What Can Integrated Analysis of Morphological and Genetic Data Still Reveal about the Anastrepha fraterculus (Diptera: Tephritidae) Cryptic Species Complex?

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.

Wolbachia strains in cryptic species of the Anastrepha fraterculus complex (Diptera, Tephritidae) along the Neotropical Region.

Infection by Wolbachia was described previously in eleven species of Anastrepha fruit flies some of which are important pests of fruticulture. One such species is the nominal Anastrepha fraterculus, the South American fruit fly, which actually comprises a complex of cryptic species. The suggestions of using Wolbachia for the control of these pest species, make imperative a more precise characterization of the existing strains of the bacteria. In this study, population samples of the A. fraterculus complex from Brazil, Argentina, Peru, Ecuador, Colombia, Guatemala and Mexico were analyzed for Wolbachia infection. The bacteria were genotyped by the MLST and WSP Typing methodologies. All samples were infected with Wolbachia of supergroup "A". For each of the five MLST genes, unique as well as already known alleles were detected. Nineteen sequence types for the concatenated sequences of the five MLST genes, and twenty wsp alleles were found in the samples. Host-specific haplotypes, shared strains among distinct hosts, and more than one strain of Wolbachia were found in some population samples. Recombination among the MLST genes and intragenic recombination between wsp haplotypes was rare. Phylogenetic analysis showed a great similarity among the Wolbachia strains in the A. fraterculus complex. However, some strains of Wolbachia are found throughout the Neotropical Region and there are specific strains in determined geographical areas.

Patterns of inner chorion structure in Anastrepha (Diptera: Tephritidae) eggs.

The inner chorion structure of Anastrepha eggs from 16 species of various infrageneric taxonomic groups is described by scanning and transmission electron microscopy. The layers of the chorion, the outer egg membrane, are structurally similar. Furthermore, an additional trabecular layer (ATL) that exists in some species, together with other characteristics, facilitates the recognition of four patterns of chorion structuring: Pattern I, in which the ATL layer is absent, is found in Anastrepha amita, the Anastrepha fraterculus complex, Anastrepha obliqua, Anastrepha sororcula, Anastrepha suspensa and Anastrepha zenildae (fraterculus group), and Anastrepha bistrigata and Anastrepha striata (striata group); Pattern II in Anastrepha serpentina (serpentina group), Anastrepha grandis (grandis group) and Anastrepha pseudoparallela (pseudoparallela group), in which the ATL presents large open spaces with pillars; Pattern III, found in Anastrepha consobrina (pseudoparallela group), in which the ATL is composed of round cavities; and Pattern IV, found in Anastrepha alveata and Anastrepha pickeli (spatulata group), where the large ATL cavities are reticulated. Comparatively, the chorion structure in Anastrepha eggs is more complex than in eggs of other fruit flies, e.g., Bactrocera, Rhagoletis and Ceratitis.

Wolbachia in guilds of Anastrepha fruit flies (Tephritidae) and parasitoid wasps (Braconidae).

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.

Morphometric study of third-instar larvae from five morphotypes of the Anastrepha fraterculus cryptic species complex (Diptera, Tephritidae).

The occurrence of cryptic species among economically important fruit flies strongly affects the development of management tactics for these pests. Tools for studying cryptic species not only facilitate evolutionary and systematic studies, but they also provide support for fruit fly management and quarantine activities. Previous studies have shown that the South American fruit fly, Anastrepha fraterculus, is a complex of cryptic species, but few studies have been performed on the morphology of its immature stages. An analysis of mandible shape and linear morphometric variability was applied to third-instar larvae of five morphotypes of the Anastrepha fraterculus complex: Mexican, Andean, Ecuadorian, Peruvian and Brazilian-1. Outline geometric morphometry was used to study the mouth hook shape and linear morphometry analysis was performed using 24 linear measurements of the body, cephalopharyngeal skeleton, mouth hook and hypopharyngeal sclerite. Different morphotypes were grouped accurately using canonical discriminant analyses of both the geometric and linear morphometry. The shape of the mandible differed among the morphotypes, and the anterior spiracle length, number of tubules of the anterior spiracle, length and height of the mouth hook and length of the cephalopharyngeal skeleton were the most significant variables in the linear morphometric analysis. Third-instar larvae provide useful characters for studies of cryptic species in the Anastrepha fraterculus complex.

Morphometric divergence in populations of Anastrepha obliqua (Diptera, Tephritidae) from Colombia and some Neotropical locations.

