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Background Spiders are predaceous arthropods that are capable of subduing and consuming relatively large prey items compared to their own body size. For this purpose, spiders have evolved potent venoms to immobilise prey and digestive fluids that break down nutrients inside the prey’s body by means of extra-oral digestion (EOD). Both secretions contain an array of active proteins, and an overlap of some components has been anecdotally reported, but not quantified. We systematically investigated the extent of such protein overlap. As venom injection and EOD succeed each other, we further infer functional explanations, and, by comparing two spider species belonging to different clades, assess its adaptive significance for spider EOD in general. Results We describe the protein composition of the digestive fluids of the mygalomorph Acanthoscurria geniculata and the araneomorph Stegodyphus mimosarum , in comparison with previously published data on a third spider species. We found a number of similar hydrolases being highly abundant in all three species. Among them, members of the family of astacin-like metalloproteases were particularly abundant. While the importance of these proteases in spider venom and digestive fluid was previously noted, we now highlight their widespread use across different spider taxa. Finally, we found species specific differences in the protein overlap between venom and digestive fluid, with the difference being significantly greater in S. mimosarum compared to A. geniculata . Conclusions The injection of venom precedes the injection with digestive fluid, and the overlap of proteins between venom and digestive fluid suggests an early involvement in EOD. Species specific differences in the overlap may reflect differences in ecology between our two study species. The protein composition of the digestive fluid of all the three species we compared is highly similar, suggesting that the cocktail of enzymes is highly conserved and adapted to spider EOD.

The larvae of some lampyrid beetles are highly specialized predators of snails. They have been observed to climb on the shells of their prey and use this exposed position to bite and inject secretions potentially originating from the midgut. Besides serving the purpose of extra-oral digestion (EOD), injected compounds also seem to have a paralyzing effect. Up to now, the toxins causing this paralyzing activity have not been identified. In the current study, we provide a first compositional analysis of the midgut secretion from lampyrid larvae, with a focus on identifying putative neurotoxins causing the observed paralyzing effect. For this purpose, we utilized a combined proteo-transcriptomic approach to characterize the compounds present in the midgut secretion of larval stages of Lampyris noctiluca. In terms of the absolute numbers of identified compounds, the midgut secretion is dominated by hydrolyzing enzymes comprising peptidases, carboxylesterases, and glycosidases. However, when considering expression levels, a few rather short cysteine-rich peptides exceed all other compounds. Some of these compounds show moderate similarity to putative neurotoxins identified in the venom of other arthropods and could be responsible for paralyzing effects. In addition to these potential toxins, we provide a list of peptides typical of the midgut secretion of L. noctiluca, supplemented by the corresponding precursor sequences.

Predatory stink bugs are important biological control agents in agricultural and forest crops. The use of these natural enemies by companies, growers, and research centers has been an important tactic in controlling larvae pest outbreaks. Brontocoris tabidus (Signoret) and Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) were kept for 24, 36, or 48 hours without food, and the time taken by these predators for the first attack and to kill a sixth-instar Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) larvae and the number of attacks during four-hour period evaluated. In addition, the size of the mouthparts (mandibular stylets and maxillary bristles), important in prey control, and the enzymatic activity of amylase, lipase, and protease in the salivary glands content of these predators were also evaluated. The efficacy of B. tabidus in killing T. arnobia larvae was higher than that of P. nigrispinus with a death of about 72% of them in the first attack, compared to 30% for P. nigrispinus. The higher success of B. tabidus is probably due to its larger mouthparts and amylase activity stimulating phytophagy. The first attack was faster for P. nigrispinus with 36 and 48 hours without food. The attack by B. tabidus was faster when this predator stayed for 48 hours without food than for 24 and 36 hours. Biofactories could keep these insects for up to 48 hours without food before releasing them in the field to improve their impact on biological control.

