Identification and characterisation of PRXamide peptides in the western flower thrips, Frankliniella occidentalis.

Insect CAPA-PVK (periviscerokinin) and pyrokinin (PK) neuropeptides belong to the PRX family peptides and are produced from capa and pyrokinin genes. We identified and characterised the two genes from the western flower thrips, Frankliniella occidentalis. The capa gene transcribes three splice variants, capa-a, -b, and -c, encoding two CAPA-PVKs (EVQGLFPFPRVamide; QGLIPFPRVamide) and two PKs (ASWMPSSSPRLamide; DSASFTPRLamide). The pyrokinin mRNA encodes three PKs: DLVTQVLQPGQTGMWFGPRLamide, SEGNLVNFTPRLamide, and ESGEQPEDLEGSMGGAATSRQLRTDSEPTWGFSPRLamide, the most extended pheromone biosynthesis activating neuropeptide (PBAN) ortholog in insects. Multiple potential endoproteolytic cleavage sites were presented in the prepropeptides from the pyrokinin gene, creating ambiguity to predict mature peptides. To solve this difficulty, we used three G protein-coupled receptors (GPCRs) for CAPA-PVK, tryptophan PK (trpPK), and PK peptides, and evaluated the binding affinities of the peptides. The binding activities revealed each subfamily of peptides exclusively bind to their corresponding receptors, and were significant for determining the CAPA-PVK and PK peptides. Our biological method using specific GPCRs would be a valuable tool for determining mature peptides, particularly with multiple and ambiguous cleavage sites in those prepropeptides. Both capa and pyrokinin mRNAs were strongly expressed in the head/thorax, but minimally expressed in the abdomen. The two genes also were clearly expressed during most of the life stages. Whole-mounting immunocytochemistry revealed that neurons contained PRXamide peptides throughout the whole-body: four to six neurosecretory cells in the head, and three and seven pairs of immunostained cells in the thorax and abdomen, respectively. Notably, the unusual PRXamide profiles of Thysanoptera are different from the other insect groups.

Insecticidal Effects of Receptor-Interference Isolated Bioactive Peptides on Fire Ant Colonies.

Receptor-interference (Receptor-i) is a novel technology used to identify bioactive peptides as agonists or antagonists against a specific receptor, primarily targeting G-protein-coupled receptors (GPCRs). Using Receptor-i methodology, we targeted the pheromone biosynthesis activating neuropeptide receptor (PBAN-R) of the red imported fire ant (Solenopsis invicta). Based on previous studies, we selected four bioactive peptides cyclized with two cysteines: CVKLGSHFC, CIQQGSHFC, CERVGSHFC, and CMARYMSAC, and we conducted small-scale feeding bioassays, measuring fire ant worker mortality. All peptides reduced ant survival; however, CMARYMSAC (MARY) and CIQQGSHFC (IQQG) were the most effective and were selected for feeding trials against large, fully functional fire ant field colonies containing queen, brood, and up to 8000 workers. At the end of the experiment, day 84, synthetic peptide MARY killed over 80% of the workers and two of four queens. IQQG killed over 70% of the workers and three of four queens. The surviving two MARY queens lost an average of 21% of their starting weight. The surviving IQQG queen lost 31% of its weight. In contrast, control colony queens gained an average of 11% of their starting weight. These results provide proof-of-concept for the Receptor-i technology and will synergize applications to other agricultural and medical pests.

Effects of nonnutritional sugars on lipid and carbohydrate content, physiological uptake, and excretion in Drosophila suzukii.