The West Indian fruit fly, Anastrepha obliqua, is one of seven species of quarantine importance of its genus and is one of the most economically important fruit fly pests in Colombia. The taxonomic status of this species is a key issue for further implementation of any pest management program. Several molecular studies have shown enough variability within Anastrepha obliqua to suggest its taxonomic status could be revised; however, there are no morphological studies supporting this hypothesis. The aim of this work was to describe the morphological variability of Colombian populations of Anastrepha obliqua, comparing this variability with that of other samples from the Neotropics. Measurements were performed on individuals from 11 populations collected from different geographic Colombian localities and were compared with populations from Mexico (2), Dominica Island (1), Peru (1) and Brazil (2). Linear morphometric analyses were performed using 23 female morphological traits, including seven variables of the aculeus, three of the thorax, and six of the wing; seven ratios among them were also considered. Discriminant function analyses showed significant morphological differentiation among the Colombian populations, separating them into two groups. Furthermore, in the comparisons between Colombian samples with those from other countries, three clusters were observed. The possibility of finding more than one species within the nominal Anastrepha obliqua population is discussed.

Variations in the sensitivity of different primers for detecting Wolbachia in Anastrepha (diptera: tephritidae)

Wolbachia are endosymbiont bacteria of the family Rickettsiacea that are widespread in invertebrates and occur between 20 percent and 60 percent of Neotropical insects. These bacteria are responsible for reproductive phenomena such as cytoplasmic incompatibility, male killing, feminization and parthenogenesis. Supergroups A and B of Wolbachia are common in insects and can be identified using primers for 16S rDNA, ftsZ and wsp; these primers vary in their ability to detect Wolbachia. The ftsZ primer was the first primer used to detect Wolbachia in Anastrepha fruit flies. The primers for 16S rDNA, ftsZ and wsp and the corresponding PCR conditions have been optimized to study the distribution of Wolbachia and their effect on the biology of Anastrepha in Brazil. In this work, we examined the ability of these primers to detect Wolbachia in Anastrepha populations from three regions in the State of São Paulo, southeastern Brazil. All of the samples were positive for Wolbachia supergroup A when screened with primers for 16S A rDNA and wsp A; the wsp B primer also gave a positive result, indicating cross-reactivity. The ftsZ primer showed a poor ability to detect Wolbachia in Anastrepha and generated false negatives in 44.9 percent of the samples. These findings indicate that reliable PCR detection of Wolbachia requires the use of primers for 16S rDNA and wsp to avoid cross-reactions and false negatives, and that the ftsZ primer needs to be redesigned to improve its selectivity.

Variations in the sensitivity of different primers for detecting Wolbachia in Anastrepha (diptera: tephritidae).

Wolbachia are endosymbiont bacteria of the family Rickettsiacea that are widespread in invertebrates and occur between 20% and 60% of Neotropical insects. These bacteria are responsible for reproductive phenomena such as cytoplasmic incompatibility, male killing, feminization and parthenogenesis. Supergroups A and B of Wolbachia are common in insects and can be identified using primers for 16S rDNA, ftsZ and wsp; these primers vary in their ability to detect Wolbachia. The ftsZ primer was the first primer used to detect Wolbachia in Anastrepha fruit flies. The primers for 16S rDNA, ftsZ and wsp and the corresponding PCR conditions have been optimized to study the distribution of Wolbachia and their effect on the biology of Anastrepha in Brazil. In this work, we examined the ability of these primers to detect Wolbachia in Anastrepha populations from three regions in the State of São Paulo, southeastern Brazil. All of the samples were positive for Wolbachia supergroup A when screened with primers for 16S A rDNA and wsp A; the wsp B primer also gave a positive result, indicating cross-reactivity. The ftsZ primer showed a poor ability to detect Wolbachia in Anastrepha and generated false negatives in 44.9% of the samples. These findings indicate that reliable PCR detection of Wolbachia requires the use of primers for 16S rDNA and wsp to avoid cross-reactions and false negatives, and that the ftsZ primer needs to be redesigned to improve its selectivity.

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects.

BACKGROUND: In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides 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 gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. RESULTS: The gene transformer-2 produces 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 in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-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 that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements 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 which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.

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.

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.

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.

Karyotype relationships among Anastrepha bistrigata, A. striata and A. serpentina (Diptera, Tephritidae)

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.

Asymmetry of frontal bristles and postocular setae in species and hybrids of the Anastrepha fraterculus complex (Diptera, Tephritidae)

Asymmetry of the frontal bristles and postocular setae was studied in samples from natural populations and laboratory colonies of Anastrepha sp. 1 aff. fraterculus, of A. sp. 2 aff. fraterculus, and in F1 hybrids obtained from laboratory reciprocal crosses. Natural populations were sampled in a zone of sympatry and in two geographically distant regions with different climatic conditions. Asymmetry was scored as the differences between the number of bristles and of setae on the right and left sides of the head, males and females analyzed independently. The two traits exhibited variability according to the model of fluctuating asymmetry (FA). No significant differences among samples were found in the FA of frontal bristles. A significant FA was observed for the postocular setae of A. sp. 1 males from a southern population (Vacaria, RS) as compared to the asymmetry exhibited by males and females of some other samples. No significant differences in FA were observed among the interspecific hybrids and the laboratory samples of both parental species. The higher FA found in the males from Vacaria was attributed to climatic conditions prevailing in that region. The absence of a higher FA in hybrids may be related to the relatively recent evolutionary history of the two species.

