A genomic analysis and transcriptomic atlas of gene expression in Psoroptes ovis reveals feeding- and stage-specific patterns of allergen expression.

BACKGROUND: Psoroptic mange, caused by infestation with the ectoparasitic mite, Psoroptes ovis, is highly contagious, resulting in intense pruritus and represents a major welfare and economic concern for the livestock industry Worldwide. Control relies on injectable endectocides and organophosphate dips, but concerns over residues, environmental contamination, and the development of resistance threaten the sustainability of this approach, highlighting interest in alternative control methods. However, development of vaccines and identification of chemotherapeutic targets is hampered by the lack of P. ovis transcriptomic and genomic resources. RESULTS: Building on the recent publication of the P. ovis draft genome, here we present a genomic analysis and transcriptomic atlas of gene expression in P. ovis revealing feeding- and stage-specific patterns of gene expression, including novel multigene families and allergens. Network-based clustering revealed 14 gene clusters demonstrating either single- or multi-stage specific gene expression patterns, with 3075 female-specific, 890 male-specific and 112, 217 and 526 transcripts showing larval, protonymph and tritonymph specific-expression, respectively. Detailed analysis of P. ovis allergens revealed stage-specific patterns of allergen gene expression, many of which were also enriched in "fed" mites and tritonymphs, highlighting an important feeding-related allergenicity in this developmental stage. Pair-wise analysis of differential expression between life-cycle stages identified patterns of sex-biased gene expression and also identified novel P. ovis multigene families including known allergens and novel genes with high levels of stage-specific expression. CONCLUSIONS: The genomic and transcriptomic atlas described here represents a unique resource for the acarid-research community, whilst the OrcAE platform makes this freely available, facilitating further community-led curation of the draft P. ovis genome.

Peptidergic control of the crop of the cabbage root fly, Delia radicum (L.) Diptera: Anthomyiidae): A role for myosuppressin.

There is much interest in targeting neuropeptide signaling for the development of new and environmentally friendly insect control chemicals. In this study we have focused attention on the peptidergic control of the adult crop of Delia radicum (cabbage root fly), an important pest of brassicas in European agriculture. The dipteran crop is a muscular organ formed from the foregut of the digestive tract and plays a vital role in the processing of food in adult flies. We have shown using direct tissue profiling by MALDI-TOF mass spectrometry that the decapeptide myosuppressin (TDVDHVFLRFamide) is present in the crop nerve bundle and that application of this peptide to the crop potently inhibits the spontaneous contractions of the muscular lobes with an IC50 of 4.4 × 10-8 M. The delivery of myosuppressin either by oral administration or by injection had no significant detrimental effect on the adult fly. This failure to elicit a response is possibly due to the susceptibility of the peptide to degradative peptidases that cleave the parent peptide to inactive fragments. Indeed, we show that the crop of D. radicum is a source of neuropeptide-degrading endo- and amino-peptidases. In contrast, feeding benzethonium chloride, a non-peptide agonist of myosuppressin, reduced feeding rate and increased the rate of mortality of adult D. radicum. Current results are indicative of a key role for myosuppressin in the regulation of crop physiology and the results achieved during this project provide the basis for subsequent studies aimed at developing insecticidal molecules targeting the peptidergic control of feeding and food digestion in this pest species.

Molecular interactions between wheat and cereal aphid (Sitobion avenae): analysis of changes to the wheat proteome.

Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of wheat to infestation by cereal aphid (Sitobion avenae) were investigated in a proteomic analysis. Approximately, 500 protein spots were reproducibly detected in the extracts from leaves of wheat seedlings after extraction and 2-DE. Sixty-seven spots differed significantly between control and infested plants following 24 h of aphid feeding, with 27 and 11 up-regulated, and 8 and 21 down-regulated, in local or systemic tissues, respectively. After 8 days, 80 protein spots differed significantly between control and aphid treatments with 13 and 18 up-regulated and 27 and 22 down-regulated in local or systemic tissues, respectively. As positive controls, plants were treated with salicylic acid or methyl jasmonate; 81 and 37 differentially expressed protein spots, respectively, were identified for these treatments. Approximately, 50% of differentially expressed protein spots were identified by PMF, revealing that the majority of proteins altered by aphid infestation were involved in metabolic processes and photosynthesis. Other proteins identified were involved in signal transduction, stress and defence, antioxidant activity, regulatory processes, and hormone responses. Responses to aphid attack at the proteome level were broadly similar to basal non-specific defence and stress responses in wheat, with evidence of down-regulation of insect-specific defence mechanisms, in agreement with the observed lack of aphid resistance in commercial wheat lines.

