Background: In the tephritidsCeratitis,BactroceraandAnastrepha, the genetransformerprovides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the genetransformer-2in the tephritids has only been undertaken inCeratitis, and it has been shown that its function is required for the female-specific splicing ofdoublesexandtransformerpre-mRNA. It therefore participates intransformerauto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genusAnastrephawas undertaken in order to throw light on the evolution oftransformer-2.Results: The genetransformer-2produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike inDrosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection ofAnastrepha transformer-2dsRNA intoAnastrephaembryos caused a change in the splicing pattern of the endogenoustransformeranddoublesexpre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the elevenAnastrephaTransformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features.Conclusions: These results indicate thattransformer-2is required for sex determination inAnastrephathrough its participation in the female-specific splicing oftransformeranddoublesexpre-mRNAs. It is therefore needed for the auto-regulation of the genetransformer. Thus, thetransformer/transfomer-2 > doublesexelements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in whichtrais just another component of the sex determination gene cascade regulated bySex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted forSex-lethal, modified it, and converted it into the key gene controlling sex determination.

Within the Neotropical genusAnastrepha, the nominal speciesAnastrepha fraterculusis widely distributed from Mexico through northern Argentina. Currently it is believed to comprises a complex of at least eight cryptic species—known as theAnastrepha fraterculuscomplex (AF complex)—three of which occur in Brazil:A. sp.1 aff.fraterculus,A. sp.2 aff.fraterculus, andA. sp.3 aff.fraterculus. In this study, we present the results of a broad integrated analysis of multiple biological attributes in samples of the three species collected in sympatric areas. Analyses of the mitotic chromosomes confirm that all of them differ in sex chromosomes, and that the relative frequency of the distinct karyotypes is associated with variation in altitude. In these sympatric areas, a single female hybrid karyotype was detected within a significant sample of individuals. Population samples were analyzed for the ribosomal transcribed spacer ITS1, confirming that the three species have specific sequence types. Observations of reproductive behavior under laboratory conditions revealed thatA. sp.1 andA. sp.2 mate early in the morning, whileA. sp.3 mates in the middle of the day. A bimodal distribution of mating time was observed in the laboratory for hybrids, obtained betweenA. sp.1 andA. sp.3. In a mating choice experiment, most of the mating pairs were homospecific. In addition, through a list of the most frequent hosts associated with geographical occurrence, a bioclimatic model of their potential distribution was generated. The set of data allowed for the construction of explanatory hypothesis about the observed geographical pattern and the differential use of host fruits. Morphometric analyses of wings clearly demonstrated differences among the three species, for both males and females. Based on a wing image of theA. fraterculus(Wiedemann, 1830) type specimen, the morphometric analysis indicated that the type specimen would correspond to a male ofA. sp.2 aff.fraterculus. The information provided by this report is not only useful for taxonomic purposes, but also reveals aspects to be considered in any reconstruction of an evolutionary scenario of theAnastrepha fraterculuscomplex.

The endosymbiont Wolbachia is efficiently transmitted from females to their progenies, but horizontal transmission between different taxa is also known to occur. Aiming to determine if horizontal transmission might have occurred between Anastrepha fruit flies and associated braconid wasps, infection by Wolbachia was screened by amplification of a fragment of the wsp gene. Eight species of the genus Anastrepha were analyzed, from which six species of associated parasitoid wasps were recovered. The endosymbiont was found in seven Anastrepha species and in five species of braconids. The WSP Typing methodology detected eight wsp alleles belonging to Wolbachia supergroup A. Three were already known and five were new ones, among which four were found to be putative recombinant haplotypes. Two samples of Anastrepha obliqua and one sample of Doryctobracon brasiliensis showed multiple infection. Single infection by Wolbachia was found in the majority of samples. The distribution of Wolbachia harboring distinct alleles differed significantly between fruit flies and wasps. However, in nine samples of fruit flies and associated wasps, Wolbachia harbored the same wsp allele. These congruences suggest that horizontal transfer of Wolbachia might have occurred in the communities of fruit flies and their braconid parasitoids.

The South American fruit fly Anastrepha fraterculus (Wiedemann) is a complex of cryptic species, the so-called “Anastrepha fraterculus complex”, for which eight morphotypes are currently recognized. A previous analysis of ITS1 in samples of the Anastrepha fraterculus complex, while revealing high distinctiveness among samples from different localities of South America, Central America, and Mexico, no direct association was made between sequence type and morphotype. In the present report, a correlated analysis of morphometry and ITS1 data involved individuals belonging to the same population samples. Although showing a low level of intra-populational nucleotide variability, the ITS1 analysis indicated numerous inter-population sequence type variants. Morphotypes identified by morphometric analysis based on female wing shape were highly concordant with ITS1 genetic data. The correlation of genetic divergence and morphological differences among the tested samples gives strong evidence of a robust dataset, thereby indicating the existence of various taxonomic species within the A. fraterculus complex. However, the data revealed genetic and morphological variations in some regions, suggesting that further analysis is still required for some geographic regions.

The species of Anastrepha are arranged into 17 intrageneric groups. Recently, it was proposed that two species of the striata group, Anastrepha striata and A. bistrigata, might be realocated to serpentina group. Anastrepha bistrigata and A. serpentina have an X1X2Y/X1X1 X2X2 sex chromosome system while A. striata has a XY/XX system. It was previously proposed that the karyotype of A. bistrigata could be derived from that of A. striata by an Y:A fusion, and that the karyotype of A. serpentina would be derived from another, hypothetical karyotype. In the present report sequential staining with DAPI and chromomycin A3 (CMA3), followed by C-banding, revealed that the C-banded heterochromatic blocks of the sex chromosomes of A. bistrigata have different affinities to fluorochromes in comparison to the chromosomes of A. striata, from which they have hypothetically derived. The chromosomes of A. serpentina show substantial differences in their cytochemical properties compared to their A. bistrigata and A. striata counterparts. The FISH technique showed that the ribosomal gene sequences are located in DAPI- or DAPI/CMA3-positive heterochromatic blocks of the sex chromosomes, one site on the Y chromosome and one site on the X chromosome (X1 in A. bistrigata and A. serpentina). The data suggest that the karyotype of A. striata and A. bistrigata could be derived from a common ancestral karyotype, while the A. serpentina karyotype probably has a distinct origin.