A chromosome scale assembly of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), genome.

OBJECTIVE: The tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is a pest damaging many cultivated crops in North America. Although partial transcriptome data are available for this pest, a genome assembly was not available for this species. This assembly of a high-quality chromosome-length genome of TPB is aimed to develop the genetic resources that can provide the foundation required for advancing research on this species. RESULTS: The initial genome of TPB assembled with paired-end nucleotide sequences generated with Illumina technology was scaffolded with Illumina HiseqX reads generated from a proximity ligated (HiC) library to obtain a high-quality genome assembly. The final assembly contained 3963 scaffolds longer than 1 kbp to yield a genome of 599.96 Mbp. The N50 of the TPB genome assembly was 35.64 Mbp and 98.68% of the genome was assembled into 17 scaffolds larger than 1 Mbp. This megabase scaffold number is the same as the number of chromosomes observed in karyotyping of this insect. The TPB genome is known to have high repetitive DNA content, and the reduced assembled genome size compared to flowcytometric estimates of approximately 860 Mbp may be due to the collapsed assembly of highly similar regions.

A synopsis of the numbers of testicular follicles and ovarioles in true bugs (Heteroptera, Hemiptera) - sixty-five years of progress after J. Pendergrast's review.

The structure of testes and ovaries can be described in its simplest form by the number of follicles and ovarioles they contain. Sixty-five years after the last review of the internal reproductive systems in true bugs (Heteroptera), the data accumulated today on the number of testicular follicles and ovarioles in their gonads are summarized. In addition, data on the number and type (mesadenia/ectadenia) of accessory glands are given. The hemipteran suborder Heteroptera constitutes one of the most diverse groups of non-homometabolous ('Hemimetabola') insects, comprising more than 40,000 described species worldwide and approximately 100 families, classified into seven infraorders. Data are available for all infraorders; however, more than 90% of studied species belong to the largest and most evolutionarily derived infraorders Cimicomorpha and Pentatomomorpha. In true bugs, in general, the number of follicles varies from one to nine (in a testis), and the number of ovarioles varies from two to 24 (in an ovary). Seven follicles per testis and seven ovarioles per ovary prevail being found in approximately 43.5% (307 species) and 24.4% (367 species) of studied species, respectively. Such a structure of testes and ovaries is considered an ancestral character state in the Heteroptera. In the evolution of this group, the number of follicles and ovarioles both increased and decreased, but the trend towards a decrease clearly prevailed.

Comparative analysis of chromosome numbers and sex chromosome systems in Paraneoptera (Insecta).

This article is part (the 4th article) of the themed issue (a monograph) "Aberrant cytogenetic and reproductive patterns in the evolution of Paraneoptera". The purpose of this article is to consider chromosome structure and evolution, chromosome numbers and sex chromosome systems, which all together constitute the chromosomal basis of reproduction and are essential for reproductive success. We are based on our own observations and literature data available for all major lineages of Paraneoptera including Zoraptera (angel insects), Copeognatha (=Psocoptera; bark lice), Parasita (=Phthiraptera s. str; true lice), Thysanoptera (thrips), Homoptera (scale insects, aphids, jumping plant-lice, whiteflies, and true hoppers), Heteroptera (true bugs), and Coleorrhyncha (moss bugs). Terminology, nomenclature, classification, and the study methods are given in the first paper of the issue (Gavrilov-Zimin et al. 2021).

Introduction to the study of chromosomal and reproductive patterns in Paraneoptera.

This paper opens the themed issue (a monograph) "Aberrant cytogenetic and reproductive patterns in the evolution of Paraneoptera", prepared by a Russian-Bulgarian research team on the basis of long-term collaborative studies. In this first part of the issue, we provide the basic introductory information, describe the material involved and the methods applied, and give terminology and nomenclature of used taxonomic names.

New data on karyotype, spermatogenesis and ovarian trophocyte ploidy in three aquatic bug species of the families Naucoridae, Notonectidae, and Belostomatidae (Nepomorpha, Heteroptera).

