Pipeline para a análise in silico de genes diferencialmente expressos durante o desenvolvimento de abelhas apis mellifera – o caso do knock-down da esterase do hormônio juvenil

Abstract

Given the great increase in the availability of nucleic acid sequencing data, an interdisciplinary area called bioinformatics has contributed greatly to research in computational analysis of DNA, RNA and protein sequences. Bioinformatics offers the means to deftly identify differentially expressed genes and enables the creation of increasingly efficient pipelines. In the honeybee Apis mellifera, caste determination is triggered by differential feeding in the third larval instar. The female larvae destined to become queens are fed royal jelly and the workers receive a lower proportion of royal jelly mixed with honey and pollen. This diet activates different endocrine responses and gene expression pathways in the castes that culminate in the development of queens and workers. One of the endocrine responses regulated by the differential feeding is the levels of juvenil hormone (JH) that are higher in the developing queen. The differences in the JH titers between queen and worker are regulated both by the synthesis of JH by the corpora allata and by its degradation mediated by juvenile hormone esterase (JHE). To determine the molecular mechanisms directly and indirectly dependent on jhe in the larval development, members of our research group performed functional assays in A. mellifera workers. The experiments consisted of knocking-down the jhe gene by interference RNA (iRNA) in the second larval instar. RNA samples were collected from treated (dsRNA-jhe) and control (naïve) individuals at the 5th larval instar and sequenced. The aim of our work was to develop an in silico analysis pipeline to identify differentially expressed genes between both control and treated bees. First, we used the FASTQC to determine the quality of the reads, which showed high phred score values. Adapter sequences identified by FASTQC and reads shorter than 10 nt in length were removed using the CUTADAPT tool. Next, the libraries were mapped on the A. mellifera genome with the BWA tool. The mean of mapping percentage of reads for all libraries was approximately 85%. The SAM files produced in the mapping step were converted in BAM files by SAMTOOLS, which were used by CUFFLINKS to identify differentially expressed genes (DEGs). A total of 458 GDEs were found, of which 151 were down regulated and 307 up regulated in the treated bees (dsRNA-jhe). The results revealed the genes that respond to the increase in endogenous JH titers in response to the knock-down of jhe in the larval development of A. mellifera. These results are similar to previous data obtained by the topic application of JH in developing worker larvae previously published by our research group. In addition, the results demonstrate the feasibility of the developed pipeline for the analysis of differential gene expression in biological systems.