Effects of Niemann-Pick type C2 (NPC2) gene transcripts silencing on behavior of Varroa destructor and molecular changes in the putative olfactory gene networks.

To understand the role of two Niemann-Pick type C2 (NPC2) transcripts, Vd40090 (NP1) and Vd74517 (NP5), in the chemosensing pathway of Varroa destructor, we evaluated the impact of NP5 silencing on mites behavior and compared the effect of silencing of either transcripts on the interaction between chemosensory transcripts. In contrast to silencing NP1, which reduced feeding and reproduction by the mite (Nganso et al., 2021), silencing of NP5 reduced significantly the host reaching ability, but it did not affect the feeding on nurse bee. However, silencing of either transcript changed dramatically the co-expression patterns among the putative chemosensory genes, binding proteins and receptors. The results suggest the role of gustatory receptors in the detection of long-range chemical cues in the chemosensory cascade of the Varroa mite.

How Crucial is the Functional Pit Organ for the Varroa Mite?

Olfaction as well as gustation, are essential for animal survival, allowing behavioral modulation according to environmental input. We focused our study on an obligate ecto-parasitic mite of honey bees, the Varroa destructor Anderson and Trueman (Parasitiformes, Mesostigmata, Varroidae). By mechanically blocking the main olfactory organ on Varroa forelegs by varnishing with nail polish, we were able to show that other sensory organs cannot significantly compensate chemosensory abilities required for mite's host selection, identification as well as reproduction. In fact, we found that mites with blocked forelegs had a significantly lower ability to reach a host bee than those with varnished idiosoma and unvarnished control. Furthermore, fewer foreleg blocked mites were feeding on the nurse bees and their reproduction in the brood cells was significantly impaired. The inhibition of reproduction was also reflected in altered expression levels of vitellogenin and vitellogenin receptor genes in foreleg-blocked mites.

Calcineurin-mediated Dephosphorylation of Acetyl-coA Carboxylase is Required for Pheromone Biosynthesis Activating Neuropeptide (PBAN)-induced Sex Pheromone Biosynthesis in Helicoverpa armigera.

Chemical signaling plays a critical role in the behavior and physiology of many animals. Female insects, as many other animals, release sex pheromones to attract males for mating. The evolutionary and ecological success of insects therefore hinges on their ability to precisely mediate (including initiation and termination) pheromone biosynthesis. Pheromone biosynthesis activating neuropeptide (PBAN) acts directly on pheromone glands to regulate sex pheromone production using Ca2+ and cyclic-AMP as secondary messengers in the majority of species. However, the molecular mechanism downstream of the secondary messengers has not yet been elucidated in heliothine species. The present study shows that calcineurin, protein kinase A (PKA) and acetyl-coA carboxylase (ACC) are key components involved in PBAN-induced sex pheromone biosynthesis in Helicoverpa armigera using PBAN-dependent phosphoproteomics in combination with transcriptomics. RNAi-mediated knockdown and inhibitor assay demonstrated that calcineurin A is required for PBAN-induced ACC activation and sex pheromone production. Calcineurin-dependent phosphoproteomics and in vitro calcineurin phosphorylation assay further revealed that calcineurin regulated ACC activity by dephosphorylating ser84 and ser92. In addition, PKA-dependent phosphoproteomics and activity analysis revealed that PKA reduces the activity of AMP-activated protein kinase (AMPK), a negative regulator of ACC by phosphorylating the conserved ser92. Taken together, our findings indicate that calcineurin acts as the downstream signal of PBAN/G-protein receptor/Ca2+ to activate ACC through dephosphorylation while inactivating AMPK via PKA to reduce ACC phosphorylation, thus facilitating calcineurin activation of ACC.

Chemosensing of honeybee parasite, Varroa destructor: Transcriptomic analysis.

