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Final Report Summary - DROSMELL (Deciphering the sense of smell in the invasive pest Drosophila suzukii)

The purpose of this project was to study in details chemoreception in Drosophila suzukii, which is an agricultural pest that causes severe damage on berries and cherries. D. suzukii is the ideal candidate for comparison studies since it belongs to the Drosophila genus, which includes Drosophila melanogaster that is a model organism of chemoreception. It also comprises other Drosophila species whose genomes have been fully sequenced and annotated. D. suzukii differs from the other Drosophila species since it has an elongated and sclerotized ovipositor that enables females to lay eggs on undamaged ripening fruits whereas other Drosophila species have a blunt ovipositor and lay eggs on fermenting or rotten substrates. Our project was aimed to set the molecular and the neuronal basis of olfactory response of D. suzukii, by following a three-step approach. The first two steps of the project were devoted to identify candidate chemosensory genes which were relevant for fresh fruit odor recognition whereas the third step was aimed to characterize their functional contribution to oviposition behavior.

The genomes of D. suzukii and of its sister species Drosophila subpulchrella had been sequenced by the host group one year before the starting of the project. The first objective was the annotation of three gene families encoding chemoreceptor genes in both species, as well as in the closely related species Drosophila biarmipes. This species belongs to suzukii subgroup and its sequenced genome was available on public databases. Genomic sequences were then compared in a fifteen-Drosophila species framework (using annotated sequences from Drosophila 12 Genomes Consortium) to infer the gene gains and losses which were unique to D. suzukii. In parallel, selection pressures on chemoreceptor genes were quantified by dN/dS metric, which compares non-synonymous to synonymous substitution rates. Our results show that the lineage leading to the D. suzukii group, which includes D. subpulchrella (that displays a ripening fruit oviposition behavior) and D. biarmipes (that has a rotten fruit oviposition behavior) had a large expansion in putative bitter gustatory receptors. However, only few changes in gustatory receptors characterized species with ripening fruit oviposition behavior. On the contrary, several gene gains happened in olfactory receptors after the split between D. biarmipes and the lineage leading to D. suzukii and D. subpulchrella. Moreover, three gene gains were specific to D. suzukii and one of these genes had been already linked to oviposition in D. melanogaster, which makes it an interesting candidate for functional analysis.

The second step was devoted to analyzed differences in transcription between mated and virgin females. Only D. suzukii females are attracted from undamaged ripening fruits which represent the ideal oviposition substrates. On the contrary, virgin females feed and mate on fermenting substrates. We first generated the first comprehensive transcriptome of D. suzukii antenna which affords a complete view of the receptors expressed in the third-antennal segment. Then, we run a differential-expression analysis which found over-expression of several olfactory receptors genes in gravid females, indicating that in D. suzukii neuronal plasticity modulates olfactory system to match response to odorants and physiological changes. We also tested the antennal response of mated and virgin D. suzukii females to a panel of eight odorants, and we observed a differential response to two of them, thus confirming the hypothesis that there is a shift in odorant response that matches reproductive changes. These results, taken together with those from genomic comparisons, provide a handful of genes encoding odorant receptors which may be responsible for D. suzukii innovative oviposition behavior. Next step will involve the characterization of proteins encoded by these candidate genes. In particular the discovery of the volatile bound by candidate odorant receptors will provide important clues on biological-relevant odors for D. suzukii.

To characterized candidate odorant receptors genes, we started two collaborations with researchers from the University of Trento and the Swedish Agricultural University. In particular we: 1) cloned the coding sequence of an odorant receptor unique to D. suzukii, the coding sequence of its paralog shared with other Drosophila species and the olfactory receptor co-receptor OrCO. Recombinant plasmids have been sent to our collaborators to be expressed in HEK cells and in D. melanogaster neurons through the empty-neuron system. The final aim is to identify volatiles specifically recognized by the unique D. suzukii receptor. 2) We obtained plasmids for the GAL-UAS system for non-model Drosophila species. We prepared reporter plasmids which express the GFP under the UAS regulatory sequence and we are trying to obtain recombinant flies, with the UAS-GFP cassette inserted in the genome.

We also studied the chemosensory system of the ovipositor of D. suzukii as side-project. Previous analysis suggested that this appendage might be able to sense sugars present in the fruits. . We first sequenced the ovipositor transcriptome in D. suzukii and in three Drosophila species characterized by progressive changes in oviposition behavior: annotation and comparison of genes encoding chemoreceptors revealed a set of gustatory and ionotropic receptors conserved among the four Drosophila species. We therefore tested the occurrence of chemosensory neurons in D. suzukii ovipositor using an anti-horseradish peroxidase antibody to target presence and shape of neuronal structures: results indicate that neurons associated with D. suzukii ovipositor spines are likely mechanosensory neurons. We think that mechanoreceptors present on the tip of the ovipositor of D. suzukii are probably used to test the stiffness of substrates. However, we cannot exclude a role of ovipositor in chemosensation, since transcription of some chemoreceptor genes seems to be a feature common to Drosophila species

Overall, outcomes of this project set the molecular and the neuronal basis for a clearer understanding of D. suzukii chemoreception. In the last years, a great effort was done to develop attractants which can be used to specifically capture D. suzukii in the field. D. suzukii attacks cause severe loss of production and no fully satisfying control methods are available so far. Moreover, alternatives to chemical pesticides are urgently needed to be integrated in more environmental-friendly agriculture approaches. A better understanding of how D. suzukii choose its oviposition substrate contributes to ameliorate control methods based on attraction. Secondly, on a broader context, deliverables of this project contributes to better knowledge of how species adapt to new environment.

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