We report the karyotype, some aspects of spermatogenesis, and ovarian trophocytes ploidy in three aquatic bug species: Ilyocoris cimicoides (Linnaeus, 1758), Notonecta glauca Linnaeus, 1758, and Diplonychus rusticus Fabricius, 1871 from previously unexplored regions - South Europe (Bulgaria) and Southeast Asia (Vietnam). Our results add considerable support for the published karyotype data for these species. In I. cimicoides, we observed achiasmate male meiosis - the first report of achiasmy for the family Naucoridae. More comprehensive cytogenetic studies in other species of the Naucoridae are required to elucidate the role of achiasmy as a character in the systematics of the family. Our observations on the association between phases of spermatogenesis and developmental stages in I. cimicoides and N. glauca differ from the previously published data. In these species, we assume that the spermatogenesis phases are not strongly associated with certain developmental stages. For further cytogenetic studies (on the Balkan Peninsula), we recommend July as the most appropriate month for collection of I. cimicoides and N. glauca. In the ovaries of both species, we studied the level of ploidy in metaphase and interphase trophocytes. In I. cimicoides, diploid and tetraploid metaphase trophocytes were found. Heteropycnotic elements, observed in interphase trophocytes of this species, represented the X chromosomes. It allowed us to determine the trophocytes ploidy at interphase (2n was repeated up to 16 times). The situation with N. glauca was different. The metaphase trophocytes were diploid and we were not able to determine the ploidy of interphase trophocytes since such conspicuous heteropycnotic elements were not found. The scarce data available suggest a tendency for a low level of trophocyte ploidy in the basal infraorders (Nepomorpha and Gerromorpha) and for a high level in the more advanced Pentatomomorpha. Data about this character in species from other infraorders are needed to confirm that tendency.

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.

A chromosomal analysis of Nepa cinerea Linnaeus, 1758 and Ranatra linearis (Linnaeus, 1758) (Heteroptera, Nepidae).

An account is given of the karyotypes and male meiosis of the Water Scorpion Nepa cinerea Linnaeus, 1758 and the Water Stick Insect Ranatra linearis (Linnaeus, 1758) (Heteroptera, Nepomorpha, Nepidae). A number of different approaches and techniques were tried: the employment of both male and female gonads and mid-guts as the sources of chromosomes, squash and air-drying methods for chromosome preparations, C-banding and fluorescence in situ hybridization (FISH) for chromosome study. We found that N. cinerea had a karyotype comprising 14 pairs of autosomes and a multiple sex chromosome system, which is X1X2X3X4Y (♂) / X1X1X2X2X3X3X4X4 (♀), whereas R. linearis had a karyotype comprising 19 pairs of autosomes and a multiple sex chromosome system X1X2X3X4Y (♂) / X1X1X2X2X3X3X4X4 (♀). In both N. cinerea and R. linearis, the autosomes formed chiasmate bivalents in spermatogenesis, and the sex chromosome univalents divided during the first meiotic division and segregated during the second one suggesting thus a post-reductional type of behaviour. These results confirm and amplify those of Steopoe (1925, 1927, 1931, 1932) but are inconsistent with those of other researchers. C-banding appeared helpful in pairing up the autosomes for karyotype assembly; however in R. linearis the chromosomes were much more uniform in size and general appearance than in N. cinerea. FISH for 18S ribosomal DNA (major rDNA) revealed hybridization signals on two of the five sex chromosomes in N. cinerea. In R. linearis, rDNA location was less obvious than in N. cinerea; however it is suggested to be similar. We have detected the presence of the canonical "insect" (TTAGG) n telomeric repeat in chromosomes of these species. This is the first application of C-banding and FISH in the family Nepidae.

Achiasmate male meiosis in two Cymatia species (Hemiptera, Heteroptera, Corixidae).

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 Cymatia 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.

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.

Comparative cytogenetic study of three Macrolophus species (Heteroptera, Miridae).

Macrolophus pygmaeus (Rambur, 1839) (Insecta, Heteroptera, Miridae) is a predator of key vegetable crop pests applied as a biocontrol agent in the Mediterranean region. Macrolophus pygmaeus and Macrolophus melanotoma (A. Costa, 1853) are cryptic species with great morphological similarity which results in their misidentification and negative consequences for the conservation of their populations on greenhouse and outdoor crops. In order to find out specific markers for their separation we studied the karyotype, male meiosis and heterochromatin composition of these species and additionally of a third species (as a reference one), Macrolophus costalis Fieber, 1858. We demonstrate here that all the three species share achiasmate male meiosis and sex chromosome pre-reduction. On the other hand, the species differ in karyotype, with 2n=28 (26+XY) in Macrolophus pygmaeus, 2n=27 (24+X1X2Y) in Macrolophus costalis, and 2n=34 (32+XY) in Macrolophus melanotoma, and heterochromatin distribution and composition. In addition, the species differ in sperm morphology: sperm cells of Macrolophus costalis are significantly longer with longer head and tail than those of Macrolophus melanotoma and Macrolophus pygmaeus, whereas sperm cells of Macrolophus melanotoma have a longer tail than those of Macrolophus pygmaeus. All these characters can be used as markers to identify the species, in particular the cryptic species Macrolophus melanotoma and Macrolophus pygmaeus.

