The salivary glands of Brontocoris tabidus (Heteroptera: Pentatomidae): Morphology and secretory cycle.

Brontocoris tabidus (Signoret) (Heteroptera: Pentatomidae) is a zoophytophagous insect used for biological control in agriculture and forest systems because its nymphs and adults feed on insects and plants. The predatory Pentatomidae insert the mouthparts into the prey, releasing saliva to paralysis and kills the insect, as well as digest body parts to be sucked in a preliminary extra-oral digestion. In a short period of time, this insect shows the ability to feed again, suggesting the existence of a constant and abundant secretory cycle in the salivary glands. This study evaluated the morphological, histochemical and ultrastructural changes of the salivary glands of B. tabidus in fed and starved insects. The salivary complex of this predatory bug has a pair of bilobed salivary glands and a pair of tubular accessory salivary glands. The accessory glands have the lumen lined by a thick non-cuticular layer rich in glycoproteins. The secretory cells of the B. tabidus principal salivary glands have constant secretory activity, with each lobe producing different substances. The physiological processes that occur in the salivary gland of B. tabidus indicate that the insect needs to feed constantly, corroborating the potential of this insect to be used in biological control programs.

Gross morphology and ultrastructure of the salivary glands of the stink bug predator Eocanthecona furcellata (Wolff).

Eocanthecona furcellata Wolff (Hemiptera: Pentatomidae) is a native generalist predator which attacks and kills its prey by first inserting its stylet into the prey's body and then injecting saliva into it. Here, we describe the histology and ultrastructure of its salivary glands. The study showed that the salivary glands were made up of pairs of principal and tubular accessory salivary glands. The principal salivary glands were bilobed and consisted of a smaller anterior lobe and a larger elongated posterior lobe. The ducts of the principal and accessory salivary glands were located in a narrow region between the anterior and posterior lobe known as the hilum. The principal salivary gland was lined with a single-layered epithelium. The cells cytoplasm was enriched with rough endoplasmic reticulum and secretory, and the nucleus showed a higher level of uncondensed chromatin. The basal region of the cell had plasma membrane infoldings. The cytoplasm of the accessory gland was rich in rough endoplasmic reticulum and many large cavities. The ducts of the principal salivary gland were made up of a single layer of flattened cells which had a thin cuticle lining the apical portion. Variation in the lumen content of the different lobes, which made up the principal gland suggested that their chemical products also varied. These results indicate that these two salivary glands produce the proteins found in the saliva.

Comparative morphology of male genitalic structures in the minute litter bugs Dipsocoromorpha (Insecta: Hemiptera: Heteroptera).

Insect male genitalia show an evolutionarily variable morphology that has proven to be valuable for both, species identifications and phylogenetic analyses at higher taxonomic levels. Accurate usage of genitalic characters in taxonomic descriptions and phylogenetic analyses depends on consistency of terminology and validity of homology hypotheses. Both areas are underdeveloped in many insect groups. We here document the morphology and advance homology hypotheses of male genitalic features for the hemipteran infraorder Dipsocoromorpha, the minute litter bugs. Genitalic structures and the pregenital abdomen in Dipsocoromorpha are strikingly modified and diverse compared to other Heteroptera. In addition to variation in the shape of phallic structures (parameres and aedeagus), minute litter bug genitalia vary in the direction and degree of asymmetry and feature a plethora of processes derived from various abdominal segments with significant variation at low taxonomic levels. Here, male genitalic structures for an extensive taxonomic sample (32 genera and 71 specimens) are documented using scanning electron and confocal microscopy, and a universal terminology for genitalic structures across minute litter bugs is established that will facilitate species discovery and evolutionary research. We conclude by proposing primary homology hypotheses across the infraorder that now can be tested in a phylogenetic framework.

Description of the pre-reductional sex chromosome during male meiosis of Pachylis laticornis (Heteroptera: Coreidae).

In Heteroptera, the division of sex chromosomes is well defined as post-reductional for most of species, i.e., the first meiotic division is equational and the second is reductional. However, in some species pre-reductional division has been observed, whereby the first meiotic division is reductional and the second is equational. These include Anisops fieberi (Notonectidae), Ectrychotes disparate (Reduviidae), Dictyonota tricornis (Tingidae), and Archimerus alternatus (Coreidae), as well as other species of the genus Pachylis, in the family Coreidae. Thus, this study aimed to characterize the meiotic behavior of Pachylis laticornis, in order to consider whether this species also undergoes pre-reduction division for the sex chromosomes. Cytogenetic analysis of meiosis in P. laticornis made it possible to characterize the holocentric nature of the chromosomes, the chromosome number of this species [2n = 15 (2m + 12A + X0)], the chromosomal system of sex X0 type, and the presence of m-chromosomes. Furthermore, the analysis of anaphase I, telophase I and II allowed pre-reductional meiotic behavior to be observed for this sex chromosome. Thus, this meiotic behavior was confirmed for another species of Heteroptera, stressing the importance of more cytogenetic studies of meiosis to increase our understanding of variation in the behavior of sex chromosomes during spermatogenesis in heteropterans. Therefore, the present study describes the chromosomal number, the system of sex determination, and meiotic behavior of P. laticornis, corroborating the relationship of this species with others of the same genus.

