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Final Report Summary - ODOGEN (Comparative genomics and gene expression profile of sexual and parthenogenetic Ischnura hastata (Insecta, Odonata))

The aims of the project are to determine the consequences that the abandonment of sexual reproduction and switch to obligate parthenogenesis in Ischnura hastata, (Insecta, Odonata - dragonflies and damselflies). This is the only species within its group known to be parthenogenetic in the wild. In order to identify the molecular mechanisms that underlie the transition from sex to parthenogenesis, three different goals were proposed:

1. Quantify the extent of relaxed selection associated with loss of gene function, specifically in the meiotic pathway, in sexual and parthenogenetic Ischnura hastata.
2. Generate a transcriptome of sexual and parthenogenetic I. hastata to identify the regulatory basis of the behavioral shift associated with parthenogenesis.
3. To identify the genomic regions associated with parthenogenesis using RAD sequencing.

Goal 1. Analysis of meiosis-specific and meiosis-related genes in odonates.

For this goal, the opportunity to extend the RNA-based aspects of the project, through the collaboration with the 1KITE project. Dr Lorenzo-Carballa has gained access to the complete transcriptome data of 103 odonate species that have been sequenced under the umbrella of this project. Furthermore the analyses have been extended to cover a number of hemimetabolous insect species from which transcriptomic data are publicly available. This has allowed to extend the study of meiotic genes beyond dragonflies and damselflies, making the potential results and interest of the work more impactful. However, it is important to note that this study of the meiotic genes has proven to be complicated, mainly due to the nature of the 1KITE datasets available: given the short transcriptional frame of the genes under study (they are only expressed in the reproductive tissues at a very specific developmental time), and the fact that the samples from which these data were obtained were quite heterogeneous (RNA was sequenced from animals collected in the field at different age/developmental stage), it has not always been possible to find the genes of interest in these data. Nevertheless, a consistent pattern of absence of two genes (Rec8 and Cortex) from the eight meiosis-specific genes (the so-called meiotic toolkit) across all the Odonata and Hemimetabolous insect species analyzed has been found. Ongoing work includes the phylogenetic analyses of these data, which will shed light on the origin and evolution of these genes across different insect groups.

Goal 2. Comparative transcriptomics of sexual and parthenogenetic Ischnura hastata.

A transcriptome has been obtained for Ischnura hastata from RNA obtained from head and abdomen tissues of sexual and parthenogenetic mature females (see additional pdf figure 1 for a schematic representation of the RNA sequencing experiment). The size of the assembly is ca. 77,000 contigs, with 72% of the reads mapped back to the assembly as proper pairs. The transcriptome is quite complete, with only 343 orthologous genes out of the 2,675 tipically found in Arthropoda missing. After clustering and removing those contigs with less than 10 reads mapped, the final assembly has ca. 65,000 contigs. A preliminary annotation of the transcriptome based on blastx searches against protein databases and InterproScan (see additional pdf figures 2a and 2b) has yielded a total of about 23,000 annotated transcripts.

The differential expression analyses have revealed a high number of transcripts that are differentially expressed between sexuals and asexuals in both the abdomen and head tissues (see figures 3a and 3b in the attached pdf). A number of genes related with oogenesis in Drosophila have been found differentially expressed between sexual and asexual females (see figure 4 in the attached pdf file). This is the first time that genes related with gamete formation are identified in odonates. Interestingly, the gene expression patterns differ completely between both types of females at the same age. Meiotic genes have not been identified in the transcriptome, which indicates that meiosis does not occur in the females at this age, and does not allow to conclude whether the mechanism of parthenogenesis in Ischnura hastata is apomictic (oocytes are formed through meiosis) or automictic (oocytes are formed by mitotic divisions).

Some interesting findings regarding the differentially expressed transcripts in the head tissues include the identification of the CREB binding protein as upregulated in the sexual females. This gene has been related to long-term and courtship memory in Drosophila. In the case of asexuals, several upregulated transcripts have been annotated as aquaporins; which are typically related to water transport and osmoregulation in insects, however certain aquaporins expressed in the brain of Drosophila and Glossina have been found related with increased fertility and fecundity.

Current analyses include the improvement of the transcriptome annotation through blastx searches using specific databases of genes of interest (e.g.reproduction or sensory genes) found in Drosophila and other arthropods. This will allow to improve the identification of genes and functional pathways involved in the transition from sex to parthenogenesis in Ischnura hastata.

Future research involves the study of patterns of gene expression throughout the development in both sexual and parthenogenetic females, and the development of rDNA probes for Fluorescent In Situ Hybridization (FISH), to identifly spatial and temporal patterns of gene expression. Also, the establishment of a collaboration with Dr Seth Bybee from BYU in the USA, with the aim to sequence the genome of I. hastata; will allow to analyze patterns of gene duplication, loss etcetera.

Goal 3. Genotyping By Sequencing of sexual and parthenogenetic Ischnura hastata - population genomics.

Genomic DNA from a total of 367 sexual and asexual individuals has been used for ddRAD sequencing. The software STACKS was used to de novo assemble a catalog of RAD loci. The total number of variable loci in the catalog is 22,189. The preliminary results of a population genetic structure analyses for the sexuals are shown in the additional pdf figure 5. Using the information from 2,161 unlinked SNPs corresponding to 178 sexual individuals, the software STRUCTURE indicated that the genetic structure of the sexual Ischnura hastata populations is best described as 4 main clusters: Galapagos, Colombia, Mexico and a cluster comprising the Caribbean and North American populations sampled (Arkansas, Kentucky, Florida, Cuba, Puerto Rico, Dominican Republic, and Jamaica). Therefore, genome wide sequencing methods have allowed to detect population differentiation in this species, which could not be previously detected using microsatellite markers (Lorenzo-Carballa et al 2012; Heredity, 108, 386-95). Interestingly, the Caribbean and North American populations that comprise the main cluster, are under the influence of the Atlantic Maritime Tropical wind current (see figure 6 in the additional pdf), which would agree with passive dispersal by the wind of this species over high geographical distances. This main wind current joins the Gulf Stream wind current which goes from the gulf of Mexico to Europe, passing over the Azores archipelago. Ongoing analyses include to identify the geographic origin of the asexual individuals, by means of assignment tests: if the asexual individuals colonized the Azores by being transported by the wind, one would expect them to be assigned with greatest probability to this cluster of populations. Last, and outlier analysis will be conducted in order to identify loci under selection.

Overall, the objectives of the project have been achieved, with minor deviations from the original plan. The data analysis is ongoing and the results will be submitted for publication in the next months. Currently, Dr Lorenzo-Carballa has now secured a job as postdoctoral research assistant in the University of Coruña in Spain, in which she will focus on differential gene expression and transcriptomics of an invasive plant species.

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