Splicing alternativo do gene feminizer e o seu envolvimento na determinação de sexo da abelha sem ferrão Melipona interrupta (Hymenoptera: Apidae)

Abstract

Sexual differentiation is a crucial step in the development of most species of eukaryotes, since it affects strongly the physiological, morphological and behavioral aspects of the organisms. The mechanisms involved in the choice of the individuals sexual fate are beginning to be understood in some groups of animals. In the eusocial bees of Apis genus, sex is determined by a cascade composed of three major regulatory genes: csd, whose genotypes vary between sexes; fem the key gene which transmits the information from the csd to the next gene; and dsx, the last gene of the cascade, which is responsible for regulating the genes involved in the generation of each sex's peculiar phenotype. However, the csd gene is not the primary signal of sex determination in other bees. The fem gene, on the other hand, has been identified in a stingless bee species Melipona interrupta, but there is no evidence of fem s involvement in the sex determination of this species. Therefore, the aim of this study was to characterize the sequences of fem mature transcripts of M. interrupta males and females, in order to test the hypothesis of regulation of fem gene by sex-specific alternative splicing and also to investigate the role of this gene in the regulation of sex determination of this species. As in eusocial organisms the sex and caste determination are interconnected processes, we also examined the possible involvement of the fem gene in the differentiation between workers and queens. Males, workers and queens were collected in the stages of pupa and adult (eighteen samples in total). Based in a consensus sequence of the fem cDNA previously described for this species, several pairs of primers were drawn up for the amplification,cloning and sequencing of the samples fem cDNAs. The sequences obtained were aligned with each other and compared with proteins database (from the polypeptides sequences predicted for the mRNAs analyzed). Six distinct transcripts were found for the fem gene,supporting the hypothesis of the regulation of this gene by alternative splicing. Two of these transcripts are correlated with sex. The female splice variant has 1970 nucleotides, 10 exons and encodes a protein with 411 amino acids. The female isoform of Fem has three conserved domains, which are also found in the female Fem of Apis mellifera and in the female Tra (encoded by genes orthologous to fem) of other hymenopteran and dipteran insects species.The male splice variant is distinguished by the presence of a short exon (59 nucleotides) that contains a stop codon. Thus, the male isoform is a short protein (176 amino acids) and probably it s not functional, confirming the results described for the proteins orthologous to Melipona s fem. The other transcripts were found less frequently. Those are not related to gender or caste and we think they could be the products of stochastic events during the process of splicing. Therefore, the results of this study support the role of fem as the key gene of sex determination in M. interrupta.