Morphology and Proteome Characterization of the Salivary Glands of the Western Chinch Bug (Hemiptera: Blissidae).

The western chinch bug, Blissus occiduus Barber, is a serious pest of buffalograss, Buchloe dactyloides (Nuttall) due to physical and chemical damage caused during the feeding process. Although previous work has investigated the feeding behaviors of chinch bugs in the Blissus complex, no study to date has explored salivary gland morphology and the associated salivary complex of this insect. Whole and sectioned B. occiduus salivary glands were visualized using light and scanning electron microscopy to determine overall structure and cell types of the salivary glands and their individual lobes. Microscopy revealed a pair of trilobed principal glands and a pair of tubular accessory glands of differing cellular types. To link structure with function, the salivary gland proteome was characterized using liquid chromatography tandem mass spectrometry. The salivary proteome analysis resulted in B. occiduus sequences matching 228 nonhomologous protein sequences of the pea aphid, Acyrthosiphon pisum (Harris), with many specific to the proteins present in the salivary proteome of A. pisum. A number of sequences were assigned the molecular function of hydrolase and oxido-reductase activity, with one specific protein sequence revealing a peroxidase-like function. This is the first study to characterize the salivary proteome of B. occiduus and the first of any species in the family Blissidae.

Dynamic BMP signaling polarized by Toll patterns the dorsoventral axis in a hemimetabolous insect.

Toll-dependent patterning of the dorsoventral axis in Drosophila represents one of the best understood gene regulatory networks. However, its evolutionary origin has remained elusive. Outside the insects Toll is not known for a patterning function, but rather for a role in pathogen defense. Here, we show that in the milkweed bug Oncopeltus fasciatus, whose lineage split from Drosophila's more than 350 million years ago, Toll is only required to polarize a dynamic BMP signaling network. A theoretical model reveals that this network has self-regulatory properties and that shallow Toll signaling gradients are sufficient to initiate axis formation. Such gradients can account for the experimentally observed twinning of insect embryos upon egg fragmentation and might have evolved from a state of uniform Toll activity associated with protecting insect eggs against pathogens.

First report on meiotic behavior of chromosomes in three species of subfamily Pentatominae (Pentatomidae: Heteroptera).

Regardless of rich biodiversity, cytogenetic reports on Pentatomidae refer to less than 400 species. This paper reports, for the first time, course of meiosis of three species of subfamily Pentatominae viz., Tolumnia antennata Distant, Cahara jugatoria (Lethierry) and Cahara confusa (Distant). Species-specific variations observed with respect to association pattern of X and Y during prophase, arrangement pattern of chromosomes at metaphase I and degree of association between X and Y in the pseudobivalent at metaphase II are explained in the present paper.Tolumnia antennata and Cahara jugatoria possess 2n=14=12A+XY while Cahara confusa possesses 2n=12=10A+XY. At least one ring bivalent is observed during diplotene in all the three species. X and Y dissociate to get well separated at diplotene in Cahara confusa, at diakinesis in Tolumnia antennata and at metaphase I in Cahara jugatoria. In Cahara jugatoria, at metaphase I, X and Y are peripheral forming a part of the ring. In Cahara confusa, at metaphase II, X is peripheral while Y lies in the centre and the two fail to associate to form a pseudobivalent, a phenomenon never recorded earlier in any pentatomid.

Mechanisms of color production in a highly variable shield-back stinkbug, Tectocoris diophthalmus [corrected] (Heteroptera: Scutelleridae), and why it matters.

