Denise Selivon

A Genetic–Morphological Characterization of Two Cryptic Species of the Anastrepha fraterculus Complex (Diptera: Tephritidae)

Abstract Biological characteristics of two entities of the Anastrepha fraterculus complex (Diptera: Tephritidae), referred to in previous publications as A. sp.1 aff. fraterculus and A. sp.2 aff. fraterculus, were further studied by a combined analysis of isozymes, karyotypes, morphometry, and crossings, in samples from 10 Brazilian populations. A survey of 16 enzymatic systems comprising 19 loci showed significant differences in the allele frequencies at four loci, FUM, ME, HEX, and LDH, allowing the recognition of two population clusters. These clusters also differ in their karyotypes, especially in the length of the sex chromosomes and in the size and location of heterochromatic regions. A morphometric analysis of wings and the aculeus in samples from five populations clearly showed a distinction between the two clusters but not between populations within each cluster. A phenetic analysis based on the Mahalanobis distance matrix also arranged the populations into the same two clusters. Crosses between populations of the same cluster showed no significant differences in egg hatching and in the adult sex ratio. However, a significant decrease in egg hatching was observed in the intercluster crosses. In crosses of cluster 1 males to cluster 2 females, a significant deviation in the sex ratio was observed according to Haldane’s rule, but not in the reciprocal crosses, indicating that a certain degree of reproductive isolation occurs between populations of cluster 1 and cluster 2. The results indicate that the two population clusters actually represent two cryptic species of the nominal species Anastrepha fraterculus, corroborating previous studies on this complex. We propose that the denominations A. sp.1 aff. fraterculus and A. sp.2 aff. fraterculus should be maintained until an appropriate taxonomic revision is made.

The Gene Transformer of Anastrepha Fruit Flies (Diptera, Tephritidae) and Its Evolution in Insects

In the tephritids Ceratitis capitata and Bactrocera oleae, the gene transformer acts as the memory device for sex determination, via an auto-regulatory function; and functional Tra protein is produced only in females. This paper investigates the evolution of the gene tra, which was characterised in twelve tephritid species belonging to the less extensively analysed genus Anastrepha. Our study provided the following major conclusions. Firstly, the memory device mechanism used by this gene in sex determination in tephritids likely existed in the common ancestor of the Ceratitis, Bactrocera and Anastrepha phylogenetic lineages. This mechanism would represent the ancestral state with respect to the extant cascade seen in the more evolved Drosophila lineage. Secondly, Transformer2-specific binding intronic splicing silencer sites were found in the splicing regulatory region of transformer but not in doublesex pre-mRNAs in these tephritids. Thus, these sites probably provide the discriminating feature for the putative dual splicing activity of the Tra-Tra2 complex in tephritids. It acts as a splicing activator in dsx pre-mRNA splicing (its binding to the female-specific exon promotes the inclusion of this exon into the mature mRNA), and as a splicing inhibitor in tra pre-mRNA splicing (its binding to the male-specific exons prevents the inclusion of these exons into the mature mRNA). Further, a highly conserved region was found in the specific amino-terminal region of the tephritid Tra protein that might be involved in Tra auto-regulatory function and hence in its repressive splicing behaviour. Finally, the Tra proteins conserved the SR dipeptides, which are essential for Tra functionality.

Cryptic Species of the Anastrepha fraterculus Complex (Diptera: Tephritidae): A Multivariate Approach for the Recognition of South American Morphotypes

ABSTRACT Although a large amount of data have been published in past years on the taxonomic status of the Anastrepha fraterculus (Wiedemann) species complex, there is still a need to know how many species this complex comprises, the distribution of each one, and their distinguishing features. In this study, we assessed the morphometric variability of 32 populations from the A. fraterculus complex, located in major biogeographical areas from the Neotropics. Multivariate techniques for analysis were applied to the measurements of 21 variables referring to the mesonotum, aculeus, and wing. For the first time, our results identified the presence of seven distinct morphotypes within this species complex. According to the biogeographical areas, populations occurring in the Mesoamerican dominion (Mexico, Guatemala, and Panama) were clustered within a single natural entity labeled as the “Mexican” morphotype; whereas in the northwestern South American dominion, samples fell into three distinct groups: the “Venezuelan” morphotype with a single population from the Caribbean lowlands of Venezuela, the “Andean” morphotype from the highlands of Venezuela and Colombia, and the third group or “Peruvian” morphotype comprised the samples from the Pacific coastal lowlands of Ecuador and Peru. Three additional groups were identified from the Chacoan and Paranaense sub-regions: the morphotype “Brazilian-1” was recognized as including the Argentinean samples with most pertaining to Brazil, and widely distributed in these biogeographical areas; the morphotype “Brazilian-2” was recognized as including two samples from the state of Sao Paulo (Ilha-Bela and São Sebastião); whereas the morphotype “Brazilian-3” included a single population from Botucatu (state of Sao Paulo). Based on data published by previous authors showing genetic and karyotypic differentiation, as well as reproductive isolation, we have concluded that such morphotypes indeed represent natural groups and distinct taxonomic entities.

