De Novo Transcriptome Analysis of the Common New Zealand Stick Insect Clitarchus hookeri (Phasmatodea) Reveals Genes Involved in Olfaction, Digestion and Sexual Reproduction

Abstract

Phasmatodea, more commonly known as stick insects, have been poorly studied at the molecular level for several key traits, such as components of the sensory system and regulators of reproduction and development, impeding a deeper understanding of their functional biology. Here, we employ de novo transcriptome analysis to identify genes with primary functions related to female odour reception, digestion, and male sexual traits in the New Zealand common stick insect Clitarchus hookeri (White). The female olfactory gene repertoire revealed ten odorant binding proteins with three recently duplicated, 12 chemosensory proteins, 16 odorant receptors, and 17 ionotropic receptors. The majority of these olfactory genes were over-expressed in female antennae and have the inferred function of odorant reception. Others that were predominantly expressed in male terminalia (n = 3) and female midgut (n = 1) suggest they have a role in sexual reproduction and digestion, respectively. Over-represented transcripts in the midgut were enriched with digestive enzyme gene families. Clitarchus hookeri is likely to harbour nine members of an endogenous cellulase family (glycoside hydrolase family 9), two of which appear to be specific to the C. hookeri lineage. All of these cellulase sequences fall into four main phasmid clades and show gene duplication events occurred early in the diversification of Phasmatodea. In addition, C. hookeri genome is likely to express γ-proteobacteria pectinase transcripts that have recently been shown to be the result of horizontal transfer. We also predicted 711 male terminalia-enriched transcripts that are candidate accessory gland proteins, 28 of which were annotated to have molecular functions of peptidase activity and peptidase inhibitor activity, two groups being widely reported to regulate female reproduction through proteolytic cascades. Our study has yielded new insights into the genetic basis of odour detection, nutrient digestion, and male sexual traits in stick insects. The C. hookeri reference transcriptome, together with identified gene families, provides a comprehensive resource for studying the evolution of sensory perception, digestive systems, and reproductive success in phasmids.

Positive selection and comparative molecular evolution of reproductive proteins from New Zealand tree weta (Orthoptera, Hemideina)

“…Among the genes with Blast hits, the most common molecular function GO terms were serine-type endopeptidase activity (GO:0004252), serine-type endopeptidase inhibitor activity (GO:0004867) and ATP binding (GO:0005524) ( S2 Fig ). Overall, the GO categories identified in the reproductive gene search are similar to categories commonly seen when studying insect SFPs [ 9 , 16 , 81 , 84 ]. Various peptidase and peptidase regulators are among the reproductive proteins identified, and are believed to be essential for the regulation of reproduction through proteolytic cascades [ 28 ].…”

“…These types of proteins constitute a large proportion of the D . melanogaster [ 80 ], Anopheles gambiae [ 85 ], Aedes aegypti [ 86 ], Lutzomyia longipalphis [ 87 ] and Clitarchus hookeri [ 84 ] identified SFP and accessory gland proteins. The reproductive proteins identified in this screen are similar when compared with other insects, however a large proportion lack Blast hits.…”

De novo transcriptome analysis of the excretory tubules of Carausius morosus (Phasmatodea) and possible functions of the midgut ‘appendices’

“…RNA-Seq analysis, which has been successfully used in Phasmatodea before [49, 66], was performed by the Max Planck-Genome-centre with the Cufflinks v2.2.1 suite of tools (http://cole-trapnell-lab.github.io/cufflinks/) by mapping the original library reads to the CAP3 de novo transcriptome with TopHat, assembling with Cufflinks, merging with Cuffmerge, and calculating differential expression between each possible pair of tissue types with Cuffdiff [67]. The expression values were calculated as RPKM (reads per kilo base per million mapped reads), and the mean RPKM for each tissue type and gene is available in S1 Table.…”

Molecular ecology of insect pests of agricultural importance: the case of aphids

“…For example, it is now possible to study the responses to different environmental factors and identify the genetic bases of phenotypes by sequencing the transcriptomes of individuals, tissues or cells and analysing the genes expression levels (e.g. Nayduch et al, 2014;Keeling et al, 2016;Wu et al, 2016;Yu et al, 2016;Braden et al, 2017). Similarly, they have also revolutionised the area of phylogeography and evolution allowing the discovery of genome-wide molecular markers that could not be used before to study genetic variation in natural populations.…”