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Identification and characterization of the sex pheromone sensitive response gene(s) in the European Corn Borer males

Periodic Reporting for period 1 - ECBStrainShift (Identification and characterization of the sex pheromone sensitive response gene(s) in the European Corn Borer males)

Reporting period: 2015-10-15 to 2017-10-14

The genetic basis of olfaction is a topic of broad significance from fundamental and applied perspectives. The sense of smell is vital for most animals in finding food, identifying mates and avoiding predators. This study gave new insights on how moth pest species may evolve new pheromone preferences, which can cause race formation and speciation. Despite the importance of pheromone communication in reproductive isolation, its genetic basis is still unknown. By identifying the gene(s) responsible for variation in male response to female pheromone, in an important moth pest species, we give a first example on how response variation can cause strain differentiation.
The European Corn Borer (ECB) pheromone strains are a unique example where sex pheromone differentiation precedes ecological differentiation. We investigated male responses in the two pheromone races of the ECB. Females of the E strain produce E11- and Z11-14:OAc in a 99:1 ratio, and females of the Z strain emit a 3:97 ratio of the same compounds. Males of each strain are attracted to the ratios emitted by the corresponding females. However, the gene(s) underlying the olfactory and behavioral shift in male response (resp) is still unknown.
We aimed to characterize the genetic change underlying male response in this species, using a multidisciplinary approach, including classical chemical ecology, molecular biology, genetics and physiology. Thanks to this approach our work is of broad interest and use within the scientific society. Elucidating the genes underlying male olfactory responses in moths is also important from an applied perspective, because many moth species are important agricultural pests, and moth sex pheromones are an important tool in integrated pest management, used for monitoring, mating disruption and mass trapping. Identifying the gene(s) causing variation in male response can help monitoring populations for possible variations, and help to develop new pest control methods. Ultimately these methods target specifically one species and respect the environment.

Objectives
1. Genetically isolate and characterize the genes that modulate the male pheromone preference.
2. Localize them in the olfactory tissues.
3. Functionally characterize them.
Our first aim in this project was to genetically isolate and characterize the candidate genes for the male response to the female pheromone emission.
The variation in male pheromone perception has been shown to be sex-linked. The candidate genes have been hypothesized to be pheromone receptors (PRs) located on the sex-chromosome. However, through a Quantitative Trait Locus analysis (QTL) we were able to exclude the PRs as the genes underlying variation in male response, as these genes did not map to the resp locus. We showed resp to be in a region of 8 cM on the sex chromosome that maps 11 to 19 cM away from the PRs and points to new candidate genes. Comparison with fully sequenced Lepidoptera genomes indicates that this region is conserved and includes 11 genes (cg1 to cg11) . The homologous genes in D. melanogaster are genes controlling neurogenesis.

The second aim of the project was to reduce the number of candidate genes for further analysis. We used high throughput sequencing, to get the full gene length and characterize the SNP variation between the two strains. The overall results on the two pheromone strains sequence comparison within the resp region showed no strain specific difference between the coding region for all 11 genes and the non-coding region for most of the genes.
To reduce the number of candidate genes and be sure that we did not exclude important genes, we used two approaches:
First, we compared the Z/E genotype to the phenotype of lines with specific recombination points within the resp region. We narrowed down the region of interest by showing a smaller region where the genotype matches the phenotype for all recombinant lines. This comparison excluded 4 genes in the region.
In the second approach, we compared expression ratios of the genes in different tissue and developmental instars in order to link gene expression to the most relevant tissues to olfaction and the key moments of the insect’s olfactory system development. RT-qPCR was used on the 7 remaining candidate genes. All genes are exclusively expressed for both strains in the adult antennae (main olfactory tissue) with the exception of cg6 which is exclusively expressed in the brain. Three genes within this list show interesting expression patterns: i) cg7 is exclusively expressed in the male adult antennae, ii) cg10 shows a very different expression pattern between the Z and E strain (legs vs antennae) and iii) cg11 shows an early expression at the pupal instar which matches the timing of neuronal development in moths. The expression levels of these genes are higher in the E strain.

The third aim of our project was to further investigate the main candidate gene cg11. First, we investigated its expression in the antennae and compared expression in different developmental instars i.e. the pupa and the adult. The expression of cg11 was compared to the expression of known olfactory genes such as the PRs and the olfactory co-receptor ORco. RT-qPCR and in situ hybridization were employed. In the 2-days old adult males we found cg11 in the olfactory sensilla, which links it to the ECB olfaction. A difference was observed in the 4 day old pupa where cg11 is expressed in the same cells as ORco. This result could mean that the cells are not yet differentiated in the 4th day old pupa. Perhaps the pupal instar is the developmental moment that cg11 plays its role in the shift of neuronal targeting (antenna to the brain) between the two strains. One other result of this experiment is that cg11 is expressed in more cells than the neighboring PRs and is expressed next to all ORco cells. This could mean that it plays a more general role in the olfactory circuitry and not only in the pheromone circuitry.
We further investigated the role of cg11 using the CRISPR/Cas9 mutagenesis method. The aim is to create stable mutant lines that could be used for several physiological, behavioral and molecular comparisons in order to understand the role of cg11 in olfact
The project included a large collaborator network of 5 European countries and two US states. According to the EU policy and objectives for collaboration and exchange within and beyond the scientific society, this project has proven its power. Besides the technological advances and proof of state of art the results have ramifications outside our direct fields of study - entomology, sexual selection and speciation - and will interest fundamental research in the field of neural processing, as well as applied research for pest management.
This project increased our group’s visibility within the scientific society. Besides investigating the role of cg11 in moth olfaction the CRISPR/Cas9 approach employed here permit a proof of art of an innovative gene targeting method with great potential in understanding the natural genetic background and applications in therapeutics. Thanks to the EU project and its advertisement in the social media the group was solicited by other groups for their expertise on CRISPR/Cas9. This is a new opportunity showing possibilities to develop innovative services. It shows the need to develop this new gene targeting method.
QTL analysis