Knockdown of cuticular transcripts in aphids: the ups and downs
Résumé
Aphids are vectors for numerous viruses, most of them being transmitted in a non-circulative manner. Viruses bind to specific receptors located in the aphid stylets, from which they can be released to infect new hosts. The acrostyle is an organ located at the tip of maxillary stylets, which contains cuticular proteins, among which are the receptors of Cauliflower mosaic virus(CaMV) and potentially those of other non-circulative viruses1,2. Long term research to characterize these receptors has allowed us to identify one cuticular protein, Stylin-1, present in the acrostyle. Additionally this is the first protein specifically identified in insect stylets. Indirect evidence indicates a probable involvement of this protein in CaMV transmission. In order to validate the role of this candidate in non-circulative transmission we decided to set up silencing experiments to decrease the expression of the stylin-1 gene in the head of aphid, where the target organs – the retort glands which synthesize the stylets prior to the aphid moutling– are located. The amount of stylin-1 and the protein in the stylets should be negatively impacted upon treatment. Associated phenotypes, if any, will then be characterized. Even though RNAi technology has been successfully implemented in many insect species, silencing genes in aphids still remains challenging. Several approaches have thus been developed in our study on two different aphid species, Acyrthosiphon pisum and Myzus persicae, to maximize the chance of achieving our goal. Double stranded RNA (dsRNA)has been delivered by either injection into the insect abdomen, or by ingestion of artificial diets. Here we discuss our approaches and the problems we have faced, such as a high variability in the transcript levels measured by RT-qPCR and variable performance of housekeeping genes in control experiments. These issues can prevent accurate interpretation of the experiments. Several parameters such as the rearing of aphids, the choice of aphid larval stage, the concentration and mode of delivery of dsRNA molecules, the choice of housekeeping genes and negative controls, as well as methods of RTqPCR analyses by individual or pools have been optimized. An overview of the experiments conducted, which have led us to successfully reducing the accumulation of Stylin-1, in both aphid species will be presented along with the challenges overcome...