The nonnutritive sugar, erythritol, has the potential to be a human-safe management tool for the small fruits and cherry pest, Drosophila suzukii, or spotted-wing drosophila. Feeding on erythritol decreases fly survival and oviposition by starving and creating an osmotic imbalance in the body. Recently, we demonstrated that erythritol combined with another nonnutritive sugar, sucralose, was fed upon more than erythritol alone and hastens D. suzukii mortality. This suggests that sucralose is a suitable nonnutritive phagostimulant alternative to sucrose. Although promising, the nutritional and physiological impacts of sucralose on D. suzukii are unknown. In this study, we investigated whether sucralose is metabolized or excreted by D. suzukii when fed various erythritol, sucrose, and sucralose formulations. We found that sucralose cannot be metabolized or converted into any nutritional substitutes or storage carbohydrates in D. suzukii. Instead, sucralose molecules were largely accumulated in the hemolymph and slowly excreted from the body, creating a significant osmotic imbalance in D. suzukii. To excrete unused sugars, flies will use their own body fluids to restore homeostasis, resulting in losing a substantial amount of body weight and becoming desiccated in the process. In summary, ingesting sucralose leads to starvation and hyperosmotic pressure in the body, causing a decrease in fitness. With confirmation of sucralose being non-metabolizable and phagostimulative to D. suzukii, the erythritol+sucralose formulation is a promising insecticide for growers to use.

Identification and functional analysis of dsRNases in spotted-wing drosophila, Drosophila suzukii.

RNAi efficiency in insects is different from species to species; some species in Coleoptera are relatively more amenable to RNA interference (RNAi) than other species. One of the major factors is the presence of dsRNA-degrading enzymes, called dsRNases, in saliva, gut, or hemolymph in insects, which degrade the double-stranded RNA (dsRNA) introduced, resulting in the low efficacy of RNAi. In this study, we report a dsRNA-degrading activity in the gut homogenates from the spotted-wing drosophila, Drosophila suzukii, by ex vivo assay. Then, we identified two Drosophila suzukii dsRNase genes, named DrosudsRNase1 and DrosudsRNase2. In silico analysis shows that the gene structures are similar to dsRNases found in other insects. When dsRNases expressed in Sf9 cells were compared for their dsRNA degrading activities, dsRNase1 was more vital than dsRNase2. Both dsRNases were expressed highly and exclusively in the gut compared to the rest of body. Also, they were highly expressed during larval and adult stages but not in embryonic and pupal stages, suggesting the dsRNases protect foreign RNA molecules received during the feeding periods. DsRNase1 was expressed at a higher level in adults, whereas dsRNase2 showed more expression in early larvae. Our study on the tissue and development-specific patterns of dsRNases provides an improved understanding of the RNAi application for the management of D. suzukii.

Sex-biased gene expression in antennae of Drosophila suzukii.

Drosophila suzukii differs from other members of the genus Drosophila in its host preference and oviposition behavior. The flies are attracted to ripening fruits, and females have a serrated ovipositor enabling eggs to be laid inside the fruit. In addition to its huge economic impact, its unique chemoecological, morphological, and physiological characteristics have garnered considerable research interests. In this study, we analyzed D. suzukii antennal transcriptomes to identify sex-biased genes by comparison of differential gene expressions between male antennae (MA) and female antennae (FA). Among 13,583 total genes of the fly genome, 11,787 genes were expressed in either MA or FA. There are only 132 genes (9 in MA, 7 in FA, and 116 in both, FPKM >1) were expressed in antennae exclusively, and 2,570 genes (9 in MA, 0 in FA, and 2,561 in both) were enriched in antennae containing 185 and 113 sex-biased genes in MA and FA, respectively. Interestingly, many immune-related genes were highly expressed in MA, whereas several chemosensory genes were at high rank in FA. We identified 27 sex-biased chemosensory genes including odorant and gustatory receptors, odorant-binding proteins, chemosensory proteins, ionotropic receptors, and cytochrome P450s, and validated the gene expressions using quantitative real-time PCR. The highly expressed sex-biased genes in antennae are likely involved in the fly specific mating, host-finding behaviors, or sex-specific functions. The molecular results demonstrated here will facilitate to find the unique chemoreception of D. suzukii, as well as on the development of new management strategies for this pest.

