiment. The results showed that populations retain signatures from past contamination events, and that heavy metal pollution can cause adaptive changes in population. Differences in inversion polymorphism between the two populations increased over generations under lead contamination in the laboratory. The inversion polymorphism of population originating from the more polluted natural environment was more stable during the experiment, both under conditions with and without lead. Therefore, results showed that inversion polymorphism as a genetic marker reflects a strong signature of adaptation to the local environment, and that historical demographic events and selection are important for both prediction of evolutionary potential and long-term viability of natural populations.

B chromosomes showing active ribosomal RNA genes contribute insignificant amounts of rRNA in the grasshopper Eyprepocnemis plorans.

The genetic inertness of supernumerary (B) chromosomes has recently been called into question after finding several cases of gene activity on them. The grasshopper Eyprepocnemis plorans harbors B chromosomes containing large amounts of ribosomal DNA (rDNA) units, some of which are eventually active, but the amount of rRNA transcripts contributed by B chromosomes, compared to those of the standard (A) chromosomes, is unknown. Here, we address this question by means of quantitative PCR (qPCR) for two different ITS2 amplicons, one coming from rDNA units located in both A and B chromosomes (ITS2(A+B)) and the other being specific to B chromosomes (ITS2(B)). We analyzed six body parts in nine males showing rDNA expression in their B chromosomes in the testis. Amplification of the ITS2(B) amplicon was successful in RNA extracted from all six body parts analyzed, but showed relative quantification (RQ) values four orders of magnitude lower than those obtained for the ITS(A+B) amplicon. RQ values differed significantly between body parts for the two amplicons, with testis, accessory gland and wing muscle showing threefold higher values than head, gastric cecum and hind leg. We conclude that the level of B-specific rDNA expression is extremely low even in individuals where B chromosome rDNA is not completely silenced. Bearing in mind that B chromosomes carry the largest rDNA cluster in the E. plorans genome, we also infer that the relative contribution of B chromosome rRNA genes to ribosome biogenesis is insignificant, at least in the body parts analyzed.

B1 was the ancestor B chromosome variant in the western Mediterranean area in the grasshopper Eyprepocnemis plorans.

We analyzed the distribution of 2 repetitive DNAs, i.e. ribosomal DNA (rDNA) and a satellite DNA (satDNA), on the B chromosomes found in 17 natural populations of the grasshopper Eyprepocnemis ploransplorans sampled around the western Mediterranean region, including the Iberian Peninsula, Balearic Islands, Sicily, and Tunisia. Based on the amount of these repetitive DNAs, 4 types of B variants were found: B1, showing an equal or higher amount of rDNA than satDNA, and 3 other variants, B2, B24 and B5, bearing a higher amount of satDNA than rDNA. The variants B1 and B2 varied in size among populations: B1 was about half the size of the X chromosome in Balearic Islands, but two-thirds of the X in Iberian populations at Alicante, Murcia and Albacete provinces. Likewise, B2 was about one-third the size of the X chromosome in populations from the Granada province but half the size of the X in the populations collected at Málaga province. The widespread geographical distribution of the B1 variant makes it the best candidate for being the ancestor B chromosome in the whole western Mediterranean region.

High diversity in CMA3/DAPI-banding patterns in Heteropterans.

Heteroptera is the most numerous and diverse suborder of Hemiptera, with about 38,000 species. This diversity also involves cytogenetic features, including chromosome number and a sex determining system. Information about heterochromatin occurrence and distribution is scarce in heteropterans, but still, there is some evidence of variability. We determined the chromosome number and CMA3/DAPI-banding pattern of 179 individuals of 25 heteropteran species from Brazil. Eight species of Pentatomidae exhibited a constant chromosome number (2n = 12 + XY), but in Coreidae (12 species), Largidae (1 species), Rhopalidae (1 species), and Pyrrhocoridae (3 species), the numbers ranged from 2n = 10 + 2m + X0 to 2n = 24 + 2m + X0. Although there were no large differences in the chromosome size between species, the CMA3/DAPI-banding patterns differed markedly. Among the genera, species of Edessa, Spartocera, Hypselonotus, Phtia,Holhymenia and Euryophthalmus showed a large accumulation of heterochromatin, while the other species exhibited few or no heterochromatic bands. In general, when heterochromatin was more accumulated, this occurred preferentially at terminal positions, except in Holhymenia histrio, which exhibited intercalary bands. This study made it possible to identify some chromosome rearrangements and to enhance our knowledge of the evolutionary mechanisms that determine karyotype differentiation in Heteroptera.