The burrower bug Scaptocoris castanea Perty, 1830 (Hemiptera: Cydnidae) is an agricultural pest feeding on roots of several crops. The histology and ultrastructure of the salivary glands of S. castanea were described. The salivary system has a pair of principal salivary glands and a pair of accessory salivary glands. The principal salivary gland is bilobed with anterior and posterior lobes joined by a hilus where an excretory duct occurs. The accessory salivary gland is tubular with a narrow lumen that opens into the hilus near the excretory duct, suggesting that its secretion is stored in the lumen of the principal gland. The cytoplasm of the secretory cells is rich in the rough endoplasmic reticulum, secretory vesicles with different electron densities and mitochondria. At the base of the accessory gland epithelium, there were scattered cells that do not reach the gland lumen, with the cytoplasm rich in the rough endoplasmic reticulum, indicating a role in protein production. Data show that principal and accessory salivary glands of S. castanea produce proteinaceous saliva. This is the first morphological description of the S. castanea salivary system that is similar to other Hemiptera Pentatomomorpha, but with occurrence of basal cells in the accessory salivary gland.

The management of the Neotropical brown stinkbug Euschistus heros (Hemiptera: Pentatomidae) in soybean fields has been heavily dependent on the application of neonicotinoid insecticides. Neonicotinoids act primarily by impairing the function of the nicotinic acetylcholine receptors of the nervous system. These compounds also target specific organs (e.g., salivary glands), which may potentiate their insecticidal efficacy. Here, we evaluated whether the exposure to the neonicotinoid imidacloprid would cause cytomorphological changes in the salivary glands of E. heros. First, we determined the lethal concentrations (LCs) of imidacloprid through contact and ingestion. Subsequently, the cytomorphology of the salivary glands were evaluated in insect groups that survived exposure to the LC5 (3.75 mg a.i./L), LC50 (112.5 mg a.i./L), or LC75 (375.0 mg a.i./L, equivalent to the recommended field rate) doses. Imidacloprid induced apoptosis and necrosis in the salivary gland cells according to the insecticide concentration and salivary gland region. All concentrations increased apoptosis and injured cells (e.g., vacuolization, chromatin condensation, swelling of organelles, and plasma membrane rupture) in the principal and accessory salivary glands. Individuals that survived exposure to the highest concentrations (i.e., LC5 and LC50) were more affected, and exhibited several necrotic cells on their main principal salivary glands. Collectively, our results indicate that imidacloprid exerts toxic effects on the non-target organs, such as the salivary glands, which increases the efficacy of this compound in the management of stink bug infestations.

Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly Dolopus genitalis using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies (Lucilia cuprina) produced rapid contractile paralysis (PD50 at 1 min = 3.1 μg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/μL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/β defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.

Saliva is known to play a crucial role in tarnished plant bug (TPB, Lygus lineolaris [Palisot de Beauvois]) feeding. By facilitating the piercing, the enzyme-rich saliva may be used for extra-oral digestion and for overcoming plant defense before the plant fluids are ingested by TPBs. To identify salivary gland genes, mRNA was extracted from salivary glands and cDNA library clones were sequenced. A de novo-assembling of 7,000 Sanger sequences revealed 666 high-quality unique cDNAs with an average size of 624 bp, in which the identities of 347 cDNAs were determined using Blast2GO. Kyoto Encyclopedia of Genes and Genomes analysis indicated that these genes participate in eighteen metabolic pathways. Identifications of large number of enzyme genes in TPB salivary glands evidenced functions for extra-oral digestion and feeding damage mechanism, including 45 polygalacturonase, two α- amylase, one glucosidase, one glycan enzyme, one aminopeptidase, four lipase, and many serine protease cDNAs. The presence of multiple transcripts, multigene members, and high abundance of cell wall degradation enzymes (polygalacturonases) indicated that the enzyme-rich saliva may cause damage to plants by breaking down plant cell walls to make nutrients available for feeding. We also identified genes potentially involved in insect adaptation and detoxifying xenobiotics that may allow insects to overcome plant defense responses, including four glutathione S-transferases, three esterases, one cytochrome P450, and several serine proteases. The gene profiles of TPB salivary glands revealed in this study provides a foundation for further understanding and potential development of novel enzymatic inhibitors, or other RNAi approaches that may interrupt or minimize TPB feeding damage.