Effects of Manduca sexta allatostatin and an analogue on the peach-potato aphid Myzus persicae (hemiptera: aphididae) and degradation by enzymes in the aphid gut.

The oral toxicity of the C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analogue δR³Î´R5Manse-AS, where R residues were replaced by their D-isomers, were tested against the peach-potato aphid Myzus persicae by incorporation into an artificial diet. Both peptides had significant dose-dependent effects on mortality, growth, and fecundity compared with control insects. The analogue, δR³Î´R5Manse-AS, had an estimated LC50 of 0.31 µg/µl diet and was more potent than Manse-AS (estimated LC50 of 0.58 µg/µl diet). At a dose of 0.35 µg δR³Î´R5Manse-AS/µl diet, 76% of the aphids were dead after 6 days and all were dead after 10 days. In comparison, three times the dose of Manse-AS was required to achieve 74% mortality after 8 days and 98% mortality after 16 days. The degradation of both peptides by extracts prepared from the gut of M. persicae was investigated. The estimated half-life of Manse-AS, when incubated with the gut extract from M. persicae, was 31 min. Degradation was due to a cathepsin L-like cysteine protease, carboxypeptidase-like activity, endoprotease activity with glutamine specificity, pyroglutamate aminopeptidase activity, and possibly trypsin-like proteases. The half-life of the δR³Î´R5 Manse-AS analogue was enhanced (73 min) with the D-isomers of R appearing to prevent cleavage around the R residues by cathepsin L-like cysteine proteases or from trypsin-like proteases. The greater stability of the analogue may explain its increased potency in M. persicae. This work demonstrates the potential use of Manse-AS and analogues, with greater resistance to enzymatic attack, in aphid control strategies.

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, Carcinus maenas.

Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.

Insecticidal spider venom toxin fused to snowdrop lectin is toxic to the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae) and the rice brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The SFI1/GNA fusion protein, comprising of snowdrop lectin (Galanthus nivalis agglutinin, GNA) fused to an insecticidal spider venom neurotoxin (Segestria florentina toxin 1, SFI1) was tested for toxicity against the rice brown planthopper Nilaparvata lugens (Stål) and the peach-potato aphid Myzus persicae (Sulzer) by incorporation into artificial diets. Significant effects on the mortality of N. lugens were observed, with 100% of the insects fed on the SFI1/GNA fusion protein diet dead by day 7. The survival of the aphid M. persicae was also reduced when fed on the SFI1/GNA fusion protein. After 14 days, only 49% of the aphids that were fed on the fusion protein were still alive compared with approximately 90% of the aphids fed on the control diet or on diet containing GNA only. The SFI1/GNA fusion protein also slowed the development of M. persicae, and the reproductive capacity of the aphids fed on the SFI1/GNA fusion protein was severely reduced. The ability of GNA to act as a carrier protein, and deliver the SFI1 neurotoxin to the haemolymph of N. lugens, following oral ingestion, was investigated. The successful delivery of intact SFI1/GNA fusion protein to the haemolymph of these insects was shown by western blotting. Haemolymph taken from the insects that were fed on the fusion protein contained two GNA-immunoreactive proteins of molecular weights corresponding to GNA and to the SFI1/GNA fusion protein.

G protein coupled receptors as targets for next generation pesticides.