Chemosensing is a primary sense in nature, however little is known about its mechanism in Chelicerata. As a model organism we used the mite Varroa destructor, a key parasite of honeybees. Here we describe a transcriptomic analysis of two physiological stages for the Varroa foreleg, the site of primary olfactory organ. The transcriptomic analysis revealed transcripts of chemosensory related genes belonging to several groups. These include Niemann-Pick disease protein, type C2 (NPC2), gustatory receptors (GRs), ionotropic receptors (IRs), sensory neuron membrane proteins (SNMPs) and odorant binding proteins (OBP). However, no insect odorant receptors (ORs) and odorant co-receptors (ORcos) were found. In addition, we identified a homolog of the most ancient IR co-receptor, IR25a, in Varroa as well as in other members of Acari. High expression of this transcript in the mite's forelegs, while not detectable in the other pairs of legs, suggests a function for this IR25a-like in Varroa chemosensing.

Circadian rhythms and endocrine functions in adult insects.

Many behavioral and physiological processes in adult insects are influenced by both the endocrine and circadian systems, suggesting that these two key physiological systems interact. We reviewed the literature and found that experiments explicitly testing these interactions in adult insects have only been conducted for a few species. There is a shortage of measurements of hormone titers throughout the day under constant conditions even for the juvenile hormones (JHs) and ecdysteroids, the best studied insect hormones. Nevertheless, the available measurements of hormone titers coupled with indirect evidence for circadian modulation of hormone biosynthesis rate, and the expression of genes encoding proteins involved in hormone biosynthesis, binding or degradation are consistent with the hypothesis that the circulating levels of many insect hormones are influenced by the circadian system. Whole genome microarray studies suggest that the modulation of farnesol oxidase levels is important for the circadian regulation of JH biosynthesis in honey bees, mosquitoes, and fruit flies. Several studies have begun to address the functional significance of circadian oscillations in endocrine signaling. The best understood system is the circadian regulation of Pheromone Biosynthesis Activating Neuropeptide (PBAN) titers which is important for the temporal organization of sexual behavior in female moths. The evidence that the circadian and endocrine systems interact has important implications for studies of insect physiology and behavior. Additional studies on diverse species and physiological processes are needed for identifying basic principles underlying the interactions between the circadian and endocrine systems in insects.

Functional impact of silencing the Helicoverpa armigera sex-peptide receptor on female reproductive behaviour.

Female Helicoverpa armigera sex pheromone production is under the control of pheromone biosynthesis-activating neuropeptide (PBAN). After mating, females undergo suppression of sex pheromone production and enhanced oviposition as a result of the transfer of male-derived seminal peptides. In a previous study we identified a putative H. armigera sex-peptide receptor (HeaSP-R) and demonstrated a significant up-regulation in gene expression levels of this receptor in brains and pheromone glands of mated females, thereby implicating a regulatory role for sex peptide in the reproductive behaviour of H. armigera. In the present study, we show that virgin females injected with Drosophila melanogaster SP (DrmSP), in addition to inhibition of pheromone production, also exhibited a suppression of calling behaviour and a significant reduction in the gene expression levels of the PBAN-receptor. In addition, RNA interference (RNAi) silencing of the HeaSP-R expression by 50-60% prevented DrmSP-suppression of pheromone production and calling behaviour. Moreover, mated, silenced females failed to increase their oviposition rates as is normally observed in mated females, and their behaviour did not differ from that of virgin females. However, sex pheromone production by mated, silenced females remained low, comparable to mated, normal females, thereby indicating the probable involvement of additional factors in the suppression of sex pheromone production after mating.

Identification and differential expression of a sex-peptide receptor in Helicoverpa armigera.