Attracting Chrysopidae With Plant Volatiles for Lace Bug (Hemiptera: Tingidae) Control in Rhododendrons and Azaleas.

The azalea lace bug (Stephanitis pyrioides Scott) (Hemiptera: Tingidae) is an invasive pest of rhododendrons and azaleas (Ericaceae: Rhododendron), which feeds on the underside of leaves causing chlorosis, reduced photosynthesis, and even plant death. While insecticides can control this pest, growers, landscape managers, and homeowners have requested softer alternatives. Augmentative release of predatory green lacewing Chrysoperla sp. (Neuroptera: Chrysopidae) eggs and larvae has reduced S. pyrioides, but large-scale implementation may not be practical nor cost-effective. Attracting naturally occurring Chrysopidae with plant volatiles may be an economical and convenient option. In this study, we tested whether volatile blends 1) attracted Chrysoperla sp., and 2) controlled S. pyrioides populations on Rhododendron spp. in farm or urban landscapes. Experimental plots contained different multicomponent lures placed aboveground next to infested plants. Adult Chrysoperla sp., other natural enemies, and S. pyrioides from egg to adult stages were monitored in both farm and urban landscapes for two summers. Overall, two out of three volatile blends consistently attracted Chrysoperla sp. to sticky traps near baited plants. Methyl salicylate + acetic acid + 2-phenylethanol (methyl salicylate blend) and acetophenone + acetic acid + 2-phenylethanol (acetophenone blend) captured more adult Chrysoperla sp. than control traps in farm landscapes. However, only the acetophenone blend was associated with a slight reduction of S. pyrioides. Additional research is needed to determine whether the phenology of the first generation of both species are synchronized for effective season biological control in the Pacific Northwest.

Neuropeptides and peptide hormones identified in codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

The codling moth, Cydia pomonella, is a worldwide pest of pome fruits. Neuropeptides regulate most physiological functions in insects and represent new targets for the development of control agents. The only neuropeptides reported from the codling moth to date are the allatostatin A family peptides. To identify other neuropeptides and peptide hormones from codling moth, we analyzed head transcriptomes, identified 50 transcripts, and predicted 120 prepropeptides for the codling moth neuropeptides and peptide hormones. All transcripts were amplified, and these sequences were verified. One of the notable findings in this study is that diapause hormones (DHs) reported from Tortricid moths, including the codling moth, do not have the WFGPRL sequence in C-terminal ends in the pban genes. The C-terminal motif is critical to characterize insect DH peptides, and always conserved in pban/dh genes in Lepidoptera and many insect orders. Interestingly, the WFGPRL sequence was produced only from the capa gene in the codling moth. The allatostatin A-family encoding transcript predicted nine peptides, seven of which, as expected, are identical to those previously isolated from the moth. We also identified new codling moth orthologs of insect neuropeptides including CCHamides, allatostatin CC, RYamides, and natalisins. The information provided in this study will benefit future codling moth investigations using peptidoproteomics to determine peptide presence and functions.

Identification and characterization of capa and pyrokinin genes in the brown marmorated stink bug, Halyomorpha halys (Hemiptera): Gene structure, immunocytochemistry, and differential expression.

