Isolation and characterization of microsatellite markers from the olive fly, Bactrocera oleae, and their cross-species amplification in the Tephritidae family

<p>Abstract</p> <p>Background</p> <p>The Tephritidae family of insects includes the most important agricultural pests of fruits and vegetables, belonging mainly to four genera (<it>Bactrocera, Ceratitis, Anastrepha </it>and <it>Rhagoletis</it>). The olive fruit fly, <it>Bactrocera oleae</it>, is the major pest of the olive fruit. Currently, its control is based on chemical insecticides. Environmentally friendlier methods have been attempted in the past (Sterile Insect Technique), albeit with limited success. This was mainly attributed to the lack of knowledge on the insect's behaviour, ecology and genetic structure of natural populations. The development of molecular markers could facilitate the access in the genome and contribute to the solution of the aforementioned problems. We chose to focus on microsatellite markers due to their abundance in the genome, high degree of polymorphism and easiness of isolation.</p> <p>Results</p> <p>Fifty-eight microsatellite-containing clones were isolated from the olive fly, <it>Bactrocera oleae</it>, bearing a total of sixty-two discrete microsatellite motifs. Forty-two primer pairs were designed on the unique sequences flanking the microsatellite motif and thirty-one of them amplified a PCR product of the expected size. The level of polymorphism was evaluated against wild and laboratory flies and the majority of the markers (93.5%) proved highly polymorphic. Thirteen of them presented a unique position on the olive fly polytene chromosomes by <it>in situ </it>hybridization, which can serve as anchors to correlate future genetic and cytological maps of the species, as well as entry points to the genome. Cross-species amplification of these markers to eleven Tephritidae species and sequencing of thirty-one of the amplified products revealed a varying degree of conservation that declines outside the <it>Bactrocera </it>genus.</p> <p>Conclusion</p> <p>Microsatellite markers are very powerful tools for genetic and population analyses, particularly in species deprived of any other means of genetic analysis. The presented set of microsatellite markers possesses all features that would render them useful in such analyses. This could also prove helpful for species where SIT is a desired outcome, since the development of effective SIT can be aided by detailed knowledge at the genetic and molecular level. Furthermore, their presented efficacy in several other species of the Tephritidae family not only makes them useful for their analysis but also provides tools for phylogenetic comparisons among them.</p

Dihydroisoxazole inhibitors of Anopheles gambiae seminal transglutaminase AgTG3

Background: Current vector-based malaria control strategies are threatened by the rise of biochemical and behavioural resistance in mosquitoes. Researching mosquito traits of immunity and fertility is required to find potential targets for new vector control strategies. The seminal transglutaminase AgTG3 coagulates male Anopheles gambiae seminal fluids, forming a ‘mating plug’ that is required for male reproductive success. Inhibitors of AgTG3 can be useful both as chemical probes of A. gambiae reproductive biology and may further the development of new chemosterilants for mosquito population control. Methods: A targeted library of 3-bromo-4,5-dihydroxoisoxazole inhibitors were synthesized and screened for inhibition of AgTG3 in a fluorescent, plate-based assay. Positive hits were tested for in vitro activity using cross-linking and mass spectrometry, and in vivo efficacy in laboratory mating assays. Results: A targeted chemical library was screened for inhibition of AgTG3 in a fluorescent plate-based assay using its native substrate, plugin. Several inhibitors were identified with IC50 < 10 μM. Preliminary structure-activity relationships within the library support the stereo-specificity and preference for aromatic substituents in the chemical scaffold. Both inhibition of plugin cross-linking and covalent modification of the active site cysteine of AgTG3 were verified. Administration of an AgTG3 inhibitor to A. gambiae males by intrathoracic injection led to a 15% reduction in mating plug transfer in laboratory mating assays. Conclusions: A targeted screen has identified chemical inhibitors of A. gambiae transglutaminase 3 (AgTG3). The most potent inhibitors are known inhibitors of human transglutaminase 2, suggesting a common binding pose may exist within the active site of both enzymes. Future efforts to develop additional inhibitors will provide chemical tools to address important biological questions regarding the role of the A. gambiae mating plug. A second use for transglutaminase inhibitors exists for the study of haemolymph coagulation and immune responses to wound healing in insects