Sex-pheromone production in the night flying female moth, Helicoverpa armigera is under neuroendocrine control due to the timely release of Pheromone Biosynthesis-Activating Neuropeptide (PBAN). Males orient to the females by upwind anemotaxis which usually leads to a successful mating. During copulation insect males transfer seminal peptides, produced in Male Accessory Glands (MAGs) which are implicated in post-mating behavioral changes of the females. These changes include the termination of pheromone biosynthesis and thus females do not re-mate. In previous studies we showed that synthetic Drosophila melanogaster Sex-Peptide (DrmSP), which is responsible for terminating receptivity in female flies, can terminate PBAN-stimulated pheromone production by pheromone glands of the female moth, H. armigera. In addition, we demonstrated that at least one fraction of the H. armigera MAG extract is both immunoreactive to DrmSP antibody and is pheromonostatic, we also showed that different sets of DrmSP-like immunoreactive peptides are up-regulated in the central nervous system of mated females. In the present study, we identify a putative receptor for sex-peptide (SP-R) in H. armigera on the basis of sequence homologies deposited in the GenBank. In addition, in an attempt to draw some light on the physiological significance of SP-like peptides in this moth, we conducted a differential expression study of this receptor comparing gene expression levels in relation to different photoperiods, sex and mating status of the moth. Photoperiod and mating influence SP-R gene expression levels and sexual dimorphic changes were observed in neural tissues due to the different physiological states. After mating SP-R transcript levels in female neural tissues and pheromone glands are up-regulated. Physiological studies in vivo confirm the up-regulation of gene expression levels in pheromone glands isolated from mated females.

Regulatory Role of PBAN in Sex Pheromone Biosynthesis of Heliothine Moths.

Both males and females of heliothine moths utilize sex-pheromones during the mating process. Females produce and release a sex pheromone for the long-range attraction of males for mating. Production of sex pheromone in females is controlled by the peptide hormone (pheromone biosynthesis activating neuropeptide, PBAN). This review will highlight what is known about the role PBAN plays in controlling pheromone production in female moths. Male moths produce compounds associated with a hairpencil structure associated with the aedaegus that are used as short-range aphrodisiacs during the mating process. We will discuss the role that PBAN plays in regulating male production of hairpencil pheromones.

Gene-silencing reveals the functional significance of pheromone biosynthesis activating neuropeptide receptor (PBAN-R) in a male moth.

The role of pheromone biosynthesis activating neuropeptide (PBAN) in the regulation of pheromone biosynthesis of several female moth species is well elucidated, but its role in the males has been a mystery for over two decades since its discovery from both male and female central nervous systems. In previous studies we have identified the presence of the gene transcript for the PBAN-G-protein coupled receptor (PBAN-R) in Helicoverpa armigera male hair-pencil-aedaegus complexes (male complexes), a tissue structurally homologous to the female pheromone gland. Moreover, we showed that this transcript is up-regulated during pupal-adult development, analogous to its regulation in the female pheromone-glands, thereby indicating a likely functional gene. Here we argue in favor of PBAN's role in regulating the free fatty-acid components (myristic, palmitic, stearic, and oleic acids) and alcohol components (hexadecanol, cis-11 hexadecanol, and octadecanol) in male complexes. We demonstrate the diel periodicity in levels of male components, with peak titers occurring during the 7th-9th h in the scotophase, coincident with female pheromone production. In addition, we show significant stimulation of component levels by synthetic HezPBAN. Furthermore, we confirm PBAN's function in this tissue through knockdown of the PBAN-R gene using RNAi-mediated gene-silencing. Injections of PBAN-R dsRNA into the male hemocoel significantly inhibited levels of the various male components by 58%-74%. In conclusion, through gain and loss of function we revealed the functionality of the PBAN-R and the key components that are up-regulated by PBAN.

Pheromone biosynthesis activating neuropeptide (PBAN): regulatory role and mode of action.

This review focuses on the endocrine regulation of reproductive behavior in moth species with particular emphasis on Helicoverpa spp. Reproductive behavior in most adult moths is dependent on the release of a unique blend of sex pheromones by the females to attract conspecific males. Mating, on the other hand, results in a loss of sexual receptivity due to the transfer of secretions from the male accessory glands, which renders females unattractive to ensuing mates. Synchronization of sexual behavior is attained by the timely release of Pheromone-Biosynthesis-Activating Neuropeptide (PBAN), a member of the PBAN/Pyrokinin neuropeptide family, characterized by a common amino acid sequence FXPRLamide motif in the C-terminus. PBAN is released into the hemolymph of females during the scotophase and is drastically reduced after mating, contributing to the loss in female receptivity. Pheromone production is age-dependent and Juvenile Hormone is involved in its regulation. PBAN activates pheromone production through its binding to a PBAN-Receptor (PBAN-R) and subsequent up-regulation of key enzymes in the biosynthetic pathway. The PBAN-R gene was identified as a member of the G-protein coupled receptor family (GPCRs), classified with the vertebrate subfamily of neuromedin U receptors. Using both biochemical and in silico mutagenesis studies, putative binding sites are predicted. Differential expression studies reveal its localization in pheromone glands, neural tissues and the male aedeagus. In the latter tissue, no activity and/or receptor-binding can be detected in response to PBAN. These results raise many questions concerning the evolutionary role of the PBAN/Pyrokinin receptors belonging to the GPCR family.

