Site-directed mutagenesis and PBAN activation of the Helicoverpa zea PBAN-receptor

Pheromone biosynthesis-activating neuropeptide (PBAN) and pyrokinins belong to a family of insect peptide hormones that have a common FXPRLamide C-terminal ending. The G-protein-coupled receptors (GPCRs) for this peptide family were first identified from a moth and Drosophila with sequence similarity to neuromedin U receptors from vertebrates. We have characterized the PBAN-receptor (PBAN-R or PR) active binding domains using chimeric GPCRs and proposed that extracellular loop 3 is critical for ligand selection. Here, we characterized the 3rd extracellular domain of PBAN-R through site-directed point mutations. Results are discussed in context of the structural features required for receptor activation using receptor activation experiments and in silico computational modeling. This research will help in characterizing these receptors towards a goal of finding agonists and/or antagonists for PBAN/pyrokinin receptors

Molecular Structure and Diversity of PBAN/pyrokinin Family Peptides in Ants

Neuropeptides are the largest group of insect hormones. They are produced in the central and peripheral nervous systems and affect insect development, reproduction, feeding, and behavior. A variety of neuropeptide families have been identified in insects. One of these families is the PBAN/pyrokinin family defined by a common FXPRLamide or similar amino acid fragment at the C-terminal end. These peptides, found in all insects studied thus far, have been conserved throughout evolution. The most well studied physiological function is regulation of moth sex pheromone biosynthesis through the pheromone biosynthesis activating neuropeptide (PBAN), although several developmental functions have also been reported. Over the past years we have extended knowledge of the PBAN/pyrokinin family of peptides to ants, focusing mainly on the fire ant, Solenopsis invicta. The fire ant is one of the most studied social insects and over the last 60 years a great deal has been learned about many aspects of this ant, including the behaviors and chemistry of pheromone communication. However, virtually nothing is known about the regulation of these pheromone systems. Recently, we demonstrated the presence of PBAN/pyrokinin immunoreactive neurons in the fire ant, and identified and characterized PBAN and additional neuropeptides. We have mapped the fire ant PBAN gene structure and determined the tissue expression level in the central nervous system of the ant. We review here our research to date on the molecular structure and diversity of ant PBAN/pyrokinin peptides in preparation for determining the function of the neuropeptides in ants and other social insects

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 EQ-LIPFPRVamide) 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 caps gene

Effect of non-nutritive sugars to decrease the survivorship of spotted wing drosophila, Drosophila suzukii

In this study, we investigated the effects of non-nutritive sugars and sugar alcohols on the survivorship of spotted wing drosophila, Drosophila suzukii, and found erythritol and erythrose as potentially insecticidal to the fly. In a dose-dependent study, erythritol and erythrose significantly reduced fly longevity, with 100% mortality with 1, 0.5, 0.1 & 0.05 M doses after feeding for 7 days. When sucrose and erythritol solutions were provided separately to flies for 7 days, there was no effect on survivorship regardless of erythritol concentrations. However, with a serial combination of sucrose and erythritol solutions, fly survivorship was significantly decreased for the same period. Also, the higher dose of erythritol regardless of the sucrose dose combined showed greater mortality. In a no-choice assay, D. suzukii ingested more erythritol than sucrose or water, indicating the fly continuously fed on erythritol for 72 h. Also under no-choice conditions, erythritol and sucrose-fed flies gained more weight than water-fed flies. However, in two-choice assays, the amount of erythritol ingested was less than sucrose or water. Total sugar and glycogen levels among erythritol and erythrose-fed flies were significantly less than mannitol, sorbitol, xylitol, and sucrose-fed flies after 48 h. This indicates that these two non-nutritive sugars can’t be used a substrate for enzymes involved in sugar metabolism. Although the metabolism of erythritol and erythrose is unknown in insects, the mortality of D. suzukii flies ingesting these sugars might be caused by two potential physiological changes. The fly is starved by feeding of non-metabolizable erythritol and erythrose, or experiences abnormally high osmotic pressure in the hemolymph with erythritol molecules diffused from the midgut. Non-nutritive sugars might be used as an insecticide alone or combined with conventional or biological insecticides to enhance efficacy. If other sugar sources are present, a palatable sugar might be mixed with erythritol to elicit feeding

The Prognostic Role of the Surgical Approach and Adjuvant Therapy in Operable Mucosal Melanoma of the Head and Neck

Objectives The aim of this study was to investigate the prognostic impact of the surgical approach and adjuvant treatment in operable malignant melanoma of head and neck (MMHN). Methods Retrospective reviews of 31 patients who underwent surgery-based treatment with curative intent, either by the endoscopic or external approach, for MMHN were performed to analyze recurrence patterns, salvage modalities, and oncological outcomes (disease-specific survival and disease-free survival). Results Overall recurrence rate was 61% (19/31). In stage III patients (n=24), 50% (12/24) developed recurrences with a median recurrence-free period of 6.0 months, and 30% (4/12) of them was successfully salvaged by reoperation with adjuvant radiotherapy. On the contrary, all stage IVA patients (n=7) developed recurrences with a median recurrence-free period of 4.4 months. Distant metastasis was the most common pattern of failure and no patients were salvaged. Among variables, age and T classification, not the surgical approach, were significant prognosticators for disease-free survival and disease-specific survival. Adjuvant radiotherapy was associated with a lower rate of local failure, compared to surgery alone (hazard ratio, 0.02; 95% confidence interval, 0.06 to 0.75; P=0.02). However, adjuvant systemic therapy was not effective in reducing the risk of failures for any pattern. Conclusion Our data suggested that meticulous surgical resection, either by the endoscopic or external approach, with adjuvant radiotherapy increases the local control rate in MMHN

