Evolução molecular e caracterização do gene defective chorion 1 em Anastrepha fraterculus e Anastrepha obliqua (Diptera, Tephritidae)

Abstract

The Tephritidae family comprises many fruit fly species with predominance of frugivorous larvae, several of which of great economic importance. The fraterculus group is the most important species group, because it includes some of the most economically important species that cause damage to fruit production in Brazil and worldwide. Some species in this group are closely related, infest several host fruits, have a wide geographical distribution, and show considerable similarity and evolutionary proximity, which renders their identification difficult. Therefore, the investigation of genes with high evolutionary rates, such as the defective chorion 1 (dec-1), may be relevant for a better understanding of their phylogeny and the underlying differentiation forces involved. The gene dec-1 is involved with the production and modification of specific proteins that participate in the egg chorion formation during oogenesis. Evidence about this gene may bring relevant information on the differentiation of Anastrepha species. This work aims to investigate the molecular structure and the evolutionary patterns of dec-1 in order to understand the differentiation process of species in the Anastrepha genus, as well as among Tephritidae. A specific fragment of the dec-1 from two species of Anastrepha (A. fraterculus and A. obliqua) was amplified from samples collected in nature and compared to transcriptome sequences from next-generation data available in our laboratory. Conventional PCR was performed with specific dec-1 primers and fragments were submitted to Sanger sequencing. For structure prediction, dec-1 gene sequence of Anastrepha ludens was used as model. A group of orthologous sequences for this gene from other related flies was retrieved from NCBI and used to test for signatures of natural selection using clade and branch-specific methods with PAML and HyPhy software. In order to characterize the ovarian development, female ovaries of the species A. fraterculus were collected at different developmental periods, measured and submitted to statistical analysis. The ovarian characterization showed that choriogenesis probably occurs in A. fraterculus flies from 10 to 14 days of adult life, a period in which dec-1 gene products would be cleaved into isoforms and protein derivatives. The data collected from the ovaries proved to be informative to base and guide studies about ovarian characterization and reproductive development of A. fraterculus populations. A combination of transcriptome data with classical sequencing procedures allowed the identification of three alternative transcripts in both A. fraterculus and A. obliqua, corroborating the pattern found in other tephritids and recovering three of the six known D. melanogaster transcripts. Overall, there is evidence of purifying selection having a preponderant role both, for gene structure as well as along evolutionary branches. Despite evidence of a significant evolutionary conservation, some codons and branches under positive selection were detected, such as in the branch that separates A.obliqua flies, as well as on the separation of A. fraterculus into three clusters, and on the separation of Tephritidae from other Diptera. Analyses on some positively selected changes revealed seven non-synonymous changes that led to radical amino acid replacements which altered physicochemical properties. These results indicate that positive selection affected these regions during a specific evolutionary time and were fixed in different lineages, perhaps because it conferred some adaptive advantage. Thus, this gene showed some positively selected changes that seem to have been important to differentiate species in the group, suggesting that it might have a possible role in their differentiation process.