CAPA and pyrokinin (PK) neuropeptides are produced from two different genes, capa and pyrokinin, respectively. In this study, we identified and characterized the capa and pyrokinin genes from the brown marmorated stink bug, Halyomorpha halys (Hemiptera). The capa gene encodes two CAPA-PVK (periviscerokinin) peptides (DAGLFPFPRVamide and EQLIPFPRVamide) and one CAPA-DH (diapause hormone; NGASGNGGLWFGPRLamide). The pyrokinin gene encodes three PK2 peptides (QLVSFRPRLamide, SPPFAPRLamide, and FYAPFSPRLamide). The whole-mounting immunocytochemistry revealed the neurons contained PRXamide-like peptides throughout the cerebral ganglia (CRG), gnathal ganglia (GNG), thoracic ganglia (TG), and abdominal ganglia (AG). A pair of neurosecretory cells in the CRG and three cell clusters in the GNG were found with the axonal projections extended through the lateral side. A pair of immunostained cells were found in the TG, while three pairs of cells were present in the fused AG. Different expression patterns of capa and pyrokinin genes were observed in the CRG-GNG, TG, and AG. The capa gene was highly expressed in the AG tissue, whereas the pyrokinin gene was strongly expressed in the CRG-GNG. Interestingly, different developmental stages showed similar expressions of both genes, with the highest from the first nymph, gradually decreasing to the female adult. Comparison of peptide sequences encoded from pyrokinin genes showed the PK1 peptide is lost in Heteroptera suborders including H. halys, but retained in other suborders. The missing PK1 from the pyrokinin gene might be compensated by CAPA-DH (=PK1-like) produced by the capa gene.

Identification and characterization of pyrokinin and CAPA peptides, and corresponding GPCRs from spotted wing drosophila, Drosophila suzukii.

The family of FXPRLamide peptides serves as a major insect hormone. It is characterized by a core active amino acid sequence conserved at the C-terminal ends, and provides various physiological roles across the Insecta. In this study we identified and characterized pyrokinin (PK) and CAPA cDNAs encoding two FXPRLamide peptides, pyrokinin and CAPA-DH (diapause hormone), and two corresponding G protein-coupled receptors (GPCRs) from spotted wing drosophila (SWD), Drosophila suzukii. Expressions of PK and CAPA mRNAs were differentially observed during all life stages except the embryo, and the detection of CAPA transcription was relatively strong compared with the PK gene in SWD. Both D. suzukii pyrokinin receptor (DrosuPKr) and CAPA-DH receptor (DrosuCAPA-DHr) were functionally expressed and confirmed through binding to PK and DH peptides. Differential expression of two GPCRs occurred during all life stages; a strong transcription of DrosuPKr was observed in the 3rd instar. DrosuCAPA-DHr was clearly expressed from the embryo to the larva, but not detected in the adult. Gene regulation during the life stages was not synchronized between ligand and receptor. For example, SWD CAPA mRNA has been up-regulated in the adult while CAPA-DHr was down-regulated. The difference could be from the CAPA mRNA translating multiple peptides including CAPA-DH and two CAPA-PVK (periviscerokinin) peptides to act on different receptors. Comparing the genes of SWD PK, CAPA, PKr and CAPA-DHr to four corresponding genes of D. melanogaster, SWD CAPA and the receptor are more similar to D. melanogaster than PK and the receptor. These data suggest that the CAPA gene could be evolutionally more conserved to have a common biological role in insects. In addition, the effect of Kozak sequences was investigated by the expression of the GPCRs with or without Kozak sequences in Sf9 insect cells. The Kozak sequenced PK receptor was significantly less active than the original (= no Kozak sequenced) receptor. Our results provide a knowledge for potential biological function(s) of PK and CAPA-DH peptides in SWD, and possibly offer a novel control method for this pest insect in the future.

Tarsi of Male Heliothine Moths Contain Aldehydes and Butyrate Esters as Potential Pheromone Components.