Sex chromosome pre-reduction in male meiosis of Lethocerus patruelis (Stål, 1854) (Heteroptera, Belostomatidae) with some notes on the distribution of the species.

The karyotype and meiosis in males of giant water bug Lethocerus patruelis (Heteroptera: Belostomatidae: Lethocerinae) were studied using standard and fluorochrome (CMA3 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, Lethocerus 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 Lethocerus patruelis is presented, the contemporary distribution of Lethocerus patruelis in Bulgaria and in the northern Aegean Islands is discussed, and the first information about the breeding and nymphal development of this species in Bulgaria is provided.

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.

Cytogenetics of the true bug infraorder Cimicomorpha (Hemiptera, Heteroptera): a review.

The Cimicomorpha is one of the largest and highly diversified infraorders of the Heteroptera. This group is also highly diversified cytogenetically and demonstrates a number of unusual cytogenetic characters such as holokinetic chromosomes; m-chromosomes; multiple sex chromosome systems; post-reduction of sex chromosomes in meiosis; variation in the presence/absence of chiasmata in spermatogenesis; different types of achiasmate meiosis. We present here a review of essential cytogenetic characters of the Cimicomorpha and outline the chief objectives and goals of future investigations in the field.

Cytogenetic characterization of the Trinidad endemic, Arachnocoris trinitatus Bergroth: the first data for the tribe Arachnocorini (Heteroptera: Cimicomorpha: Nabidae).

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 ofA. trinitatus.

First evidence of sex chromosome pre-reduction in male meiosis in the Miridae bugs (Heteroptera).

The karyotype and male meiosis of Macrolophus costalis Fieber (Insecta, Heteroptera, Miridae) were studied using C-banding, AgNOR-banding and DNA sequence specific fluorochrome staining. The chromosome formula of the species is 2n = 28(24+X1X2X3Y). Male meiotic prophase is characterized by a prominent condensation stage. At this stage, two sex chromosomes, "X" and Y are positively heteropycnotic and always appeared together, while in autosomal bivalents homologous chromosomes were aligned side by side along their entire length, that is, meiosis is achiasmatic. At metaphase I, "X" and Y form a pseudobivalent and orient to the opposite poles. At early anaphase I, the "X" chromosome disintegrates into three separate small chromosomes, X1, X2, and X3. Hence both the autosomes and sex chromosomes segregate reductionally in the first anaphase, and separate equationally in the second anaphase. This is the first evidence of sex chromosome pre-reduction in the family Miridae. Data on C-heterochromatin distribution and its composition in the chromosomes of this species are discussed.

The origin of the achiasmatic XY sex chromosome system in Cacopsylla peregrina (Frst.) (Psylloidea, Homoptera).

The status of an extra univalent, if it is a B chromosome or an achiasmatic Y chromosome, associating with the X chromosome in male meiosis of Cacopsylla peregrina (Frst.) (Homoptera, Psylloidea) was analysed. One extra univalent was present in all males collected from three geographically well separated populations, it was mitotically stable, and showed precise segregation from the X chromosome. These findings led us to propose that the univalent represents in fact a Y chromosome. The behaviour of the X and Y chromosomes during meiotic prophase suggested that their regular segregation was based on an achiasmatic segregation mechanism characterised by a 'touch and go' pairing of segregating chromosomes at metaphase I. To explain the formation of the achiasmatic Y within an insect group with X0 sex chromosome system, it was suggested that the Y chromosome has evolved from a mitotically stable B chromosome that was first integrated into an achiasmatic segregation system with the X chromosome, and has later become fixed in the karyotype as a Y chromosome.

C-heterochromatin and extra (B) chromosome distribution in six species of the Nabis (Heteroptera, Nabidae) with the modal male karyotype 2n = 16 + XY.

The basic male karyotype of the six Nabis species (Heteroptera, Nabidae) is confirmed as being 2n=16+XY. The chromosomes are holokinetic while male meiosis is achiasmatic. The sex chromosomes undergo postreduction and in second metaphase show distance pairing, registered in all nabid species examined so far. Using C-banding technique for the first time in the family Nabidae, the heterochromatin was revealed on chromosomes of six species. The species showed different amount and distribution of C-heterochromatin. Only in Nabis (Dolichonabis) limbatus did the C-bands distribution make possible the identification of every chromosome pair in the karyotype. In other species, C-bands were found in some of the autosomes and the X, localized either interstitially or at telomeres. Only the Y usually showed relative stability ofthe C-banding pattern. In four of six species, extra (B) chromosomes were observed and their behaviour in meiosis described.