The gene doublesex of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

The doublesex (dsx) gene of several Anastrepha species was isolated and characterised. Its molecular organisation was found to be the same in all the species examined. This gene is composed of four exons: Exons 1 and 2 are common to both sexes, exon 3 is female specific, and exon 4 is male specific. It codes for both the female DsxF and male DsxM proteins, corresponding to the sex-specific splicing product of its primary transcript; male-specific splicing is the default mode. A comparison of the Dsx proteins of different Anastrepha species with those of other insects showed them to be very similar. Molecular evolutionary analysis (both at the nucleotide and amino acid levels) of dsx in different insects revealed a topology in good agreement with their owners’ taxonomic relationships. The great majority of the nucleotide changes detected in the dsx gene of the analysed species were significantly synonymous, evidence that strong purifying selection has acted on dsx so that the functional structure of the Dsx proteins is preserved. However, the common region of DsxF and DsxM proteins appeared to be the main target for selection acting upon the long-term evolution of gene dsx.

Haldane's rule and other aspects of reproductive isolation observed in the Anastrepha fraterculus complex (Diptera: Tephritidae)

Some aspects of reproductive isolation between allopatric populations of two closely related species of the Anastrepha fraterculus complex (A. fraterculus sp. 1 and sp. 2) were evaluated in laboratory conditions. Most of the crosses were fertile in each species as well as between sp. 2 females and sp. 1 males. In the reciprocal cross only 41.7% of the matings yielded viable progeny. Egg hatching occurred at similar rates within the two species, but was significantly lower in the crosses between the species. Adult emergence did not differ significantly among crosses. The sex ratio of adult progeny within each species, as well as in the hybrid progeny derived from sp. 1 females crossed to sp. 2 males, did not differ from the expected 1:1 ratio. However, in the crosses between sp. 2 females to sp. 1 males, a significant deviation in the sex ratio in favor of females was observed, according to the Haldane’s rule. The results reinforce previous data which indicated that A. fraterculus sp. 1 and A. fraterculus sp. 2 are distinct biological entities.

Cytological characterization of sex chromosomes and ribosomal DNA location in Anastrepha species (Diptera, Tephritidae).

This paper reports a comparative analysis of heterochromatin organization in the sex chromosomes of the fruit fly Anastrepha. Mitotic chromosomes of eight Anastrepha species from different taxonomic groups were stained with DAPI and chromomycin A3 fluorochromes followed by C-banding. A specific sex-chromosome banding pattern was obtained for each of the analyzed species. Fluorescence in situ hybridization (FISH) was performed to investigate the chromosomal location of rDNA loci. In all cases the rDNA sequences were found to localize exclusively to the sex chromosomes. The results further extend the chromosomal knowledge of Anastrepha and allow a precise species identification.

Karyotype characterization of Anastrepha fruit flies (Diptera: Tephritidae)

The study comprises the description of the C-banded mitotic chromosomes of five species of Anastrepha, A. amita Zucchi, A. turpiniae Stone, A. zenildae Zucchi, A. grandis (Macquart) and A. leptozona Hendel , and a reanalysis of the chromosomes of three other species, A. distincta Greene, A. obliqua (Macquart) and A. sororcula Zucchi. The species have a diploid number of 12, while A. leptozona has 2n = 10. Heteromorphism of the sex chromosomes is present in all of the species, which have a XY/XX sex chromosome system. A. grandis and A. leptozona have meta and submetacentric autosomes while all of the chromosomes in the other species are acrocentric. The species can be characterized unequivocally by the length and the pattern of blocks of heterochromatin in the sex chromosomes.

Occurrence of Wolbachia in Brazilian samples of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

Wolbachia bacteria were detected by PCR followed by sequencing of a fragment of the 16S ribosomal gene, in a natural population sample and in two laboratory colonies of the medfly, C. capitata (Wied.), from Brazil. Sequencing revealed that the fragment was compatible with the Wolbachia type A group found in several insect species. This is the first description of a natural Wolbachia infection in C. capitata, since several other samples from different regions so far analyzed seemed to be free of infection.

Wolbachia in Anastrepha Fruit Flies (Diptera: Tephritidae)

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and cause a variety of reproductive abnormalities, such as cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing, and host feminization. In this study, we used three sets of Wolbachia-specific primers (16S rDNA, ftsZ, and wsp) in conjunction with the polymerase chain reaction (PCR), cloning and sequencing to study the infection of fruit flies (Anastrepha spp. and Ceratitis capitata) by Wolbachia. The flies were collected at several localities in Brazil and at Guayaquil, Ecuador. All of the fruit flies studied were infected with Wolbachia supergroup A, in agreement with the high prevalence of this group in South America. Phylogenetic analysis showed that the wsp gene was the most sensitive gene for studying the relationships among Wolbachia strains. The Wolbachia sequences detected in these fruit flies were similar to those such as wMel reported for other fruit flies. These results show that the infection of Anastrepha fruit flies by Wolbachia is much more widespread than previously thought.

Rose, an Eye Color Mutation in a Species of the Anastrepha fraterculus Complex (Diptera: Tephritidae)

Abstract The rose eye color mutation is described in Anastrepha sp.1 aff. fraterculus. It changes the color of the wild-type eyes from reddish with a blue-green iridescence to yellow-orange with a rosy iridescence. The wild-type and rose phenotypes are distinguishable in dried individuals at least 2–3 wk after death. Rose is transmitted as a recessive autosomal mutation. Disruption of the ommatidial pattern was observed as an alignment irregularity and enlargement of some individual ommatidia. Mutant individuals have reduced fitness relative to the wild-type. The rose mutation, the first one described in Anastrepha, has potential use in release-recapture studies and as a marker for mass-reared flies.