The Noctuidae are one of the most speciose moth families and include the genera Helicoverpa and Heliothis. Females use (Z)-11-hexadecenal as the major component of their sex pheromones except for Helicoverpa assulta and Helicoverpa gelotopoeon, both of which utilize (Z)-9-hexadecenal. The minor compounds found in heliothine sex pheromone glands vary with species, but hexadecanal has been found in the pheromone gland of almost all heliothine females so far investigated. In this study, we found a large amount (0.5-1.5 µg) of hexadecanal and octadecanal on the legs of males of four heliothine species, Helicoverpa zea, Helicoverpa armigera, H. assulta, and Heliothis virescens. The hexadecanal was found on and released from the tarsi, and was in much lower levels or not detected on the remaining parts of the leg (tibia, femur, trochanter, and coxa). Lower amounts (0.05-0.5 µg) of hexadecanal were found on female tarsi. This is the first known sex pheromone compound to be identified from the legs of nocturnal moths. Large amounts of butyrate esters (about 16 µg) also were found on tarsi of males with lower amounts on female tarsi. Males deposited the butyrate esters while walking on a glass surface. Decapitation did not reduce the levels of hexadecanal on the tarsi of H. zea males, indicating that hexadecanal production is not under the same neuroendocrine regulation system as the production of female sex pheromone. Based on electroantennogram studies, female antennae had a relatively high response to hexadecanal compared to male antennae. We consider the possible role of aldehydes and butyrate esters as courtship signals in heliothine moths.

The Biochemical Adaptations of Spotted Wing Drosophila (Diptera: Drosophilidae) to Fresh Fruits Reduced Fructose Concentrations and Glutathione-S Transferase Activities.

Spotted wing drosophila, Drosophila suzukii Matsumura, is an invasive and economically damaging pest in Europe and North America. The females have a serrated ovipositor that enables them to infest almost all ripening small fruits. To understand the physiological and metabolic basis of spotted wing drosophila food preferences for healthy ripening fruits, we investigated the biological and biochemical characteristics of spotted wing drosophila and compared them with those of Drosophila melanogaster Meigen. We found that the susceptibility to oxidative stressors was significantly increased in spotted wing drosophila compared with those of D. melanogaster. In addition, we found that spotted wing drosophila had significantly reduced glutathione-S transferase (GST) activity and gene numbers. Furthermore, fructose concentrations found in spotted wing drosophila were significantly lower than those of D. melanogaster. Our data strongly suggest that the altered food preferences of spotted wing drosophila may stem from evolutionary adaptations to fresh foods accompanied by alterations in carbohydrate metabolism and GST activities.

Immune-Related Gene Profiles and Differential Expression in the Grey Garden Slug Deroceras reticulatum Infected with the Parasitic Nematode Phasmarhabditis hermaphrodita.

The grey garden slug (Deroceras reticulatum), a common terrestrial slug native to Europe with a global distribution including North America, is commonly considered the most severe slug pest in agriculture. The nematode Phasmarhabditis hermaphrodita, which has been used in the U.K. and Europe as a commercial biocontrol agent since 1994, has also recently been collected in Oregon and California and has long been considered a candidate biocontrol agent for slug management in the U.S. In this study, we report differential gene expressions in nematode-infected slugs using RNA-seq to identify slug immune-related genes against nematodes. Comparison of gene expression levels between the whole bodies of a nematode-infected slug (N-S) and an uninfected control slug (C-S) revealed that there were a total of 39,380 regulated unigenes, of which 3084 (3%) were upregulated and 6761 (6%) were downregulated at greater than 2-fold change (FC > 2) in the nematode-infected slug. To further investigate the biological functions of differentially expressed genes (DEGs), gene ontology (GO) and functional enrichment analysis were performed to map the DEGs to terms in the GO, eukaryotic ortholog groups of proteins (KOG) and Kyoto Encyclopedia of Genes and Genome Pathway (KEGG) databases. Among these DEGs, approximately 228 genes associated with immunity or immune-related pathways were upregulated 2-fold or more in the N-S compared to C-S. These genes include toll, Imd, JNK, scavenger receptors (SCRs), C-type lectins (CTLs), immunoglobulin-like domains, and JAK/STAT63 signaling pathways. From the RNA-seq results, we selected 18 genes and confirmed their expression levels by qRT-PCR. Our findings provide insights into the immune response of slugs during nematode infection. These studies provide fundamental information that will be valuable for the development of new methods of pest slug control using pathogenic nematodes in the field.

Successful transmission of Solenopsis invicta virus 3 to Solenopsis invicta fire ant colonies in oil, sugar, and cricket bait formulations.

