Functional analysis of the long noncoding ovary-1 (lncov1) gene and the transcriptome of worker ovaries of the honey bee, Apis mellifera L.

• Grant number: 14/05757-5
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: July 01, 2014
• End date: June 30, 2017
• Field of knowledge: Biological Sciences - Genetics - Animal Genetics
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Klaus Hartmann Hartfelder
• Grantee: Gustavo Jacomini Tibério
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 11/03171-5 - Causal analysis of Apis mellifera development: regulatory genes and hierachical networks of gene expression in the specification of tissue and organs, AP.TEM

Abstract

The most notable feature of eusocial insects is the presence of castes with division of labor, constituting the fundamental characteristic of highly social organization. In Apis mellifera, workers and queens exhibit morphological and physiological differences related to their roles in the colony. Ovary size is the main difference between these morphs and is the anatomical basis underlying division of labor. The availability of the genome sequence of Apis facilitates the characterization of genes involved in developmental processes, including programmed cell death, which occurs in ovaries during larval development of worker bees. In the early larval stages, both castes still have 150 or more ovariole primordia in each ovary. This number is retained in queens while the workers lose 90-99 % of these ovarioles before pupation, which allows adult queens to perform reproduction function, while workers are functionally sterile. Recently we identified a candidate gene for a key role in ovary differentiation between queen and worker honeybees, the long non-coding RNA lncov1. It is overexpressed in the ovaries of workers, showing an increase in the level of transcripts in the feeding phase of the last larval stage (L5F3), which is the developmental stage of showing intense autophagic cell death. Furthermore, the localization of lncov1 RNA by FISH corresponded to TUNEL-positive cells undergoing programmed cell death. Besides its expression in the ovary, the transcript was detected in the development of other important tissues in which queens and workers differ in terms of morphological and physiological aspects. Since long non-coding RNAs have regulatory functions in gene expression by forming scaffolds with functional proteins, other RNAs and/or DNA, the objective of this project is investigate the function of this RNA during the development of workers, in particular its interaction with certain proteins. The method of choice are pull down assays that will be performed using extracts from the ovaries of workers in L5F3 phase, followed by the sequencing and identification of proteins that interact with lncov1. Furthermore, we will seek to further extend our knowledge about the role of this RNA in the development of the worker ovary by employing RNAi fllowed by RNA Seq to obtain differential transcriptomes. RNA will be extracted from ovaries of worker larvae treated with dslncov1 (double-stranded RNA) and of ovaries treated with a control dsRNA. The two transcriptomes will then be compared to identify differentially expressed genes. By these experimental approaches we intend to study aspects that elucidate lncov1 function(s) in the context of ovarian development in Apis mellifera. (AU)