There is an on-going need for the discovery and development of new pesticides due to the loss of existing products through the continuing development of resistance, the desire for products with more favourable environmental and toxicological profiles and the need to implement the principles of integrated pest management. Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behaviour, including reproduction, osmoregulation, growth and development. Modifying normal receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness or reproductive capacity to reduce pest populations. Hence GPCRs offer potential targets for the development of next generation pesticides providing opportunities to discover new chemistries for invertebrate pest control. Such receptors are important targets for pharmaceutical drugs, but are under-exploited by the agro-chemical industry. The octopamine receptor agonists are the only pesticides with a recognized mode of action, as described in the classification scheme developed by the Insecticide Resistance Action Committee, that act via a GPCR. The availability of sequenced insect genomes has facilitated the characterization of insect GPCRs, but the development and utilization of screening assays to identify lead compounds has been slow. Various studies using knock-down technologies or applying the native ligands and/or neuropeptide analogues to pest insects in vivo, have however demonstrated that modifying normal receptor function can have an insecticidal effect. This review presents examples of potential insect neuropeptide receptors that are potential targets for lead compound development, using case studies from three representative pest species, Tribolium castaneum, Acyrthosiphon pisum, and Drosophila suzukii. Functional analysis studies on T. castaneum suggest that GPCRs involved in growth and development (eclosion hormone, ecdysis triggering hormone and crustacean cardioacceleratory peptide receptors) as well as the dopamine-2 like, latrophilin-like, starry night, frizzled-like, methuselah-like and the smoothened receptors may be suitable pesticide targets. From in vivo studies using native ligands and peptide analogues, receptors which appear to have a role in the regulation of feeding in the pea aphid, such as the PISCF-allatostatin and the various "kinin" receptors, are also potential targets. In Drosophila melanogaster various neuropeptides and their signalling pathways have been studied extensively. This may provide insights into potential pesticide targets that could be exploited in D. suzukii. Examples include the sex peptide receptor, which is involved in reproduction and host seeking behaviours, and those responsible for osmoregulation such as the diuretic hormone receptors. However the neuropeptides and their receptors in insects are often poorly characterized, especially in pest species. Although data from closely related species may be transferable (e.g. D. melanogaster to D. suzukii), peptides and receptors may have different roles in different insects, and hence a target in one insect may not be appropriate in another. Hence fundamental knowledge of the roles and functions of receptors is vital for development to proceed.

Genomic and peptidomic analyses of the neuropeptides from the emerging pest, Drosophila suzukii.

Drosophila suzukii is a highly polyphagous invasive pest which has been recently introduced into Europe and North America, where it is causing severe economic losses through larval infestations of stone and berry fruits. The peptidome of the selected nervous tissues of adult D. suzukii was investigated as a first step in identifying potential targets for the development of novel insecticides. Through in silico analyses of the D. suzukii genome databases 28 neuropeptide families, comprising more than 70 predicted peptides were identified. Using a combination of liquid chromatography and mass spectrometry of tissue extracts, 33 predicted peptides, representing 15 different peptide families were identified by their molecular masses and a total of 17 peptide sequences were confirmed by ion fragmentation. A comparison between the peptides and precursors of D. suzukii and D. melanogaster shows they are highly conserved, with differences only identified in the amino acid sequences of the peptides encoded in the FMRFamide, hugin and ecydysis triggering hormone precursors. All other peptides predicted and identified from D. suzukii appear to be identical to those previously characterized from D. melanogaster. Adipokinetic hormone was only identified in the corpus cardiacum, other peptides present included short neuropeptide F, a pyrokinin and myosuppressin, the latter of which was the only peptide identified from the crop nerve bundle. Peptides present in extracts of the brain and/or thoracico-abdominal ganglion included allatostatins, cardioacceleratory peptide 2b, corazonin, extended FMRFamides, pyrokinins, myoinihibitory peptides, neuropeptide-like precursor 1, SIFamide, short neuropeptide F, kinin, sulfakinins and tachykinin related peptides.

The pathogenicity of Vairimorpha necatrix (Microspora: Microsporidia) against the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae) and its potential use for the control of lepidopteran glasshouse pests.

