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.

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.

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.

Phenotypic Effects of PBAN RNAi Using Oral Delivery of dsRNA to Corn Earworm (Lepidoptera: Noctuidae) and Tobacco Budworm Larvae.

Insect neuropeptides represent more than 90% of all insect hormones. The pheromone biosynthesis activating neuropeptide (PBAN)/pyrokinin family is a major group of insect neuropeptides. These neuropeptides regulate a variety of biological functions from embryo to adult in moths including, sex pheromone biosynthesis and diapause. Other functions are yet to be determined. The identification of suitable target genes is most important for the successful application of RNA interference (RNAi) for pest insect control. Insect neuropeptide genes including PBAN are known to have multiple functions and could be a good target for RNAi suppression. In this study, we selected the PBAN gene and its neuropeptide products as an RNAi target for two economically important moth species, the corn earworm, Helicoverpa zea (Boddie), and the tobacco budworm, Heliothis virescens (Fabricius). We investigated RNAi effects on immature moths that had ingested the specific double-stranded RNA (dsRNA) starting at the first instar larva through pupation. We report that RNAi treatments resulted in delay of larval growth, interference of pupal development, and mortality in the two pest moths. In addition, we selected small interfering RNAs (siRNAs) to determine if they have negative phenotypic effects similar to their full-length RNAi parents. This is one of the few examples of negative RNAi effects on lepidopteran pests via feeding and suggests possible RNAi-based control of pest moths.

Neuropeptides predicted from the transcriptome analysis of the gray garden slug Deroceras reticulatum.

The gray garden slug, Deroceras reticulatum (Gastropoda: Pulmonata), is one of the most common terrestrial molluscs. Research for this slug has focused mainly on its ecology, biology, and management due to the severe damage it causes on a wide range of vegetables and field crops. However, little is known about neuropeptides and hormonal signalings. This study, therefore, aimed to establish the transcriptome of D. reticulatum and to identify a comprehensive repertoire of neuropeptides in this slug. Illumina high-throughput sequencing of the whole body transcriptome of D. reticulatum generated a total of 5.9 billion raw paired-end reads. De novo assembly by Trinity resulted in 143,575 transcripts and further filtration selected 120,553 unigenes. Gene Ontology (GO) terms were assigned to 30,588 unigenes, composed of biological processes (36.9%), cellular components (30.2%) and molecular functions (32.9%). Functional annotation by BLASTx revealed 39,987 unigenes with hits, which were further categorized into important functional groups based on sequence abundance. Neuropeptides, ion channels, ribosomal proteins, G protein-coupled receptors, detoxification, immunity and cytoskeleton-related sequences were dominant among the transcripts. BLAST searches and PCR amplification were used to identify 65 putative neuropeptide precursor genes from the D. reticulatum transcriptome, which include achatin, AKH, allatostatin A, B and C, allatotropin, APGWamide, CCAP, cerebrin, conopressin, cysteine-knot protein hormones (bursicon alpha/beta and GPA2/GPB5), elevenin, FCAP, FFamide, FVamide (enterin, fulicin, MIP and PRQFVamide), GGNG, GnRH, insulin, NdWFamide, NKY, PKYMDT, PRXamide (myomodulin, pleurin and sCAP), RFamide (CCK/SK, FMRFamide, FxRIamide, LFRFamide, luqin and NPF), and tachykinin. Over 330 putative peptides were encoded by these precursors. Comparative analysis among different molluscan species clearly revealed that, while D. reticulatum neuropeptide sequences are conserved in Mollusca, there are also some unique features distinct from other members of this species. This is the first transcriptome-wide report of neuropeptides in terrestrial slugs. Our results provide comprehensive transcriptome data of the gray garden slug, with a more detailed focus on the rich repertoire of putative neuropeptide sequences, laying the foundation for molecular studies in this terrestrial slug pest.

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.

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.

Identification and expression of capa gene in the fire ant, Solenopsis invicta.

Recent genome analyses suggested the absence of a number of neuropeptide genes in ants. One of the apparently missing genes was the capa gene. Capa gene expression in insects is typically associated with the neuroendocrine system of abdominal ganglia; mature CAPA peptides are known to regulate diuresis and visceral muscle contraction. The apparent absence of the capa gene raised questions about possible compensation of these functions. In this study, we re-examined this controversial issue and searched for a potentially unrecognized capa gene in the fire ant, Solenopsis invicta. We employed a combination of data mining and a traditional PCR-based strategy using degenerate primers designed from conserved amino acid sequences of insect capa genes. Our findings demonstrate that ants possess and express a capa gene. As shown by MALDI-TOF mass spectrometry, processed products of the S. invicta capa gene include three CAPA periviscerokinins and low amounts of a pyrokinin which does not have the C-terminal WFGPRLa motif typical of CAPA pyrokinins in other insects. The capa gene was found with two alternative transcripts in the CNS. Within the ventral nerve cord, two capa neurons were immunostained in abdominal neuromeres 2-5, respectively, and projected into ventrally located abdominal perisympathetic organs (PSOs), which are the major hormone release sites of abdominal ganglia. The ventral location of these PSOs is a characteristic feature and was also found in another ant, Atta sexdens.

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.

Role of extracellular domains in PBAN/pyrokinin GPCRs from insects using chimera receptors.

Pheromone biosynthesis-activating neuropeptide (PBAN) is a peptide used by a variety of moths to regulate pheromone production. Pyrokinins are peptides that activate muscle contraction in a variety of insects. These peptides have a common FXPRLamide C-terminal ending that is required for activity. Receptors have been identified from a moth and Drosophila as belonging to the rhodopsin family of G-protein coupled receptors (GPCRs) with sequence similarity to neuromedin U receptors from vertebrates. No insect GPCR has been characterized with regard to role of extracellular domains required for peptide binding and receptor activation. To begin characterizing these GPCRs we created chimera receptors using a PBAN-receptor from a moth and pyrokinin-receptors from Drosophila where extracellular domains were swapped. The N-terminal of the moth GPCR has two N-glycosylation sites that when replaced with glutamines, activity was reduced but not absent, indicating these sites contribute to receptor stability. Activity was greatly reduced by replacing the 2nd extracellular loop that has an N-glycosylation site and a cysteine that can form a disulfide bridge with a cysteine at the beginning of the 3rd transmembrane domain. Exchange of the 3rd extracellular loop between the moth and Drosophila receptor resulted in differential activation by PBAN or a diapause hormone peptide. This result indicates that the 3rd extracellular loop is directly involved in peptide ligand recognition. Results are discussed in context of the structural features of insect GPCRs that are required for receptor activation as compared to vertebrate receptors.