Tests were conducted to evaluate whether Solenopsis invicta virus 3 (SINV-3) could be delivered in various bait formulations to fire ant colonies and measure the corresponding colony health changes associated with virus infection in Solenopsis invicta. Three bait formulations (10% sugar solution, cricket paste, and soybean oil adsorbed to defatted corn grit) effectively transmitted SINV-3 infections to S. invicta colonies. Correspondingly, viral infection was shown to be detrimental to colony health and productivity. By day 32, all ant colonies exposed to a single 24h pulse treatment of SINV-3 became infected with the virus regardless of the bait formulation. However, the SINV-3 sugar and cricket bait-treated colonies became infected more rapidly than the oil-treated colonies. Sugar and cricket-treated colonies exhibited significant declines in their brood ratings compared with the untreated control and oil bait-treated colonies. Measures of colony health and productivity evaluated at the end of the study (day 47) showed a number of differences among the bait treatments and the control group. Statistically significant and similar patterns were exhibited among treatments for the quantity of live workers (lower), live brood (lower), total colony weight (lower), worker mortality (higher), proportion larvae (lower), and queen weight (lower). Significant changes were also observed in the number of eggs laid by queens (lower) and the corresponding ovary rating in SINV-3-treated colonies. The study provides the first successful demonstration of SINV-3 as a potential biopesticide against fire ants.

Selection and Comparative Gene Expression of Midgut-Specific Targets for Drosophila suzukii.

Spotted-wing drosophila (SWD), Drosophila suzukii, is a destructive and invasive pest that attacks most small fruits and cherries. The current management for SWD involves the use of conventional insecticides. In an effort to develop a biologically based control option, the application of RNA interference (RNAi) has been investigated. To develop an RNAi approach, suitable targets must be identified, and an efficient delivery method must be developed for introducing the double-stranded RNA (dsRNA) in the midgut. In D. suzukii, we previously found that dsRNA nucleases actively degrade dsRNA molecules in the midgut. In this study, we focused on identifying biological targets focused on the midgut membrane. The profile of midgut-specific genes was analyzed and compared with the genes expressed in the whole-body using transcriptome analysis. Differential gene expression analysis revealed that 1921 contigs were upregulated and 1834 contigs were downregulated in the midgut when compared to genes from other body tissues. We chose ten midgut-specifically upregulated genes and empirically confirmed their expressions. We are particularly interested in the midgut membrane proteins, including G protein-coupled receptors (GPCRs) such as diuretic hormone 31 (DH31) receptor, neuropeptide F (NPF) recepror, toll-9, adhesion receptors, methuselah (mth), and gustatory receptor, because insect GPCRs have been offered great potential for next-generation pest management.

Erythritol sprays reduce Drosophila suzukii infestation without impacting honey bee visitation nor fruit quality.

BACKGROUND: Spotted-wing drosophila, Drosophila suzukii, is an economic pest of small fruits and cherries. Insecticides primarily control this pest while alternative controls are in development. Laboratory studies show that erythritol is insecticidal to D. suzukii and other pests while approved for human consumption. Moreover, erythritol combined with sucrose or non-caloric sucralose can stimulate feeding and quicken mortality. Before growers can use erythritol, the impact on crop protection, non-target insects, and fruit quality need evaluation. RESULTS: In three blueberry and cherry field cage trials, oviposition on fruit sprayed with erythritol:sucrose or erythritol:sucralose formulations was lowered by 59%-81% compared with unsprayed controls. Fly infestation (larval or adult counts from fruit) was 90% lower in a greenhouse blueberry trial, and 49% lower in an open field blueberry trial with 2 m erythritol : 0.5 m sucrose. Infestation was also 57% lower in an open field cherry trial with 1.5 m erythritol:0.5 m sucrose. Other field trials with very low pest pressure or frequent rains revealed no differences from controls. Field trials consistently revealed that honey bees did not preferentially visit plants sprayed with either erythritol formulation, although yellow jackets visited plants sprayed with erythritol:sucrose more frequently. Erythritol formulations consistently led to more leaf spotting, but there was no reduction in the quality of treated blueberries or cherries in terms of mold development, firmness, diameter, epidermal penetration force, and Brix° (total soluble solids) at harvest. CONCLUSION: Eleven trials conducted over four years show that erythritol formulations can reduce D. suzukii pressure without attracting foraging honey bees nor negatively impacting fruit quality. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Insecticidal Properties of Erythritol on Four Tropical Tephritid Fruit Flies, Zeugodacus cucurbitae, Ceratitis capitata, Bactrocera dorsalis, and B. latifrons (Diptera: Tephritidae).