A droplet feeding technique was used to feed known amounts of Vairimorpha necatrix (Kramer) spores to larvae of the tomato moth, Lacanobia oleracea (L) in order to assess the susceptibility of this lepidopteran pest to the pathogen. All first- to fourth-instar larvae died as a result of ingesting 1000 or more V necatrix spores. Two forms of death were observed, which were dependent on the dose and the age of the insect when treated. For first-instar larvae, rapid death (within 6days of dosing) occurred after ingestion of 2000 spores, whereas lower doses resulted in a proportion of larvae dying from chronic infection (microsporidiosis). For more advanced stages, increasing spore doses were required to give rapid death, such that a dose of 200,000 spores was needed to give 80% mortality within 6 days for third-instar larvae. Rapid death was not observed in fourth- to sixth-instar larvae. In all cases successful pupation and adult emergence were much reduced compared with non-infected larvae. Suspensions of V necatrix were sprayed on to tomato (Lycopersicon esculentum Mill) plants maintained in small glasshouses prior to infestation of the plants with L oleracea larvae. The numbers and biomass of pest larvae retrieved from the plants sprayed with V necatrix were significantly reduced by up to 40% and 70%, respectively, compared with plants sprayed with water (control). Similarly, plants sprayed with V necatrix showed a reduction in damage of up to 45% compared with the control plants.

Oral activity of FMRFamide-related peptides on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut.

Insect myosuppressins and myosuppressin analogues were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Acyrthosiphon pisum myosuppressin (Acypi-MS) and leucomyosuppressin (LMS) had significant dose-dependent effects (0.1-0.5µg peptide/µl diet) on feeding suppression, mortality, reduced growth and fecundity compared with control insects, but Acypi-MS was more potent than LMS. One hundred percent of aphids had died after 10days of feeding on 0.5µg Acypi-MS/µl diet whereas 40% of aphids feeding on 0.5µg LMS/µl diet were still alive after 13days. Myosuppressins were degraded by aphid gut enzymes; degradation was most likely due to a carboxypeptidase-like protease, an aminopeptidase and a cathepsin L cysteine protease. The estimated half-life of Acypi-MS in a gut extract was 30min, whereas LMS was degraded more slowly (t½=54min). No toxicity was observed when the analogues δR(9) LMS and citrolline(9) Acypi-MS or FMRFamide were fed to the pea aphid. These findings not only help to better understand the biological effects of myosuppressins in aphids but also demonstrate the potential use of myosuppressins in a strategy to control aphid pests.

Neuropeptides associated with the central nervous system of the cabbage root fly, Delia radicum (L).

The peptidome of the central nervous system of adult cabbage root fly, Delia radicum (L) was investigated using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Over twenty neuropeptides were identified from three different tissue sources, the combined brain/suboesophageal ganglion (SOG), the retrocerebral complex, and the thoracic-abdominal ganglion (TAG). A number of peptides were identified in all three tissues, including allatostatins, short neuropeptide F-like peptides, corazonin, a pyrokinin, and a myosuppressin. Adipokinetic hormone was restricted to the retrocerebral complex. Other peptides, including FMRFamides and sulfakinins were detected only in the brain/SOG and TAG. Some peptides, notably myoinhibitory peptides and tachykinins, which have been identified in other fly species, were not detected in any tissue sample. This study has structurally characterized for the first time, the neuropeptides from adult D. radicum.

Effects of Manduca sexta allatostatin and an analog on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut.

