Transcriptomics and identification of candidate genes for studies in Coleoptera

Abstract

The molecular evolution of genes, joined to the differences between sexes, is a matter of interest poorly investigated across the class Insecta from a global perspective. The abundance of insect species, the high diversity of some of its orders, its ancient evolutionary origin, the current studies focused in a few model species, among other reasons, leads to a remarkable gap in studies in insect evolution and diversification, especially on beetles.

In this doctoral thesis, we sequenced by the first time five testis transcriptomes of four species of chrysomelids (Calligrapha confluens, Calligrapha aff. floridana, Calligrapha multipunctata and two specimens of Calligrapha philadelphica from two distant localities (PA and QC)) available from project CGL2011-23820, supporting this thesis. After the de novo assembly of the transcript contigs, functionally annotated 32,8-44,6% of them using the more suitable platform for de novo annotation of non-model species: Blas2GO.

We subsequently explored the Gene Ontology Consortium, searching for candidates for gene-finding. We selected 44 sperm individualization genes (GO:0007291) and explored their sex-biased condition in the insect model species Drosophila melanogaster through Flybase. Seven exhibited male-biased expression (blanks, Cyt-c-d, gudu, hmw, klhl10, nsr and Prosalpha6T) while one exhibited female-biased (scat).

Afterwards, we searched 20 beetle transcriptomes (19 of them from the 1KITE insect database), retrieving the putative orthologs of the 44 sperm individualisation genes. First, we confirmed the orthology of CG9313, Tektin-A and tomboy40 in Calligrapha transcriptomes. Subsequently, we inferred gene trees of 41 sperm individualisation genes (three of them excluded) across the class Insecta (up to 119 species). We identified duplications in some lineages of the 41 gene trees, as well as putative events of gene loss.

Moreover, we estimated the evolutionary rates for each of the 41 sperm individualisation genes. For amino acid sequences of insect species, it resulted, in average, 0.00239 ± 0.003012 subs./l./Ma (ranging from 0.000237 in orb2 to 0.009667 in hmw). Regarding nucleotide sequences of beetle species, it resulted, in average, 0.00452 ± 0.002083 subs./l./Ma (ranging from 0.00208 subs./l./Ma in nes to 0.01190 subs./l./Ma in Cul3; the time constraint for Coleoptera: 277.4-315.2

Ma).

We also searched for patterns of evolution of the 41 sperm individualisation genes. We found faster rates of evolution in proteins of sex-biased and clock- constrained genes (except for hmw, which is far from a molecular clock). However, we did not find faster evolution rates for the nucleotide sequences of the same genes for the conditions of sex-biased, presence of duplications or their position in an interaction network.

We also analysed a possible effect of gene interaction on the evolutionary rates of genes. Although we did not find significant differences in rate evolution between groups of interacting genes, we found significance in the edge Dronc-shi separating groups, as well as in the edges Dronc-Dredd and Chc-Past1.

In summary, through this doctoral thesis we have enlarged the genetic pool of insect species available on public databases of genes, enriching the representativeness of Coleoptera species; we have provided important information about the functionality of new sequenced testes-expressed genes; we have reported bioinformatic tools for gene-finding (by the use, development or improvement of them); we have contributed a considerable amount of gene trees reflecting the molecular evolution of sperm individualisation genes in insects; and, finally, we have investigated the patterns of evolution associated to different gene traits.

However, we still find an undoubtable missing data in our investigations; the new sequencing of insect species is slow so far, although it has increased in the last recent years. Also, studies on gene expression are currently lacking out of model species, either of sex, tissue or stage of development although a great effort is being done creating new databases of insect expression data. Altogether, further analyses are required to improve the picture of evolution of insect sex- specific expressed genes.