COMPOSITIONS FOR CONTROLLING MOSQUITO POPULATIONS

Methods are disclosed herein for at least one of (i) substantially reducing Plasmodium and/or oocyst development, (ii) substantially reducing mating success, (iii) substantially abolishing egg development after blood feeding, (iv) substantially reducing the mean survival rate; (v) substantially reducing at least one of the transmission of mosquito borne pathogens; and (vi) substantially reducing the propensity for mosquito biting comprising contacting adult, female mosquitoes with a composition comprising an effective amount of one or more non-steroidal ecdysone agonists. The one or more non-steroidal ecdysone agonists can be comprised indoor or outdoor surface

Decoding the Reproductive System of the Olive Fruit Fly, Bactrocera oleae

In most diploid organisms, mating is a prerequisite for reproduction and, thus, critical to the maintenance of their population and the perpetuation of the species. Besides the importance of understanding the fundamentals of reproduction, targeting the reproductive success of a pest insect is also a promising method for its control, as a possible manipulation of the reproductive system could affect its destructive activity. Here, we used an integrated approach for the elucidation of the reproductive system and mating procedures of the olive fruit fly, Bactrocera oleae. Initially, we performed a RNAseq analysis in reproductive tissues of virgin and mated insects. A comparison of the transcriptomes resulted in the identification of genes that are differentially expressed after mating. Functional annotation of the genes showed an alteration in the metabolic, catalytic, and cellular processes after mating. Moreover, a functional analysis through RNAi silencing of two differentially expressed genes, yellow-g and troponin C, resulted in a significantly reduced oviposition rate. This study provided a foundation for future investigations into the olive fruit fly’s reproductive biology to the development of new exploitable tools for its control

Mosquito biology. Evolution of sexual traits influencing vectorial capacity in anopheline mosquitoes

The availability of genome sequences from 16 anopheline species provides unprecedented opportunities to study the evolution of reproductive traits relevant for malaria transmission. In Anopheles gambiae, a likely candidate for sexual selection is male 20-hydroxyecdysone (20E). Sexual transfer of this steroid hormone as part of a mating plug dramatically changes female physiological processes intimately tied to vectorial capacity. By combining phenotypic studies with ancestral state reconstructions and phylogenetic analyses, we show that mating plug transfer and male 20E synthesis are both derived characters that have coevolved in anophelines, driving the adaptation of a female 20E-interacting protein that promotes oogenesis via mechanisms also favoring Plasmodium survival. Our data reveal coevolutionary dynamics of reproductive traits between the sexes likely to have shaped the ability of anophelines to transmit malaria

Data from: Evolution of sexual traits influencing vectorial capacity in anopheline mosquitoes

The availability of genome sequences from 16 anopheline species provides unprecedented opportunities to study the evolution of reproductive traits relevant for malaria transmission. In Anopheles gambiae, a likely candidate for sexual selection is male 20-hydroxyecdysone (20E). Sexual transfer of this steroid hormone as part of a mating plug dramatically changes female physiological processes intimately tied to vectorial capacity. By combining phenotypic studies with ancestral state reconstructions and phylogenetic analyses, we show that mating plug transfer and male 20E synthesis are both derived characters that have coevolved in anophelines, driving the adaptation of a female 20E-interacting protein that promotes oogenesis via mechanisms also favoring Plasmodium survival. Our data reveal coevolutionary dynamics of reproductive traits between the sexes likely to have shaped the ability of anophelines to transmit malaria

Fig S1 Species phylogeny based on single copy orthologs_protein sequence alignment

The concatenated protein sequence alignments of nine anopheline and two outgroup dipteran species (created using MUSCLE and trimmed with trimAl) of 4,829 relaxed single-copy orthologs (a maximum of three paralogs allowed in no more than two species, longest protein selected) determined by OrthoDB orthology delineation (available at http://cegg.unige.ch/orthodbmoz2), which resulted in 3,400,861 amino acid columns (with 906,126 distinct alignment patterns)

Implementing the new p&amp;rtnership CVCP's review of action following the Dearing Report

SIGLEAvailable from British Library Document Supply Centre-DSC:98/10017 / BLDSC - British Library Document Supply CentreGBUnited Kingdo