Theory suggests that aposematism, specifically the learned avoidance of unprofitable prey via memorable color patterns, should result in selection for pattern uniformity. However, many examples to the contrary are seen in nature. Conversely, honest sexual signals are likely to exhibit greater variation because they reflect underlying variation in mate quality. Here we aim to characterize and quantify the mechanistic causes of color in Tectocoris diophthalmus [corrected] to shed light on the costs of color production, and thus the potential information content of its color signals. We use Tectocoris diophthalmus [corrected] because it is a weakly-defended stinkbug, and presents elements that have classically been studied in the context of aposematism (red coloring), and sexual selection (sexual dichromatism and iridescent coloring). Pigment analysis reveals that variation in orange coloration is due to the amount of erythropterin pigment, stored in intracellular granules. This pigment is common in Heteroptera, and as an endogenously produced excretory byproduct is unlikely to reflect mate quality or variation in unprofitability of the bug. Electron microscopy reveals the iridescent patches are caused by an epicuticular multilayer reflector, and the hue and patch size are directly related to the layer widths and extent of coverage of this layering. Furthermore, we identified melanin as an essential component of the multilayer reflector system; therefore, the quality of the iridescent patches may be affected by aspects of rearing environment and immunocompetence. We posit that T. diophthalmus [corrected] has co-opted the melanic patches of a 'typical' red and black aposematic signal, transforming it into a complex and variable iridescent signal that may enhance its capacity to display individual quality.

Role of the brain in photoperiodic regulation of juvenile hormone biosynthesis in the brown-winged green bug Plautia stali.

We investigated the roles of the brain in photoperiodic regulation of juvenile hormone (JH) biosynthesis in Plautia stali (Heteroptera: Pentatomidae). Within 4days after transferring the adults from short-day to long-day conditions, the biosynthesis of juvenile hormone III skipped bisepoxide became higher than that under short-day conditions. When the corpus cardiacum-corpus allatum complex (CC-CA) was coincubated with different regions of the brain, JH biosynthetic activities were inhibited by a middle part of the brain, i.e., the brain excluding the optic lobes, extirpated from short-day and long-day females. Backfills from the CC-CA revealed three groups of somata in the middle part of the brain: 15-18 pairs of somata were stained in the pars intercerebralis, 9-10 pairs were stained in the pars lateralis, and five pairs were stained on the nervi corporis cardiaci 2. These results suggest that inhibitory molecules of JH biosynthesis are produced in the middle part of the brain, regardless of photoperiod, but their release is suppressed under long-day conditions, leading to an increase in JH biosynthetic activities. Further, some neurons with somata in the pars intercerebralis, in the pars lateralis, or on the nervi corporis cardiaci 2 might be involved in photoperiodic regulation of JH biosynthesis.

Karyological and meiotic studies in seven species of Coreinae (Hemiptera: Heteroptera: Coreidae) from North India.

Coreinae, the largest subfamily of Coreidae, is distributed worldwide but is most abundant in the tropics. Cytogenetic data is available for 122 species, which include 40 from India. In the present paper, 7 species of Coreinae have been cytogenetically investigated for the first time for their diploid chromosome complement and for meiosis. Also, this is the first cytogenetic report for the genera Prionoloinia and Petalocnemis. The modal diploid chromosome complement of the family Coreidae is 2n=21=18A+2m+X0. The present study on 7 species shows the diploid chromosome complement to vary from 15 to 27. Microchromosomes are present in 6 and absent only in I species. The sex determining mechanism is X0 in 5 and X1X20 in 2 species. The decrease in autosome number (14A) has been accompanied by 2 pairs of extremely large autosomes (Anoplocienis binotata Distant and Cletus borealis Blöte) and the increase in autosome number (22A, 24A) has been accompanied by distinctly small autosomes (Petalocnemis obscura and Prionolomia sp.) in the complement. The behavior of chromosomes during meiosis is typical of Coreidae. Deviations wherever recorded are discussed. During diplotene, chiasmata are located terminally and there is a predominance of a single chiasma per bivalent. However, in Anoplocnemis binotata and Cletus borealis, one or both of the large bivalents show two terminal or subterminal chiasmata in diplotene. A regular arrangement of chromosomes is observed during both metaphase I and II: all the autosomes form a ring with microchromosomes lying inside and sex chromosomes lying outside the ring.

Cytogenetic evidence for de novo synthesis of rRNA and involvement of nucleolar material in the organization of cell structures during spermiogenesis of Chariesterus armatus (Heteroptera, Coreidae).