Large-scale periodicity of nucleosome positioning signal in pericentric regions of chromosomes (Drosophila melanogaster).

Nucleosome positioning signal (NPS) in heterochromatin is not uniform. We suggest the analysis of its heterogeneity by correlation with periodic function (analog of Furrier analysis). It was established the periodical repetition of the nucleosome clusters of large size in pericentric regions in a discontinuous manner. In the 3L pericentric region, it was revealed the domination of 78-85 kbp wavelength in the correlation coefficient profile and also strong presentation of 50 kbp signal. In further to centromere position, the 69 kbp value strongly dominates as well as the 50 kbp value in the closest proximity. In addition to the long wavelength signals, there are plenty of short wavelengths signals especially in the closest vicinity to centromere. In some positions throughout pericentric region of 2L chromosome, there are two sizes of repeated intermingled correlation signals (50, and 75 kbp) with dominating value of 75 kbp in proximity and 50 kbp distantly to centromere, the situation for 2R is analogous. Some genes with long introns support these quantitative characteristics of NPSs and to some extent their dominating character in each region. The characteristic repeat periods for 3L pericentric region coincide with the distances between heterochromatin epigenetic mark clusters and their distribution throughout this region for fly embryos, larvae, and some cell lines.

Estimation of nuclear genome size of the genus Mycetophylax Emery, 1913: evidence of no whole-genome duplication in Neoattini.

Genome size estimates and their evolution can be useful for studying the phylogenetic relationships and taxonomy of a particular group. In the present study, the genome sizes of the three species that comprise the Mycetophylax genus were estimated by flow cytometry (FCM). There was little variation in genome size among them. The mean haploid genome size value of male and female individuals of Mycetophylax morschi was 312.96 Mbp (0.32 pg) and that of Mycetophylax conformis and Mycetophylax simplex females were 312.96 Mbp (0.32 pg) and 381.42 Mbp (0.39 pg), respectively. At first glance, this variation could be related with the heterochromatin content. Our results, together with other previous reports, have contributed to our knowledge about Attini genome size and will be useful to improve the understanding of the evolution of this tribe. It will help select potential model species in Attini for future genomic and sequencing projects.

Fluorescent in situ hybridization on mitotic chromosomes of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions. Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti and Cx. quinquefasciatus, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti, the accumulation of multiple chromosomal rearrangements in cell line chromosomes makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Three-dimensional segmentation of nuclei and mitotic chromosomes for the study of cell divisions in live Drosophila embryos.

Drosophila embryogenesis is an established model to investigate mechanisms and genes related to cell divisions in an intact multicellular organism. Progression through the cell cycle phases can be monitored in vivo using fluorescently labeled fusion proteins and time-lapse microscopy. To measure cellular properties in microscopic images, accurate and fast image segmentation methods are a critical prerequisite. To quantify static and dynamic features of interphase nuclei and mitotic chromosomes, we developed a three-dimensional (3D) segmentation method based on multiple level sets. We tested our method on 3D time-series images of live embryos expressing histone-2Av-green fluorescence protein. Our method is robust to low signal-to-noise ratios inherent to high-speed imaging, fluorescent signals in the cytoplasm, and dynamic changes of shape and texture. Comparisons with manual ground-truth segmentations showed that our method achieves more than 90% accuracy on the object as well as voxel levels and performs consistently throughout all cell cycle phases and developmental stages from syncytial blastoderm to postblastoderm mitotic domains.

Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila.

The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.

[What gene and chromosomes say about the origin and evolution of insects and other arthropods].

At the turn of the 21st century, the use of molecular and molecular cytogenetic methods led to revolutionary advances in systematics of insects and other arthropods. Analysis of nuclear and mitochondrial genes, as well as investigation of structural rearrangements in the mitochondrial chromosome convincingly supported the Pancrustacea hypothesis, according to which insects originated directly from crustaceans, whereas myriapods are not closely related to them. The presence of the specific telomeric motif TTAGG confirmed the monophyletic origin of arthropods (Arthropoda) and the assignment of tongue worms (Pentastomida) to this type. Several different types of telomeric sequences have been found within the class of insects. Investigation of the molecular organization of these sequences may shed light on the relationships between the orders Diptera, Siphonaptera, and Mecoptera and on the origin of such enigmatic groups as the orders Strepsiptera, Zoraptera and suborder Coleorrhyncha.