Reproductive Genomics in the Stick Insect Clitarchus hookeri

Abstract

Stick insects (Phasmatodea) are orthopteroid insects that are famous for their natural camouflage to the foliage of the plant on which they feed. Parthenogenesis is common among these insects, with 10% of the taxa scattered in different families being completely or partially parthenogenetic. These insects are poorly studied at the molecular level compared with other insects that are of economic importance, thereby impeding our understanding of their biology and evolution. Clitarchus hookeri (White) is one of the most common stick insect species in New Zealand and is distributed throughout large areas of the North and South Islands. Interestingly, it reproduces by geographical parthenogenesis, where sexual reproduction dominates on the upper half of the North Island, while, on the lower half of the North Island and the South Island, parthenogenesis is the only reproductive mode. In order to enrich the current genetic resources and develop an understanding of several key traits for stick insects, I utilised C. hookeri as a model species. The application of next-generation sequencing techniques, including RNA-Seq and whole-genome sequencing, was used to investigate the genetic basis underlying olfactory reception, digestion and most importantly reproduction, as well as to explore genomic components contributing to large genome size. The olfactory gene repertoire revealed C. hookeri had a fully developed olfactory reception system. Expressed genes encompassed a wide variety of gene families, including odorant binding proteins (OBPs), chemosensory proteins, odorant receptors, ionotropic receptors and gustatory receptors. Among them, OBPs have three recently duplicated copies (OBP4, OBP5 and OBP6) that may underlie key adaptive events during sensory evolution. The annotation of digestive enzymes from both the midgut transcriptome and genome assembly revealed nine cellulase GH9 gene duplicates, four of which were absent in the midgut transcriptome, indicating they might have evolved new roles rather than digesting plant cell walls. Other digestive enzymes such as GH28, beta-galactosidase, chitinase, glucosylceramidase, maltase and trehalase were also found, indicating C. hookeri utilised a variety of enzymes to digest plant material. Sequencing and assembling the C. hookeri genome revealed a large repetitive genome assembly comprising 4.2 Gigabases. The genome was enriched with DNA transposons, comprising of MITE which is not observed in the Timema cristinae genome. The C. hookeri gene models resulted from in silico prediction showed longer intron length compared with most of other insects. In sexual populations, similar to other obligate sexual animals, females and males express distinct groups of genes in the gonad that play important roles in oogenesis and spermatogenesis, respectively. In addition, males also expressed seminal fluid proteins that are known to regulate female reproductive functions in Drosophila and are important for determining male reproductive success. I found that, southern parthenogens, compared with sexual females, differentially expressed a large set of genes identified from ovariolar transcriptomes, indicating a molecular shift from sexual reproduction to parthenogenesis in the production of oocytes. Parthenogens down-regulated genes involved in Gene Ontology terms such as behaviour, response to stimulus, developmental process and rhythmic process, but up-regulated many genes without homology matches. This suggests, without utilising some genes expressed in sexual females, parthenogens are likely to express novel genes. Several extensively down-regulated genes in parthenogens were observed. Many of these have essential functions in Drosophila oogenesis, such as hsp83 (heat shock protein 83) involved in centrosome function and maintaining signal transduction, Crol (crooked legs) having a role in cell cycle progression, Nrg (neuroglian) accumulated in the border cells of early developing follicles for stabilising epithelial polarity and endos (endosulfine) playing a role in oocyte meiotic regulation and maturation. In addition, the analysis of eukaryotic-conserved meiotic genes (104 genes) revealed a few of them were differentially expressed and they were involved in nearly all the main meiotic stages. Two genes associated with crossover showed down-regulation, and together with the analysis of SNPs revealing parthenogens were genetic clones derived from a single origin, is suggestive of a reduction of crossovers in the parthenogens. An extremely low level of heterozygosity within the parthenogens suggests they are likely to be derived from genome duplication of haploid gamete.