The nucleolar material of Chariesterus armatus was analyzed during spermiogenesis in cell preparations impregnated with silver nitrate. Nucleolar corpuscles were observed in spermatids at the beginning of the process, showing that this organoid is also maintained after meiosis. In addition, nucleoli were seen in the round spermatids connected to the X-chromosome (bearer of the nucleolar organizer in C. armatus), indicating de novo synthesis of nucleolar material. This differs from the reorganization of ribosomal granules, transported from meiotic spermatocytes to round spermatids, where they would support protein synthesis, which is reported for other species. We also observed connections of nucleolar corpuscles to the nuclear membrane regions where the tail and the acrosome will be formed, suggesting close involvement of the nucleolar material in the formation of these structures. In addition to the nucleolar bodies, we detected silver-positive structures, which will require new approaches to clarify their role. One of these structures, observed in the cytoplasm, appears to correspond to the chromatoid body, which has been found in several organisms, but is still poorly understood; another is a complex structure to which the tail appears to be connected. We conclude that C. armatus is an appropriate model for understanding not only the synthesis of rRNA in the spermiogenesis, but also the functional meaning of the close relationship of nucleolar material with other structures during this process.

Meiotic behavior and karyotypic variation in Acledra (Pentatomidae, Heteroptera).

Acledra comprises 15 taxonomically identified species, most of which are crop pests. This is the first cytogenetic study of species of this genus. Acledra kinbergii and A. modesta showed the modal number of the Pentatomidae (2n = 14 = 12 + XY), while A. bonariensis had a reduced complement (2n = 12 = 10 + XY), with a markedly larger autosomal pair. Meiotic behavior follows the general pattern of the family; the autosomes divide pre-reductionally, the sex chromosomes are achiasmatic and divide post-reductionally, and at metaphase II the autosomes show a ring-shaped configuration with the pseudobivalent at the center. However, the configuration at metaphase I varies; A. modesta shows the typical arrangement (ring of bivalents with the sex chromosomes lying at its center). In A. kinbergii, the sex chromosomes are part of the ring or only the Y chromosome is at the center. In A. bonariensis, the ring arrangement is not well defined. There are also differences at the diffuse stage; chromatin strands of different width are observed in A. bonariensis and A. modesta, whereas bivalents do not entirely lose their identity in A. kinbergii. In A. bonariensis, the reduced complement may have originated from the fusion of the two larger non-homologous autosomes, which could characterize the ancestral karyotype of this genus. The presence of secondary constrictions in the larger pair of A. modesta and A. bonariensis may support this hypothesis. Since secondary constrictions are uncommon in the holokinetic chromosomes of heteropterans, their presence in these species may indicate that it is a plesiomorphic character of the genus.

Distribution and functional morphology of photomechanic infrared sensilla in flat bugs of the genus Aradus (Heteroptera, Aradidae).

Globally the flat bug genus Aradus comprises about 200 species. About half a dozen Aradus species can be primarily found on burnt areas and, therefore, have been called pyrophilous. Bugs and their offspring feed on fungi growing on burnt wood. Recently, prothoracic infrared (IR) receptors have been described in the pyrophilous Australian species Aradus albicornis. In our study we investigated 10 Aradus species, once again including A. albicornis, and found prothoracic as well as hitherto unknown mesothoracic IR sensilla in A. albicornis, Aradus lugubris and Aradus fuscicornis. In Aradus flavicornis only prothoracic IR receptors were found. Currently the latter two species are not known as pyrophilous. However, there is considerable evidence that these flat bugs also approach forest fires. In all four species where IR receptors were identified, the dome-shaped IR sensilla look very similar. An IR sensillum consists of an internal exocuticular sphere reinforced by consecutive layers of chitin fibres. In the center of the sphere, a microfluidic core is located which consists of a cup-shaped plug of cuticle and an underlying fluid filled annular channel surrounding the tip of the dendrite of a mechanosensitive neuron. Like the IR receptors of buprestid beetles of the genus Melanophila, the IR sensilla found in Aradus species can be classified as photomechanic IR receptors.

Structural polarity and dynamics of male germline stem cells in an insect (milkweed bug Oncopeltus fasciatus).

Knowing the structure opens a door for a better understanding of function because there is no function without structure. Male germline stem cells (GSCs) of the milkweed bug (Oncopeltus fasciatus) exhibit a very extraordinary structure and a very special relationship with their niche, the apical cells. This structural relationship is strikingly different from that known in the fruit fly (Drosophila melanogaster) -- the most successful model system, which allowed deep insights into the signaling interactions between GSCs and niche. The complex structural polarity of male GSCs in the milkweed bug combined with their astonishing dynamics suggest that cell morphology and dynamics are causally related with the most important regulatory processes that take place between GSCs and niche and ensure maintenance, proliferation, and differentiation of GSCs in accordance with the temporal need of mature sperm. The intricate structure of the GSCs of the milkweed bug (and probably of some other insects, i.e., moths) is only accessible by electron microscopy. But, studying singular sections through the apical complex (i.e., GSCs and apical cells) is not sufficient to obtain a full picture of the GSCs; especially, the segregation of projection terminals is not tangible. Only serial sections and their overlay can establish whether membrane ingrowths merely constrict projections or whether a projection terminal is completely cut off. To sequence the GSC dynamics, it is necessary to include juvenile stages, when the processes start and the GSCs occur in small numbers. The fine structural analysis of segregating projection terminals suggests that these terminals undergo autophagocytosis. Autophagosomes can be labeled by markers. We demonstrated acid phosphatase and thiamine pyrophosphatase (TPPase). Both together are thought to identify autophagosomes. Using the appropriate substrate of the enzymes and cerium chloride, the precipitation of electron-dense cerium phosphate granules indicates the presence of enzymes and their location. Because the granules are very fine, they can be easily assigned to distinct cell organelles as the autophagosomes. Two methods, electron microscopy and immunocytochemistry, have pointed out a structural polarity and dynamics that are unprecedented for stem cells. We propose that these dynamics indicate a novel type of signal exchange and transduction between stem cells and their niche.