Tephritid fruit flies are among the most destructive agricultural pests of fruits and vegetables worldwide and can impose trade barriers against the movement of fresh tropical commodities. Primary pre-harvest control methods for these flies rely on the spraying of conventional chemical insecticides or bait sprays. However, resistance to these control methods has been reported in fruit flies. Erythritol is a non-nutritive sugar alternative for human consumption, which has been tested and confirmed for its insecticidal properties against various insect pest species. In this study, using laboratory bioassays, we evaluated the insecticidal effect of erythritol alone or various erythritol formulations containing sucrose and/or protein on four tropical fruit fly species established in Hawaii (e.g., melon fly, Mediterranean fruit fly, oriental fruit fly, and Malaysian fruit fly). In addition, the effects of other non-nutritive hexose and pentose sugar alcohols, such as sorbitol, mannitol, and xylitol, were tested. Among the different standalone and combinatory treatments tested, 1M erythritol and a combinatory formulation of 2M erythritol + 0.5M sucrose appeared to be the most detrimental to the survival of all four species of tested flies, suggesting the potential of using erythritol as a non-toxic management tool for the control of tropical tephritid fruit flies.

A novel physiological function of pheromone biosynthesis-activating neuropeptide in production of aggregation pheromone.

The western flower thrips, Frankliniella occidentalis, is an insect pest, and its aggregation pheromone (AP) plays a crucial role in the recruitment of both sexes. A novel pheromone biosynthesis-activating neuropeptide (PBAN)-like gene is encoded in F. occidentalis genome, but its physiological function has yet to be elucidated. This study hypothesized the physiological role played by PBAN in mediating AP production. AP has been known to be produced only by male adults in F. occidentalis. Surprisingly, our extraction of headspace volatiles contained two AP components in females as well as in males with similar composition. PBAN injection elevated the AP production whereas RNA interference (RNAi) of the gene expression suppressed the AP production in both sexes. A biosynthetic pathway to produce AP components were predicted and the enzymes catalyzing the main steps were confirmed in their expressions. Individual RNAi treatments of these genes significantly suppressed AP production. RNAi of PBAN gene downregulated the expressions of these biosynthesis-associated genes in both sexes. These results suggest that the novel neuropeptide acts as PBAN mediating AP production through stimulating its biosynthetic machinery in F. occidentalis.

Diuretic hormone 31 activates two G protein-coupled receptors with differential second messengers for diuresis in Drosophila suzukii.