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.

Bactérias Wolbachia foram detectadas por PCR seguido de seqüenciamento de um segmento do gene ribossomal 16S em uma amostra de população natural e em duas colônias de laboratório de Ceratitis capitata (Wied.) do Brasil. O seqüenciamento do fragmento amplificado mostrou que este é compatível com os de Wolbachia tipo A, encontrada em numerosas espécies de insetos. Esta é a primeira descrição de uma infecção natural desse hospedeiro por Wolbachia desde que, em linhagens de C. capitata provenientes de outras regiões, não foram encontradas essas bactérias.

The gene doublesex of the fruit fly Anastrepha obliqua (Diptera, Tephritidae).

The gene doublesex of Anastrepha obliqua is composed of four instead of the usual six exons. It is transcribed in both sexes and its primary transcript undergoes sex-specific splicing, producing female Dsx(F) and male Dsx(M) proteins, which have in common the amino-terminal region but which differ at the carboxyl-terminal region.

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.

O estudo apresenta a descrição dos cromossomos mitóticos, através do bandamento-C, de cinco espécies de Anastrepha, A. amita Zucchi, A. turpiniae Stone, A. zenildae Zucchi, A. grandis (Macquart) and A. leptozona Hendel e uma reanálise dos carótipos de três outras espécies, A. distincta Greene, A. obliqua (Macquart) and A. sororcula Zucchi, segundo a mesma técnica. As espécies apresentam número diplóide igual a 12 cromossomos, exceto A. leptozona com 2n = 10. Os cromossomos sexuais são heteromórficos e com sistema XX/XY. Os autossomos são meta ou submetacêntricos em A. grandis and A. leptozona e acrocêntricos nas demais espécies. Os cromossomos sexuais apresentam comprimento e padrão de blocos de heterocromatina característicos para cada espécie.

Evaluation of egg morphology from ethanol preserved females of Anastrepha sp.2 aff. fraterculus (Diptera: Tephritidae) / Avaliação da morfologia dos ovos de fêmeas de Anastrepha sp.2 aff. fraterculus (Diptera: Tephritidae) preservadas em etanol

A morfologia dos ovos de Anastrepha sp.2 aff. fraterculus foi avaliada em amostras de ovaríolos maduros obtidos de fêmeas preservadas em etanol, de fêmeas recém-mortas e comparadacom a de ovos recém-depositados. As características da ornamentação do córion, assim como da estrutura interna dos ovos recém-depositados também puderam ser observadas nos ovos dissecados das fêmeas. Fêmeas preservadas em etanol podem ser utilizadas para análise da morfologia dos ovos, possibilitando a inclusão de espécies, cujos ovos não são obtidos facilmente, nesse tipo de análise.

Features of eggshell morphology in Anastrepha sp.2 aff. fraterculus were evaluated in mature ovariole ova dissected from females preserved in ethanol, and from ovaries of fresh killedfemales in comparison to that of freshly laid eggs. The characteristics of sculpturing as well as of internal structure of the chorion in oviposited eggs could be observed in both samples of mature ovariole ova. Ethanol preserved females may be used as a source of eggs for morphological studies allowing this kind of analysis to species whose eggs are not readily obtained.

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

Neste trabalho foram avaliados alguns aspectos do isolamento reprodutivo entre populaçöes alopátricas de duas espécies do complexo Anastrepha fraterculus (A. fraterculus sp. 1 e sp. 2) em condiçöes de laboratório. A maioria dos cruzamentos intraespecíficos, assim como aqueles entre fêmeas da sp. 2 e machos de sp. 1, foram férteis. No cruzamento recíproco, somente 41.7 por cento resultaram em progênies viáveis. A taxa de eclosäo das larvas foi similar para as duas espécies, mas significativamente menor para os cruzamentos intraespecíficos. A proporçäo sexual da progênie adulta foi de 1:1 tanto nos cruzamentos intraespecíficos quanto naqueles de fêmeas de sp. 1 com machos de sp. 2. Entretanto, nos cruzamentos entre fêmeas de sp. 2 com machos de sp. 1 houve um desvio significativo na proporçäo sexual das progênies, com um excesso de fêmeas. Esta observaçäo está de acordo com a regra de Haldane, cuja ocorrência reflete a existência de isolamento reprodutivo pós-zigótico. Os resultados reforçam a sugestäo da existência de dois taxa distintos em A. fraterculus.