Estudos da expressão e da função do gene sintase de quitina em Atta sexdens

Abstract

In Brazil, leaf-cutting ants belonging to the genus Atta and Acromyrmex and are considered as pests in agricultural and forestry regions that can lead to great economic losses. The aim of this work was to identify the transcripsts sequence that codify to chitin synthase in Atta sexdens (AsCHS), which has been described as the main enzyme in the metabolism of chitin synthesis. Evaluate the gene expression of AsCHS during insect developmental stage (larva, pupa and medium worker) and different body parts from medium worker (head, mesosome and worker without gaster), validate reference genes for quantitative PCR (qPCR) and evaluate the gene function by gene silencing through RNAi-mediated. The sequence coding to AsCHS was amplified by PCR based on conserved regions for insect CHSs, since the genome of that ant is not available. Two isoforms, from alternative splicing of the same gene were identified and sequenced. For gene expression analysis through qPCR, the reference gene for A. sexdens were determined, ef1-alpha and rpl18 for developmental stages and rpl18 and ef1-beta for different body parts from medium worker. The AsCHS is expressed in a different level during ant development, being pupa the developmental stage with the highest level of transcripts and the mesosome and worker without gaster, among the body parts from medium worker. The gene function was evaluated by gene silencing through RNAi-mediated in pupae. A reduction of 18 % of the CHS transcripts level was verified 168 h (7 days) after the injection of 300 ng of siRNA for AsCHS compared to the controls, resulting in pupae with morphological changes and an almost 50 % of ant’s mortality. This result show that AsCHS is an important enzyme for the development of A. sexdens. To the best of our knoledge, this is the first work to use the RNA interference techniques and quantification of transcripts by qPCR in leaf-cutting ants.