Coordinated RNA-Seq and peptidomics identify neuropeptides and G-protein coupled receptors (GPCRs) in the large pine weevil Hylobius abietis, a major forestry pest

Hylobius abietis (Linnaeus), or large pine weevil (Coleoptera, Curculionidae), is a pest of European coniferous forests. In order to gain understanding of the functional physiology of this species, we have assembled a de novo transcriptome of H. abietis, from sequence data obtained by Next Generation Sequencing. In particular, we have identified genes encoding neuropeptides, peptide hormones and their putative G-protein coupled receptors (GPCRs) to gain insights into neuropeptide-modulated processes. The transcriptome was assembled de novo from pooled paired-end, sequence reads obtained from RNA from whole adults, gut and central nervous system tissue samples. Data analysis was performed on the transcripts obtained from the assembly including, annotation, gene ontology and functional assignment as well as transcriptome completeness assessment and KEGG pathway analysis. Pipelines were created using Bioinformatics tools and techniques for prediction and identification of neuropeptides and neuropeptide receptors. Peptidomic analysis was also carried out using a combination of MALDI-TOF as well as Q-Exactive Orbitrap mass spectrometry to confirm the identified neuropeptide. 41 putative neuropeptide families were identified in H. abietis, including Adipokinetic hormone (AKH), CAPA and DH31. Neuropeptide F, which has not been yet identified in the model beetle T. castaneum, was identified. Additionally, 24 putative neuropeptide and 9 leucine-rich repeat containing G protein coupled receptor-encoding transcripts were determined using both alignment as well as non-alignment methods. This information, submitted to the NCBI sequence read archive repository (SRA accession: SRP133355), can now be used to inform understanding of neuropeptide-modulated physiology and behaviour in H. abietis; and to develop specific neuropeptide-based tools for H. abietis control

Metabarcoding of insect-associated fungal communities: a comparison of internal transcribed spacer (ITS) and large-subunit (LSU) rRNA markers

Full taxonomic characterisation of fungal communities is necessary for establishing ecological associations and early detection of pathogens and invasive species. Complex communities of fungi are regularly characterised by metabarcoding using the Internal Transcribed Spacer (ITS) and the Large-Subunit (LSU) gene of the rRNA locus, but reliance on a single short sequence fragment limits the confidence of identification. Here we link metabarcoding from the ITS2 and LSU D1-D2 regions to characterise fungal communities associated with bark beetles (Scolytinae), the likely vectors of several tree pathogens. Both markers revealed similar patterns of overall species richness and response to key variables (beetle species, forest type), but identification against the respective reference databases using various taxonomic classifiers revealed poor resolution towards lower taxonomic levels, especially the species level. Thus, Operational Taxonomic Units (OTUs) could not be linked via taxonomic classifiers across ITS and LSU fragments. However, using phylogenetic trees (focused on the epidemiologically important Sordariomycetes) we placed OTUs obtained with either marker relative to reference sequences of the entire rRNA cistron that includes both loci and demonstrated the largely similar phylogenetic distribution of ITS and LSU-derived OTUs. Sensitivity analysis of congruence in both markers suggested the biologically most defensible threshold values for OTU delimitation in Sordariomycetes to be 98% for ITS2 and 99% for LSU D1-D2. Studies of fungal communities using the canonical ITS barcode require corroboration across additional loci. Phylogenetic analysis of OTU sequences aligned to the full rRNA cistron shows higher success rate and greater accuracy of species identification compared to probabilistic taxonomic classifiers

A review of Agrilus biguttatus in UK forests and its relationship with acute oak decline

Agrilus biguttatus Fab. (Coleoptera: Buprestidae) is a European bark-boring beetle whose larvae feed in the vascular tissue of oak trees. Until recently, it was considered rare in Britain, but sightings have become more frequent and it is often found on weakened trees suffering from Acute Oak Decline (AOD). This rapidly acting syndrome is characterized by patches of dark sticky fluid exuding from cracks on the trunk, with areas of necrotic tissue beneath, probably caused by a pathogenic bacterial component. However, the frequent association of AOD with the larval galleries and distinctive adult exit holes of A. biguttatus has raised concerns that the beetle may be contributing to the AOD syndrome or hastening the mortality of affected trees. This review evaluates the potential role of A. biguttatus in the AOD complex. Information on the beetle's life cycle and ecology is assessed along with the apparent increase in its abundance and distribution in the UK, and likely mechanisms of host selection. Oak tree defences against the beetle are discussed, as well as risk factors influencing susceptibility. Research on related Agrilus species is reviewed so that insights into the relationship between the beetle, the bacteria and the host tree can be made through comparisons with more extensively studied species. Possible management options in an AOD context are considered, and priority areas for future research are identified

The lifecycle of Agrilus biguttatus : the role of temperature in its development and distribution, and implications for Acute Oak Decline

he two spotted oak buprestid, Agrilus biguttatus Fabricus, is implicated in oak decline events across Europe, and is strongly linked to Acute Oak Decline in the U.K., although its role in the syndrome remains under investigation. In the U.K., the beetle is restricted to south and central England. The present study aimed to improve our understanding of the beetle's life history and thermal requirements, intending to explain its U.K. distribution, and to collect data for lifecycle modelling. Novel methods were developed to collect and culture the beetle in the laboratory, which enabled experiments to be carried out, providing data on the beetle's sex ratio, longevity and fecundity, and the development rates of eggs, larvae and pupae at constant temperatures. On average, females lived for 63 days and laid 82 eggs. Larvae developed through four instars. Sex ratio varied by site, with no overall trend apparent. The development rates of eggs, larvae and pupae (to adult emergence) had linear relationships with temperature, with lower developmental thresholds of 12.1, 11.9 and 15.1 °C, respectively. For each life stage, degree-day values were calculated. Beetles appeared to have an obligatory prepupal diapause at all temperatures studied, up to and including 25 °C. The implications of the developmental findings for the beetle's current distribution, as well as the possible effects of climate change, are discussed. The beetle appears to be thermally limited in the U.K. and, if so, its distribution, and perhaps that of Acute Oak Decline, may alter under climate change

Tourist species bias estimates of extrapolated species density in dispersive taxa: a case study from a litter beetle assemblage in temperate woodland

Extrapolative nonparametric estimators of species density are commonly used in community ecology. However, they are dependent on either (1) their use on non-dispersive taxa, or (2) the ability to separate tourists from residents in dispersive taxa. We undertook ten years of leaf litter sampling in an ancient woodland in the New Forest, Southern England. We identi- fied all the beetles from those samples and assigned them a residency status (residents, stratum tourists, and habitat tourists). Extrapolations, using the Chao 2, first- and second-order jackknife, and bootstrap approaches, of all sampled beetles all showed large overestimates of species richness when compared with extrapolations based on just residents. We recommend that the estimators should be used with caution as estimates of actual species density for dispersive taxa unless the natural history of most species in a community is well known. This applies especially to tropical ecosystems where many species have not been described. This reinforces the need for more descriptive natural history