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Insect chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals from air to olfactory receptors in the lymph of antennal chemosensilla. They may represent a new class of soluble carrier protein involved in insect chemoreception. However, their specific functional roles in insect chemoreception have not been fully elucidated. In this study, we report for the first time three novel CSP genes (AlinCSP1-3) of the alfalfa plant bug Adelphocoris lineolatus (Goeze) by screening the antennal cDNA library. The qRT-PCR examinations of the transcript levels revealed that all three genes (AlinCSP1-3) are mainly expressed in the antennae. Interestingly, these CSP genes AlinCSP1-3 are also highly expressed in the 5(th) instar nymphs, suggesting a proposed function of these CSP proteins (AlinCSP1-3) in the olfactory reception and in maintaining particular life activities into the adult stage. Using bacterial expression system, the three CSP proteins were expressed and purified. For the first time we characterized the types of sensilla in the antennae of the plant bug using scanning electron microscopy (SEM). Immunocytochemistry analysis indicated that the CSP proteins were expressed in the pheromone-sensitive sensilla trichodea and general odorant-sensitive sensilla basiconica, providing further evidence of their involvement in chemoreception. The antennal activity of 55 host-related semiochemicals and sex pheromone compounds in the host location and mate selection behavior of A. lineolatus was investigated using electroantennogram (EAG), and the binding affinities of these chemicals to the three CSPs (AlinCSP1-3) were measured using fluorescent binding assays. The results showed several host-related semiochemicals, (Z)-3-hexen-1-ol, (E)-2-hexen-1-al and valeraldehyde, have a high binding affinity with AlinCSP1-3 and can elicit significant high EAG responses of A. lineolatus antennae. Our studies indicate the three antennae-biased CSPs may mediate host recognition in the alfalfa plant bug A. lineolatus.

Despite the remarkable evolutionary success of insects at colonizing every conceivable terrestrial and aquatic habitat, only five Halobates (Heteroptera: Gerridae) species (~0.0001% of all known insect species) have succeeded at colonizing the open ocean – the largest biome on Earth. This remarkable evolutionary achievement likely required unique adaptations for them to survive and thrive in the challenging oceanic environment. For the first time, we explore the morphology and behavior of an open-ocean Halobates germanus and a related coastal species H. hayanus to understand mechanisms of these adaptations. We provide direct experimental evidence based on high-speed videos which reveal that Halobates exploit their specialized and self-groomed body hair to achieve extreme water repellence, which facilitates rapid skating and plastron respiration under water. Moreover, the grooming behavior and presence of cuticular wax aids in the maintenance of superhydrophobicity. Further, reductions of their body mass and size enable them to achieve impressive accelerations (~400 ms −2 ) and reaction times (~12 ms) to escape approaching predators or environmental threats and are crucial to their survival under harsh marine conditions. These findings might also inspire rational strategies for developing liquid-repellent surfaces for drag reduction, water desalination, and preventing bio-fouling.

It is now clear in many species that male and female genital evolution has been shaped by sexual selection. However, it has historically been difficult to confirm correlations between morphology and fitness, as genital traits are complex and manipulation tends to impair function significantly. In this study, we investigate the functional morphology of the elongate male intromittent organ (or processus) of the seed bug Lygaeus simulans, in two ways. We first use micro-computed tomography (micro-CT) and flash-freezing to reconstruct in high resolution the interaction between the male intromittent organ and the female internal reproductive anatomy during mating. We successfully trace the path of the male processus inside the female reproductive tract. We then confirm that male processus length influences sperm transfer by experimental ablation and show that males with shortened processi have significantly reduced post-copulatory reproductive success. Importantly, male insemination function is not affected by this manipulation per se. We thus present rare, direct experimental evidence that an internal genital trait functions to increase reproductive success and show that, with appropriate staining, micro-CT is an excellent tool for investigating the functional morphology of insect genitalia during copulation.

The morphology and distribution of sensilla on the surface of the antennae of the naucorids’ species were studied via scanning electron microscopy. Eleven types of sensilla were identified regarding specific sensory modalities, based on their cuticular morphology. Cuticle morphology identifies five types of sensilla trichodea, four types of sensilla basiconica, one type of sensillum coeloconicum and sensillum ampullaceum. Three new types of mechanosensitive sensilla were found. Moreover, the morphological diversity between the antennae allowed the distinction of ten different antennal types that correspond to different sensillar sets. The sensilla found in Naucoridae share similarities with the sensilla of other nepomorphan taxa, as well as of terrestrial insects. However, no sensillar synapomorphy was found between Naucoridae and Aphelocheiridae.

