Abstract 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.

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.

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.

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.

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.

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.

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.

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.

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 comparada com 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 killed females 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.

Neste trabalho foram avaliados alguns aspectos do isolamento reprodutivo entre populações alopátricas de duas espécies do complexo Anastrepha fraterculus (A. fraterculus sp. 1 e sp. 2) em condições 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% resultaram em progênies viáveis. A taxa de eclosão das larvas foi similar para as duas espécies, mas significativamente menor para os cruzamentos interespecíficos. A proporção 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 proporção sexual das progênies, com um excesso de fêmeas. Esta observação está de acordo com a regra de Haldane, cuja ocorrência reflete a existência de isolamento reprodutivo pós-zigótico. Os resultados reforçam a sugestão da existência de dois taxa distintos em A. fraterculus.

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.

Técnicas descritas anteriormente por Canovai et al. (Caryologia 47: 241-247, 1994), que produzem bandas C e ASG nos cromossomos mitóticos de Ceratitis capitata, foram testadas com os cromossomos de várias espécies de Anastrepha. O número de metáfases analisáveis aumentou significativamente quando, além dos gânglios cerebrais, discos imaginais foram utilizados nas preparações. O bandamento tipo C produzido foi bastante conspícuo, permitindo inclusive a resolução de pequenos blocos de heterocromatina. O método ASG produziu uma fração significativa de metáfases cujos cromossomos apresentavam um nítido bandamento do tipo G, que permitiu diferenciar cada cromossomo do cariótipo. Essas técnicas simples, aplicadas sem a necessidade de equipamento especial, podem ser úteis para estudos de variabilidade cariotípica intra ou inter-específica

Methods previously described by Canovai et al. (Caryologia 47: 241-247, 1994) which produced C and ASG bands in mitotic chromosomes of Ceratitis capitata were applied to the chromosomes of several Anastrepha species. Metaphase plate yield was substantially increased by use of imaginal disks together with cerebral ganglia. The C-bands were quite prominent allowing the resolution of tiny blocks of heterochromatin. The ASG method produced G-like banded chromosomes, which permitted recognition of each individual chromosome. These simple techniques do not require special equipment and may be valuable for karyotype variability studies in fruit flies and other Diptera

As duas espécies crípticas de Anastrepha fraterculus, tipo I e tipo II, diferiram quanto ao tamanho dos ovos, os quais são menores no tipo I. Em ambos pode ocorrer a extrusão de vitelo pelas extremidades dos embriões, observando-se quatro classes: 1) embriões com massas extrudadas por ambos os pólos; 2) extrusão apenas pelo pólo anterior; 3) extrusão pelo pólo posterior e 4) embriões que não eliminam as massas. As freqüências dessas classes são similares entre populações de um mesmo tipo, mas diferem entre as duas formas crípticas. Diferentes fêmeas podem produzir diferentes classes de embriões, mas para cada fêmea o padrão é constante e não varia durante um longo período do ciclo de oviposição. O diâmetro das massas extrudadas é variável, mas de igual magnitude para os dois tipos. Fêmeas distintas podem produzir massas de diferentes diâmetros que não se alteram em suas progênies. Os resultados sugerem que componentes genéticos estão envolvidos no controle da extrusão de vitelo pelos embriões de Anastrepha. As larvas, de ambos os tipos, apresentam comportamento não usual pouco antes da eclosão: ingerem a massa de vitelo do pólo anterior, giram dentro do ovo ficando com a cabeça no pólo posterior, sugam a massa de vitelo do pólo posterior e eclodem perto desse pólo

Variations in egg length were observed for two populations of cryptic species of Anastrepha fraterculus (Wiedemann). The eggs of type I flies were smaller than those of type II individuals. For both types, in regard to yolk mass extrusion, four classes of embryos were detected. Class 1: embryos that extrude masses at both extremities; class 2: embryos in which extrusion occurs only at the anterior pole; class 3: embryos that eliminate mass only at the posterior pole, and class 4: embryos that do not extrude any mass. Embryo class frequencies were similar for populations belonging to the same type, but different between types. Individual females may produce eggs from different embryo classes, but for any given female the pattern remains constant during a long period of oviposition. Variation in size of the extruded masses was similar for both populations. Individual females produced embryos with a small range of mass diameters, and different females produced masses of different mean size. However, individual mass size remained constant during oviposition. The results suggest the existence of genetic components involved in the control of this unusual process. Larvae of both types presented, just before eclosion, similar unusual behaviors: they ingest the anterior extruded mass, rotate 180°, absorb the posterior mass and eclose near the posterior pole. Data show that cryptic A. fraterculus type I and type II differs in regard to egg size as well as to the phenomenon of yolk mass extrusion

Existem poucos estudos envolvendo testes de isolamento reprodutivo entre espécies do gênero Anastrepha. Neste trabalho apresentamos os resultados de testes feitos em laboratório com duas espécies relacionadas, A. striata e A. bistrigata. Nos cruzamentos interespecíficos foram observadas tentativas de cópula, mas o exame das espermatecas mostrou que não houve transferência de espermatozóides. Não ocorreram cruzamentos mesmo quando o período de contato entre as duas espécies foi prolongado. Os resultados sugerem a existência de isolamento prézigótico entre as espécies. A análise do padrão de atividade de cópula ao longo do dia, em condições de laboratório, pela primeira vez descrito para A. bistrigata, mostrou a existência de diferenças acentuadas, sendo que em A. striata as atividades estão concentradas no período da tarde, enquanto que, em A. bistrigata, no período da manhã. O isolamento reprodutivo entre estas duas espécies, aqui caracterizado, pode ser explicado pelas diferenças no comportamento e nos períodos de atividade de cópula

The reproductive isolation between two closely related species, Anastrepha bistrigata and A. striata, was studied in the laboratory. Interespecific copulation attempts were observed, but examination of the spermathecae showed that sperm transference did not occur, even after a prolonged period of contact between the mating pairs. These results indicate prezygotic isolation. The analysis of the hourly distribution of mating activities under laboratory conditions, here described for the first time for A. bistrigata, clearly showed differences for the two species, the activities being concentrated in the afternoon period for A. striata and in the morning for A. bistrigata