Identification and Biosynthetic Studies of the Hydrocarbon Sex Pheromone in Utetheisa ornatrix

The type II class of sex pheromones found in moths is composed of polyene hydrocarbons and their epoxides. Analysis of Utetheisa ornatrix females by gas chromatography-mass spectrometry and measurement of responses of male moths by coupled gas chromatography-electroantennogram detection confirmed the presence of large amounts of (Z,Z,Z)-1,3,6,9-heneicosatetraene (1,3,6,9-21:Hy) and smaller amounts of (Z,Z,Z)-3,6,9-heneicosatriene (3,6,9-21:Hy). Both compounds were detected in pheromone glands of newly emerged adults, with low amounts found in the late pupal stage, indicating that sex pheromone biosynthesis started in the late pupal stage. In our population of females (several hundred sampled), approximately 90% produced the tetraene, 1,3,6,9-21:Hy, as the major component, while the other 10% produced only a large amount (1500–2000 ng) of 3,6,9-21:Hy, with no detectable amount of the tetraene. This result could indicate that two distinct populations are present in our original collection site in Florida. Decapitated female moths accumulated 3,6,9-21:Hy and 1,3,6,9-21:Hy compared to the same age normal females, indicating that female moths continuously produce pheromone. A pheromone biosynthesis activating neuropeptide (PBAN)-like neuropeptide did not affect sex pheromone production as indicated by injection of synthetic PBAN and decapitation of U. ornatrix female adults. When the labeled precursor, D4-9,12,15-18:acid, was injected into the early pupal stage, the most abundantly labeled hydrocarbons were 3,6,9-21:Hy and 1,3,6,9-21:Hy in the female adults. This result indicated that 3,6,9-21:Hy could be biosynthesized from linolenic acid through chain elongation and decarboxylation. To determine how 1,3,6,9-21:Hy is produced, D4-3,6,9-21:Hy was injected into pupae and monitored for incorporation of label. No label was incorporated into 1,3,6,9-21:Hy, although a large amount of triene, 3,6,9-21:Hy, was recovered in the pheromone gland. This indicates that U. ornatrix females do not use 3,6,9-21:Hy to produce 1,3,6,9-21:Hy, and the terminal double bond is introduced earlier in the biosynthetic pathway

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

Assessment of the Biological Control Potential of Common Carabid Beetle Species for Autumn- and Winter-Active Pests (Gastropoda, Lepidoptera, Diptera: Tipulidae) in Annual Ryegrass in Western Oregon

Many studies have shown that ground beetles feed on different agricultural pests, but little is known about their species communities from US cropping systems. We assessed the biological control potential of the most common carabid beetle species in Oregon annual ryegrass grown for seed by investigating spatial and temporal overlap of the most common species with those of the most damaging autumn- and winter-active pests (slugs, caterpillars and cranefly larvae) and determined the number of field-collected specimens that had fed on the respective pests using molecular gut content analysis. Only the non-native Nebria brevicollis was abundant during pest emergence and tested positive for all three pest groups. While the other common carabid beetle species—Agonum muelleri, Calosoma cancellatum and Poecilus laetulus—were also found to have consumed pests, they were active only during spring and summer, when crop damage by pests is less critical. We also show that disk tilling did not affect any of the four common carabid beetle species and that only N. brevicollis was significantly associated with a vegetated field margin. This study contributes to expanding our knowledge on conservation biological control in a system where chemical pesticides are still the mainstay of control against invertebrate pests.Keywords: conservation biological control; slugs; qPCR; molecular gut content analysis; Nebria brevicollis; Noctua pronuba; Deroceras reticulatu

Fatal systemic disorder caused by biallelic variants in FARSA

Background : Aminoacyl tRNA transferases play an essential role in protein biosynthesis, and variants of these enzymes result in various human diseases. FARSA, which encodes the α subunit of cytosolic phenylalanyl-tRNA synthetase, was recently reported as a suspected causal gene for multiorgan disorder. This study aimed to validate the pathogenicity of variants in the FARSA gene. Results : Exome sequencing revealed novel compound heterozygous variants in FARSA, P347L and R475Q, from a patient who initially presented neonatal-onset failure to thrive, liver dysfunction, and frequent respiratory infections. His developmental milestones were nearly arrested, and the patient died at 28 months of age as a result of progressive hepatic and respiratory failure. The P347L variant was predicted to disrupt heterodimer interaction and failed to form a functional heterotetramer by structural and biochemical analyses. R475 is located at a highly conserved site and is reported to be involved in phenylalanine activation and transfer to tRNA. The R475Q mutant FARSA were co-purified with FARSB, but the mutant enzyme showed an approximately 36% reduction in activity in our assay relative to the wild-type protein. Additional functional analyses on variants from previous reports (N410K, F256L, R404C, E418D, and F277V) were conducted. The R404C variant from a patient waiting for organ transplantation also failed to form tetramers but the E418D, N410K, F256L, and F277V variants did not affect tetramer formation. In the functional assay, the N410K located at the phenylalanine-binding site exhibited no catalytic activity, whereas other variants (E418D, F256L and F277V) exhibited lower ATPase activity than wild-type FARSA at low phenylalanine concentrations. Conclusions : Our data demonstrated the pathogenicity of biallelic variants in FARSA and suggested the implication of hypomorphic variants in severe phenotypes.This study was supported by a research program funded by the Korea Disease Control and Prevention Agency (Grant Nos. 2021-ER0701-00 and 2020-ER6902-00)