Pheromone biosynthetic pathways: PBAN-regulated rate-limiting steps and differential expression of desaturase genes in moth species.

We combine the use of labeled precursors with enzyme inhibitors to decipher the biosynthetic pathway of pheromone biosynthesis and the rate-limiting step/s that are regulated by pheromone biosynthesis activating neuropeptide (PBAN). We demonstrate that Plodia interpunctella is able to utilize hexadecanoic acid, and to a lesser extent tetradecanoic acid, for the biosynthesis of the main pheromone component (Z,E)-9,12-tetradecadienyl acetate. This indicated that the main pathway involves a Delta11 desaturase, chain shortening, followed by a Delta12 desaturase, but that a functional Delta9 desaturase could also be utilized. Using reverse transcription-quantitative real-time polymerase chain reaction (RT-QPCR) we distinguish two out of nine possible desaturase gene transcripts in P. interpunctella that are expressed at the highest levels. The rate-limiting step for PBAN-stimulation was studied in two moth species so as to compare the biosynthesis of a diene (P. interpunctella) and a monoene (Helicoverpa armigera) main pheromone component. In both species, incorporation of label from the (13)C sodium acetate precursor was activated by PBAN whereas no stimulatory action was observed in the incorporation of the precursors: (13)C malonyl coenzyme A; hexadecanoic 16,16,16-(2)H(3) or tetradecanoic 14,14,14-(2)H(3) acids. The acetyl coenzyme A carboxylase (ACCase) inhibitor, Tralkoxydim, inhibited the PBAN-stimulation of incorporation of stable isotope whereas the fatty-acyl reductase inhibitor, Mevastatin, failed to influence the stimulatory action of PBAN. These results provide irrefutable support to the hypothesis that PBAN affects the production of malonyl coenzyme A from acetate by the action of ACCase in the pheromone glands of these moths.

Molecular modeling of the binding of pheromone biosynthesis activating neuropeptide to its receptor.

Moth sex-pheromone biosynthesis follows a circadian cycle, which is cued by the release of the neurohormone pheromone biosynthesis activating neuropeptide (PBAN) to the hemolymph. PBAN binds to a G protein-coupled receptor (GPCR), in pheromone glands, (PG) initially identified by us in Helicoverpa zea moths (HezPBAN-R). In this study, the sequences of the seven transmembrane helices of HezPBAN-R were identified, built, packed and oriented correctly after multiple sequence alignment of the HezPBAN-R and several other GPCRs using the X-ray structure of rhodopsin as a template. Molecular dynamics simulations were run on three different beta-turn types of the C-terminal hexapeptide of PBAN and the results clustered into 12 structurally distinct groups. The lowest energy conformation from each group was used for computer-simulated docking with the model of the HezPBAN-R. Highest scoring complexes were examined and putative binding sites were identified. Experimental studies, using in vitro PG, revealed lower levels of pheromonotropic activity when challenged with pyrokinin-like peptides than with HezPBAN as ligand. Thus, the Drosophila melanogaster pyrokinin-1 receptor (CG9918) was chosen to create chimera receptors by exchanging between the three extracellular loops of the HezPBAN-R and the CG9918 for in silico mutagenesis experiments. The predicted docking model was validated with experimental data obtained from expressed chimera receptors in Sf9 cells.

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.

The presence of Drosophila melanogaster sex peptide-like immunoreactivity in the accessory glands of male Helicoverpa armigera.

