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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

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

<p>Abstract</p> <p>Background</p> <p>In the tephritids <it>Ceratitis</it>, <it>Bactrocera </it>and <it>Anastrepha</it>, the gene <it>transformer </it>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 <it>transformer-2 </it>in the tephritids has only been undertaken in <it>Ceratitis</it>, and it has been shown that its function is required for the female-specific splicing of <it>doublesex </it>and <it>transformer </it>pre-mRNA. It therefore participates in <it>transformer </it>auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus <it>Anastrepha </it>was undertaken in order to throw light on the evolution of <it>transformer-2</it>.</p> <p>Results</p> <p>The gene <it>transformer-2 </it>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 <it>Drosophila</it>. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of <it>Anastrepha transformer-2 </it>dsRNA into <it>Anastrepha </it>embryos caused a change in the splicing pattern of the endogenous <it>transformer </it>and <it>doublesex </it>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 <it>Anastrepha </it>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.</p> <p>Conclusions</p> <p>These results indicate that <it>transformer-2 </it>is required for sex determination in <it>Anastrepha </it>through its participation in the female-specific splicing of <it>transformer </it>and <it>doublesex </it>pre-mRNAs. It is therefore needed for the auto-regulation of the gene <it>transformer</it>. Thus, the <it>transformer/transfomer-2 > doublesex </it>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 <it>tra </it>is just another component of the sex determination gene cascade regulated by <it>Sex-lethal</it>). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for <it>Sex-lethal</it>, modified it, and converted it into the key gene controlling sex determination.</p

Running title: Distribution of Anastrepha fraterculus complex

19 p.-7 fig.-4 tab.Three species of the Anastrepha fraterculuscomplex (AF complex) occur in eastern Brazil, namely Anastrepha sp.1aff. fraterculus, Anastrepha sp.2aff. fraterculus, and Anastrepha sp.3aff. fraterculus. Along their extensive distribution, which differs in relation to geographic topography, they present differences in the host fruit usage in some zones of sympatry. The dynamics underlying the extant distribution pattern of the three species of the AF complex in eastern Brazil was herein analysed by a novel application of the generalized logical formalism methodology. The present report illustrates that two factors, the uni-directional incompatibility between sp1and sp2, with the former overriding the second, besides a yet unknown environmental factor related to altitude, playan instructional role in the generation of the extant distribution pattern observed in nature. Simulations about temporal order of invasion of species in a given area showed that the outcome is independent of the order of arrival of the species. This kind of approach has also a potential to make predictions about the population dynamics after the introduction of species, or the eradication of species in a given area.The study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP2016/00782-7) to DS. LS received a financial support from the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES/PROEX-2016) for a short stay at the Instituto de Biociências, Universidade de São Paulo, Brazil.Peer reviewe

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 A. 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 A. fraterculus complex