Diuretic hormones (DHs) bind to G protein-coupled receptors (GPCRs), regulating water and ion balance to maintain homeostasis in animals. Two distinct DHs are known in insects: calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. In this study, we identified and characterized DH31 and two DH31 GPCR variants, DH31-Ra and DH31-Rb, from spotted-wing drosophila, Drosophila suzukii, a globally prevalent vinegar fly causing severe damage to small fruits. Both GPCRs are active, but DH31-Ra is the dominant receptor based on gene expression analyses and DH31 peptide binding affinities. A notable difference between the two variants lies in 1) the GPCR structures of their C-termini and 2) the utilization of second messengers, and the amino acid sequences of the two variants are identical. DH31-Ra contains 12 additional amino acids, providing different intracellular C-terminal configurations. DH31-Ra utilizes both cAMP and Ca2+ as second messengers, whereas DH31-Rb utilizes only cAMP; this is the first time reported for an insect CT-like DH31 peptide. DH31 stimulated fluid secretion in D. suzukii adults, and secretion increased in a dose-dependent manner. However, when the fly was injected with a mixture of DH31 and CAPA, an anti-diuretic hormone, fluid secretion was suppressed. Here, we discuss the structures of the DH31 receptors and the differential signaling pathways, including second messengers, involved in fly diuresis. These findings provide fundamental insights into the characterization of D. suzukii DH31 and DH31-Rs, and facilitate the identification of potential biological targets for D. suzukii management.

Nonnutritive Sugars for Spotted-Wing Drosophila (Diptera: Drosophilidae) Control Have Minimal Nontarget Effects on Honey Bee Larvae, a Pupal Parasitoid, and Yellow Jackets.

Drosophila suzukii Matsumura, spotted-wing drosophila, is a major pest of small fruits and cherries and often managed with conventional insecticides. Our previous work found that erythritol, a nonnutritive polyol, has insecticidal properties to D. suzukii. Two formulations of erythritol (1.5M), with 0.5M sucrose or 0.1M sucralose, are most effective at killing D. suzukii. In this study, we investigated the nontarget effects of these erythritol formulations on honey bee Apis mellifera Linnaeus larvae, a pupal parasitoid of D. suzukii, Pachycrepoideus vindemiae Rondani, and western yellow jacket, Vespula pensylvanica Saussure. We directly exposed honey bee larvae by adding a high dose (2 µl) to larval cells and found no significant mortality from either formulation compared to the water control. Pachycrepoideus vindemiae may encounter erythritol in field settings when host plants of D. suzukii are sprayed. The erythritol+sucralose formulation was more detrimental than erythritol+sucrose to P. vindemiae, however, this effect was greatly reduced within a 21-d period when a floral source was present. Since yellow jackets are a nuisance pest and were attracted to the erythritol formulations in recent field trials, we tested adult V. pensylvanica survival with continuous consumption of these formulations in the laboratory. We found no detectable detriment from either formulation, compared to the sucrose control. Overall, both erythritol formulations caused minimal nontarget effects on honey bee larvae, P. vindemiae parasitoids, and western yellow jackets.

GPCR-Based Bioactive Peptide Screening Using Phage-Displayed Peptides and an Insect Cell System for Insecticide Discovery.

The discovery of new insecticides improves integrated pest management (IPM), but is usually a long high-risk process with a low probability of success. For over two decades, insect neuropeptides (NPs) and their G-protein coupled receptors (GPCRs) have been considered as biological targets for insect pest control, because they are involved in almost all physiological processes associated with insect life stages. A key roadblock to success has been the question of how large volume chemical libraries can be efficiently screened for active compounds. New genomic and proteomic tools have advanced and facilitated the development of new approaches to insecticide discovery. In this study, we report a novel GPCR-based screening technology that uses millions of short peptides randomly generated by bacteriophages, and a method using an insect Sf9 cell expression system. The fire ant is a good model system, since bioactive peptides have been identified for a specific GPCR. The novel small peptides could interfere with the target GPCR-ligand functions. Therefore, we refer to this new mechanism as "receptor interference" (RECEPTORi). The GPCR-based bioactive peptide screening method offers multiple advantages. Libraries of phage-displayed peptides (~109 peptides) are inexpensive. An insect cell-based screening system rapidly leads to target specific GPCR agonists or antagonists in weeks. Delivery of bioactive peptides to target pests can be flexible, such as topical, ingestion, and plant-incorporated protectants. A variety of GPCR targets are available, thus minimizing the development of potential insecticide resistance. This report provides the first proof-of-concept for the development of novel arthropod pest management strategies using neuropeptides, and GPCRs.