In this study a highly specific polyclonal antibody to DrmSP was produced and used to develop and standardize a sensitive direct ELISA. Structure-activity studies revealed that the antiserum is specific to the N-terminal of DrmSP. This ELISA was used for the detection of DrmSP-like immunoreactivity in the reproductive tissues of male Helicoverpa armigera moths at femtomole levels. Two positive immunoreactive peaks were found in HPLC purified extracts of male accessory glands. The immunoreactive peak, which contained a higher amount of immunoreactivity, was also found to be pheromonostatic in PBAN-injected decapitated females as well as in intact female moths during their peak pheromone production. Lower levels of DrmSP-like immunoreactivity were found in younger males (1-2 day-old) when compared to older males (3-7 day-old).

Identification of a G protein-coupled receptor for pheromone biosynthesis activating neuropeptide from pheromone glands of the moth Helicoverpa zea.

Pheromone biosynthesis-activating neuropeptide (PBAN), a peptide produced by the subesophageal ganglion, is used by a variety of moths to regulate pheromone production. PBAN acts directly on pheromone gland cells by using calcium and cAMP as second messengers. We have identified a gene encoding a G protein-coupled receptor (GPCR) from pheromone glands of the female moth Helicoverpa zea. The gene was identified based on sequence identity to a group of GPCRs from Drosophila that are homologous to neuromedin U receptors in vertebrates. The full-length PBAN receptor was subsequently cloned, expressed in Sf9 insect cells, and shown to mobilize calcium in response to PBAN. This response was dose-dependent (EC50 = 25 nM) with a maximum response at 300 nM and a minimal observable response at 10 nM. Four additional peptides produced by the PBAN-encoding gene were also tested for activity, and it was determined that three had similar activity to PBAN and the other was slightly less active. Peptides belonging to the same family as PBAN, namely pyrokinins, as well as the vertebrate neuromedin U peptide also induced a calcium response. We have identified a GPCR for the PBAN/pyrokinin family of peptides with a known function of stimulating pheromone biosynthesis in female moths. It is related to several receptors from insects (Drosophila and Anopheles) and to neuromedin U and ghrelin receptors from vertebrates.

Activation of octopaminergic receptors by essential oil constituents isolated from aromatic plants: possible mode of action against insect pests.

As a result of screening a large number of essential oils from Israeli aromatic plants and their biologically active constituents, we isolated two oils with high activity against several stored-product insects. In this study the effect of these compounds on the acetylcholinesterase and the octopamine systems in insects was studied in order to elucidate their mode of action. Inhibition of acetylcholinesterase activity in vitro was evident only at high concentrations (10(-3) M) and could not account effectively for the low-dose mortality for some stored-product insects observed in vivo. However, the essential oil constituents were found to cause a significant increase in the levels of the intracellular messenger, cyclic AMP of abdominal epidermal tissue in the model insect, Helicoverpa armigera Hübn. The effect was significant even at low, physiological concentrations (10(-8) M) when tested directly on abdominal epidermal tissue preparations in vitro. This intracellular response was found to resemble closely the significant increases in the levels of the cyclic AMP of abdominal epidermal tissue due to treatment with the neurotransmitter/neuromodulator, octopamine. Subsequent treatment with the octopaminergic antagonist, phentolamine, effectively inhibited the cyclic AMP levels induced by essential oil treatment, indicating possible competitive activation of octopaminergic receptors by essential oil constituents.

Neuroendocrine control of pheromone biosynthesis in moths.

Prevalent among the Lepidoptera, as in many other insect orders, species-specific pheromones are synchronously produced and released for mate finding. Pheromone biosynthesis activating neuropeptide (PBAN) is a neuropeptide widespread throughout the class Insecta. Although its role in the several different orders of insects has not been fully elucidated, its regulatory role in Lepidopteran pheromone biosynthesis has been strongly implicated. The biosynthesis, gene expression, distribution, and release of PBAN have been studied in several moth species. This review discusses PBAN's mode of action as a pheromonotropic neurohormone at the organism, tissue, and cellular levels. The discussion includes an overview on PBAN structure-activity relationships, its target tissue identification, its putative receptor proteins, and the second messengers involved in signal transduction and the key regulatory enzymes in the pheromone biosynthetic pathway that may be influenced by PBAN. Finally, the review includes a discussion of various mediators and inhibitors of the pheromonotropic action due to PBAN.