Synapsis with and without recombination in the male meiosis of the leaf-footed bug Holhymenia rubiginosa (Coreidae, Heteroptera).

In organisms with chiasmatic meiosis two different relationships have been described between crossing over and synapsis: in one group of organisms synapsis depends on the initiation of meiotic recombination while in the other group it is independent of this initiation. These patterns have been observed mainly in organisms where all meiotic bivalents in the set have similar behaviors. In some heteropteran insects a pair of chromosomes named m chromosomes is known to behave differently from autosomes regarding synapsis and recombination. Here we used immunodetection of a synaptonemal complex component and acid-fixed squashes to investigate the conduct of the small m chromosome pair during the male meiosis in the coreid bug Holhymenia rubiginosa. We found that the m chromosomes form a synaptonemal complex during pachytene, but they are not attached by a chiasma in diakinesis. On the other hand, the autosomal bivalents synapse and recombine regularly. The co-existence of these variant chromosome behaviors during meiosis I add further evidence to the absence of unique patterns regarding the interdependence of synapsis and recombination.

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.

Study of chromosomal and nucleolar aspects in testes of Nysius californicus (Heteroptera: Lygaeidae).

In Nysius californicus (family Lygaeidae, subfamily Orsillinae), a pest commonly known as the seed bug, the chromosome complement is 2n = 16 (12A + 2m + XY), testes are formed by seven seminiferous tubules covered by an orange-colored membrane, and spermatogenesis is cystic. At prophase, sex chromosomes are heteropycnotic and autosomes usually show a chiasma. At metaphase, sex chromosomes along with microchromosomes may be seen located at the center of a ring formed by the remaining autosomes. A characteristic specific of N. californicus was the presence of nucleolar material observed from the cystic cell to the completely differentiated spermatozoon. Variations in size, shape and location of the nucleolar material occur during this process, denoting a variable degree of activity in the different stages.

Study of chromosomal and nucleolar aspects in testes of Nysius californicus (Heteroptera: Lygaeidae)

In Nysius californicus (family Lygaeidae, subfamily Orsillinae), a pest commonly known as the seed bug, the chromosome complement is 2n = 16 (12A + 2m + XY), testes are formed by seven seminiferous tubules covered by an orange-colored membrane, and spermatogenesis is cystic. At prophase, sex chromosomes are heteropycnotic and autosomes usually show a chiasma. At metaphase, sex chromosomes along with microchromosomes may be seen located at the center of a ring formed by the remaining autosomes. A characteristic specific of N. californicus was the presence of nucleolar material observed from the cystic cell to the completely differentiated spermatozoon.Variations in size, shape and location of the nucleolar material occur during this process, denoting a variable degree of activity in the different stages.

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.

F-actin, beta-tubulin, aldolase, and fructose-1,6-bisphosphatase in heteropteran ovarioles--I. Immunocytochemical investigations of whole-mounted ovarioles.

The distribution of F-actin, beta-tubulin, aldolase, and fructose-1,6-bisphosphatase (FBPase) in ovarioles of four heteropteran species (Ilyocoris cimicoides, Coreus marginatus, Lygus pratensis, and Notostira elongata) was investigated biochemically and immunocytochemically. Aldolase was found to be uniformly distributed in the cytoplasm of trophocytes and follicular cells, with the highest concentration in prefollicular cells. Its concentration in follicular cells increased during differentiation and reached a peak in ovarian follicles at the stage of late choriogenesis. FBPase was observed in the cytoplasm (weak reaction) and on cell borders (strong reaction) of both germ line and somatic cells. No FBPase or aldolase signal was observed on the F-actin trophic core mesh or on stress fibers.