The C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analog delta R(3)delta R(5)Manse-AS, where R residues were replaced by their d-isomers, were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Both peptides had significant dose-dependent feeding suppression effects, resulting in mortality, reduced growth and fecundity compared with control insects. The delta R(3)delta R(5)Manse-AS analog had an estimated LC(50) of 0.18 microg/microl diet, and was more potent than Manse-AS. At a dose of 0.35 microg delta R(3)delta R(5)Manse-AS/microl diet, 98% of aphids were dead within 3 days, at a rate similar to those aphids that had been starved (no diet controls). On comparison, it required 13 days and three times the dose of Manse-AS fed to aphids to attain 96% mortality. It is possible that the feeding suppression effects of Manse-AS on aphids are due to the inhibition of gut motility. The estimated half-life of Manse-AS when incubated with a gut extract from A. pisum was 54 min. Degradation was most likely due to cathepsin L cysteine and/or trypsin-like proteases, by an unidentified glutamine-specific protease and by a carboxypeptidase-like enzyme. The d-isomers of R in the Manse-AS analog appeared to prevent hydrolysis by cathepsin L cysteine and trypsin-like enzymes, and enhance its half-life (145 min). However delta R(3)delta R(5)Manse-AS was cleaved by enzymes with carboxypeptidase-like and chymotrypsin-like activity. The increased stability of the Manse-AS analog may explain its enhanced feeding suppression effects when continually fed to aphids, and demonstrates the potential use of Manse-AS in a strategy to control aphid pests.

Infection by the microsporidium Vairimorpha necatrix (Microspora: Microsporidia) elevates juvenile hormone titres in larvae of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae).

The effects of infection by a microsporidium, Vairimorpha necatrix (Kramer), on the endogenous levels of juvenile hormones in tomato moth (Lacanobia oleracea L.) larvae were investigated. Levels of juvenile hormone II (JH II) were 10-fold greater in the infected larvae on day two of the sixth stadium but no significant difference was observed on day seven. Juvenile hormone I (JH I) was also detected in day two and day seven sixth instar infected larvae but was not detected in non-infected larvae. The duration of the fifth and sixth stadia was significantly longer for infected larvae when compared with non-infected larvae. No evidence was found to suggest that supernumerary moults are a feature of infection by V. necatrix in L. oleracea larvae. Experiments were performed to determine whether the elevation in JH levels, which probably prevents pupation, is an adaptive mechanism of the microsporidium for extending the growth phase of the host, thereby allowing increased spore production. A proportion of infected larvae were collected on days 9 and 24 of the sixth stadium and spore extracts prepared from each larva. These days represent the average duration of the sixth stadium required for uninfected larvae to reach pupation, and the average number of days that V. necatrix-infected larvae survive in the sixth stadium before dying from infection. The mean spore yields from infected larvae 24 days into the sixth stadium were significantly higher than the spore yields obtained from day nine sixth instar larvae. The hypothesis that V. necatrix manipulates host endocrinology (i.e. prolong the host larval state to maximise spore yield) is discussed in context with the results obtained.

Tritrophic interactions between transgenic potato expressing snowdrop lectin (GNA), an aphid pest (peach-potato aphid; Myzus persicae (Sulz.) and a beneficial predator (2-spot ladybird; Adalia bipunctata L.).

Tritrophic interactions between transgenic potato expressing the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA), an aphid pest, Myzus persicae (Sulz.), and a beneficial predator, the 2-spot ladybird (Adalia bipunctata L.) were investigated. Clonal plants expressing GNA at 0.1-0.2% total soluble protein in leaves were used. No significant effects on development and survival of ladybird larvae fed on aphids from these transgenic plants were observed, with larval survival in the experimental group being 90% compared to 89% for controls. There were also no effects on subsequent female or male longevity. Female fecundity was also investigated. Although no significant differences (p > 0.05) were observed in egg production between control and experimental groups, a 10%, reduction (p < 0.01) in egg viability (determined by % hatch) occurred in ladybirds fed aphids reared on transgenic plants. Additional studies were carried out using aphids fed on artificial diet containing GNA, to deliver quantified levels of the protein to ladybird adults. GNA had no deleterious effects upon adult longevity, but resulted in a consistent trend for improved fecundity. Egg production was increased by up to 70% and egg viability also increased significantly. The results suggest that GNA is not deleterious to ladybirds. Results from these studies highlight the need to discriminate between direct and indirect effects when studying tritrophic interactions between plants/pests/natural enemies. Furthermore, it emphasises the importance of demonstrating 'cause and effect'.