Traumatic insemination (TI) is an extraordinary style of mating behavior wherein the female integument is pierced by the male extragenital structure to transfer the spermatozoa into the female's body through wounding. Flower bugs of the genus Orius belong to the family Anthocoridae (Heteroptera), which is referred to as the \"TI family\". Males possess sharp shaped extragenitalia, and females receive the extragenitalia using the copulatory tubes, which are specialized extragenital structures in Orius species. Since TI is not well studied in insects possessing the copulatory tube, we examined the genital structures and copulatory processes of three species, Orius strigicollis, O. sauteri, and O. minutus. Scanning electron microscopic observations revealed the positions of male extragenital structures during copulation. A needle-like flagellum was deeply inserted into the female intersegment between the abdominal VII and VIII segments, while the curved part of a sickle-like cone forced the intersegment to expand. No scars were detected around the copulation region after copulation. The copulatory tube adhered to the interior of segment VII, and the interior integument around the copulatory tube remained intact after copulation. On the basis of these results, TI does not occur in these Orius species. A pair of seminal conceptacles, which exists in typical TI insects, was found at the base of the oviducts in O. strigicollis. The distal end of the copulatory tube connected to a closed bag with a double-membrane, termed the sperm pouch. The sperm pouch was filled with filamentous structures after copulation and structures with equivalent forms were observed in adult male testis. These structures, considered to be spermatozoa, persisted in the pouch for at least two weeks after copulation, suggesting that the pouch is a long-term spermatozoa storage organ.

The exceptional abilities of stink bugs (Hemiptera: Pentatomidae) to colonize a diverse group of plants have been attributed to the feeding behaviors and the functions of the salivary complex of these insects. Here, we describe the ultrastructure of the salivary glands of the Neotropical brown stink bug, Euschistus heros, which is a major component of the pentatomid pest complex on soybeans, Glycine max, in the neotropics. Our results revealed a salivary gland complex consisting of two lobes (i.e., anterior and posterior), with a constriction between them (i.e., the hilum), in which the salivary and accessory gland ducts are inserted. The principal gland epithelium has a single layer of cells lining an enlarged lumen filled with saliva, and these cells are cuboidal, rich in rough endoplasmic reticulum and secretory vesicles, with well-developed nuclei, all of which are typical features of protein-secreting cells. We report, for the first time in insects, the presence of a layer of muscle cells surrounding the columnar hilum epithelium. The accessory salivary gland cells are cuboidal with nuclei containing condensed chromatin and cytoplasm rich in vacuoles and rough endoplasmic reticulum, indicating the potential involvement of these glands in water transport/secretion. The lumen content of each lobe of the principal gland suggests that the lobes produce different compounds. Thus, our results suggest that the E. heros salivary complex might have unconventional mechanisms to mix/release saliva, which might help explain the polyphagous abilities of these insects.

Plant-sucking stinkbugs are especially associated with mutualistic gut bacterial symbionts. Here, we explored the symbiotic relationship of a pistachio stinkbug, Acrosternum heegeri Fieber by histological, fluorescence in situ hybridization (FISH), real-time PCR and molecular phylogenetic techniques. Furthermore, the effects of the symbiont on the resting/wandering behaviors of the newborn nymphs, pre-adult survival rates, and stage compositions were investigated. Transmission electron microscopy and real-time PCR analyses showed that a rod-shaped gammaproteobacterium was persistently located within the posterior midgut crypts. Molecular phylogenetic and FISH techniques strongly suggested that this symbiont should be placed in the genus Pantoea of the Enterobacteriales. Scanning electron microscopy confirmed the presence of the bacterial cells on the egg surface which the surface sterilization of the eggs resulted in the successful removal of the symbiont from the eggs. Symbiotic and aposymbiotic A. heegeri showed no significant differences in the wandering behaviors of the first nymphal stages, while the symbiont-free insects suffered retarded growth and lower survivability. Together, the results highlight the habitat and acquisition features of Pantoea symbiont and its contribution in A. heegeri biology that might help us for better pest management in the future.

Heteroptera have diverse feeding habits with phytophagous, zoophagous, and haematophagous species. This dietary diversity associated with the monophyly of Heteroptera makes these insects a good object for comparative studies of the digestive tract. This work compares the ultrastructure of the middle midgut region in the phytophagous Coptosoma scutellatum (Plataspidae), Graphosoma lineatum (Pentatomidae), Kleidocerys resedae (Lygaeidae), and zoophagous Rhynocoris iracundus (Reduviidae), Nabis rugosus (Nabidae), and Himacerus apterus (Nabidae), to verify if diet affects midgut cells in phylogenetically related insects. The middle region of the midgut was used for comparison because it is the main site for digestion and absorption of the midgut. The digestive cell ultrastructure was similar in the six species, with features of secretory, absorptive, transport, storage, and excretory cells, suggesting a stronger correlation of middle digestive cell ultrastructure with the phylogeny of these species than with the different heteropteran feeding habits.

The organization of the symbiotic system (i.e., distribution and ultrastructure of symbionts) and the mode of inheritance of symbionts in two species, Nysius ericae and Nithecus jacobaeae belonging to Heteroptera: Lygaeidae, are described. Like most hemipterans, Nysius ericae and Nithecus jacobaeae harbor obligate prokaryotic symbionts. The symbiotic bacteria are harbored in large, specialized cells termed bacteriocytes which are localized in the close vicinity of the ovaries as well as inside the ovaries. The ovaries are composed of seven ovarioles of the telotrophic type. Bacteriocytes occur in each ovariole in the basal part of tropharium termed the infection zone. The bacteriocytes form a ring surrounding the early previtellogenic oocytes. The cytoplasm of the bacteriocytes is tightly packed with large elongated bacteria. In the bacteriocytes of Nysius ericae, small, rod-shaped bacteria also occur. Both types of bacteria are transovarially transmitted from one generation to the next.

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.