Podisus distinctus (Hemiptera: Pentatomidae) is a zoophytophagous insect with significant potential for use as a biological control agent in agriculture and forestry because their nymphs and adults actively prey on diverse insect species. The saliva of this insect possesses active substances that cause paralysis and death of the prey. As the first step in identifying compounds of P. distinctus saliva, this study describes the ultrastructure of the salivary glands of this predator. The salivary system of P. distinctus possesses a pair of main salivary glands with a short anterior lobe, a long posterior lobe, and a pair of tubular accessory glands. The main salivary gland of P. distinctus has no associated muscles, suggesting that the saliva-release mechanism occurs with the help of certain thorax muscles. The main salivary gland epithelium has a single layer of cells (varying from cubical to columnar) with cytoplasm rich in rough endoplasmic reticulum, spherical granules of different sizes, a nucleus with a predominance of decondensed chromatin, and nucleolus. The apical cell region has a few short microvilli and the basal region has plasma membrane infoldings. The epithelium of the accessory salivary glands possesses a single-layered epithelium of cubic cells delimiting a narrow lumen. The apical cell region has a high density of microvilli and pleomorphic mitochondria, whereas the central cell region is rich in rough endoplasmic reticulum with a well-developed nucleus and decondensed chromatin. The basal cell region is characterized by the presence of several basal plasma membrane infoldings associated with mitochondria and numerous openings to the hemocoel forming large channels. The ultrastructural characteristics suggest that the main salivary glands and accessory salivary glands play a vital role in protein synthesis for saliva production and that the accessory glands are involved in transport of materials of the hemolymph.

(Say) is a generalist predator attacking many insect species from different orders. The bug injects saliva into its prey's body. The ingested hemolymph and liquefied internal tissues pass through the bug's alimentary tract. Collagenase working on peptide bonds of collagen and basement membrane proteins, leads to the disintegration of the prey's internal organs. As yet, there is an almost complete lack of knowledge on the collagenase activity in . The collagenase activity of the salivary glands and midgut was optimum at pH 8.0 which was congruent with the optimal pH of the total proteolytic activity of the salivary glands. More collagenolytic activity was determined in the posterior lobe of the salivary glands and anterior midgut. Significant inhibition of collagenolytic activity by ethylenediaminetetraacetic acid (EDTA) revealed the enzyme is a metalloproteinase. The collagenase activity notably decreased when the bug went hungry. The salivary gland collagenase is a vital enzyme in extra-oral digestion and facilitates the action of other digestive enzymes. The midgut collagenase may be involved in the digestion of the ingested muscle fibers. The collagenase probably acts as an intoxicating agent in the saliva (venom) of . Paralysing toxins are present in the salivary gland secretion.

Rhynocoris marginatus Fabricius (Hemiptera: Reduviidae) is an important biological control agent against more than 25 insect pests in India. For a better understanding of the feeding adaptation of this bug, the gross morphology and histology of its head and salivary apparatus were studied using both a light microscope and scanning electron microscope. The head is more or less elongate, mobile, and immersed into the eyes. R. marginatus has a three-segmented curved rostrum; the middle segment is longer than the other two segments. The terminal rostral segment bears spines and trichobothria externally. Stylet bundles bear two pairs of maxillary and mandible stylets in the curved rostrum with serrations. The stylets help to penetrate into the tissue and directly pump the toxic venomous saliva deep into the prey. The principal gland is bi-lobed (anterior lobe and posterior lobe), whereas the accessory gland is uni-lobed, exhibiting distinct functional and histological differences. These glands receive tracheal and nerve supply. Mononucleated, binucleated, trinucleated and polynucleated cells are distributed both in anterior and posterior lobes of the principal gland. The cytoplasm has collecting vacuoles with secretions. Therefore, this predator is highly equipped with well-developed mouthparts that are attached to the salivary apparatus.