Antonios A. Augustinos

Analysis of olive fly invasion in California based on microsatellite markers

The olive fruit fly, Bactrocera oleae, is the main pest of the olive fruit and its expansion is exclusively restricted to the cultivation zone of the olive tree. Even though olive production has a century-old history in California, the olive fly was first detected in the Los Angeles area in 1998. Within 5 years of the first observation, the insect was reported from all olive cultivation areas of the state. Field-collected flies from five locations in California and another from Israel were analyzed on the basis of microsatellite polymorphisms in 10 microsatellite loci. These results were integrated with those of a previous study of olive fly populations around the European part of the Mediterranean basin. The analysis pointed to the eastern part of the Mediterranean as the putative source of the observed invasion. Moreover, samples from California were quite different from Mediterranean samples implying the participation of phenomena such as genetic drift during the invasion and expansion of the olive fly in California.

Microsatellite analysis of olive fly populations in the Mediterranean indicates a westward expansion of the species.

Bactrocera oleae is the major insect pest of the olive fruit. Twelve microsatellite loci isolated from the genome of this insect were used in a Mediterranean-wide population analysis. These loci were highly polymorphic with a mean number of alleles per locus of 10.42 and a mean effective number of alleles of 2.76. The analysis was performed on a sample of 671 flies collected from nineteen locations around the European part of the Mediterranean basin. Despite the high level of gene flow across the Mediterranean, results support the notion of a differentiation of three subpopulations: one of the Iberian Peninsula, one of Greece and Italy and one of Cyprus. In addition, the gradual decrease of heterozygosity from the Eastern to the Western part of the Mediterranean indicates a westward expansion of the species.

Exploitation of the Medfly Gut Microbiota for the Enhancement of Sterile Insect Technique: Use of Enterobacter sp. in Larval Diet-Based Probiotic Applications

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a pest of worldwide substantial economic importance, as well as a Tephritidae model for sterile insect technique (SIT) applications. The latter is partially due to the development and utilization of genetic sexing strains (GSS) for this species, such as the Vienna 8 strain, which is currently used in mass rearing facilities worldwide. Improving the performance of such a strain both in mass rearing facilities and in the field could significantly enhance the efficacy of SIT and reduce operational costs. Recent studies have suggested that the manipulation of gut symbionts can have a significant positive effect on the overall fitness of insect strains. We used culture-based approaches to isolate and characterize gut-associated bacterial species of the Vienna 8 strain under mass rearing conditions. We also exploited one of the isolated bacterial species, Enterobacter sp., as dietary supplement (probiotic) to the larval diet, and we assessed its effects on fitness parameters under the standard operating procedures used in SIT operational programs. Probiotic application of Enterobacter sp. resulted in improvement of both pupal and adult productivity, as well as reduced rearing duration, particularly for males, without affecting pupal weight, sex ratio, male mating competitiveness, flight ability and longevity under starvation.

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

BackgroundThe Tephritidae family of insects includes the most important agricultural pests of fruits and vegetables, belonging mainly to four genera (Bactrocera, Ceratitis, Anastrepha and Rhagoletis). The olive fruit fly, Bactrocera oleae, 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 insects 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.ResultsFifty-eight microsatellite-containing clones were isolated from the olive fly, Bactrocera oleae, 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 in situ 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 Bactrocera genus.ConclusionMicrosatellite 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.

Isolation, annotation and applications of expressed sequence tags from the olive fly, Bactrocera oleae

The olive fruit fly, Bactrocera oleae, is the major pest of the olive tree. Despite its importance, very little genetic and molecular knowledge is available. The present study is a first attempt to identify and characterize B. oleae expressed sequence tags (ESTs). One hundred and ninety-five randomly selected cDNA clones were isolated and the obtained sequences were annotated through BLASTX similarity searches. A set of 159 unique putative transcripts were functionally assigned using Gene Ontology terms in broad categories of biological process, molecular function and cellular component based on D. melanogaster matches. Moreover, the cytogenetic location of 35 ESTs was determined by in situ hybridization to B. oleae polytene chromosomes. The resulting low-resolution EST map more than doubles the available entry points to the insect’s genome and can assist syntenic comparisons with other distant species. The deduced codon usage of the isolated ESTs suggested a conserved pattern of B. oleae with its closest relatives. Additionally, the comparative analysis of B. oleae ESTs with the homologous D. melanogaster genes led to the development of 17 nuclear EPIC-PCR markers for the amplification of intron sequences of 11 Tephritidae species. Sequencing analysis of several cross-amplified intron sequences revealed a high degree of conservation among Bactrocera species and a varying transferability of the generated markers across the examined genera, suggesting that this method can provide a useful tool for the clarification of phylogenetic relationships among different species, particularly in cases of species complexes.

Mitotic and polytene chromosomes analysis of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

The Oriental fruit fly, Batrocera dorsalis s.s. (Hendel) is one of the most destructive agricultural pests, belonging to a large group of difficult to distinguish morphologically species, referred as the B. dorsalis complex. We report here a cytogenetic analysis of two laboratory strains of the species and provide a photographic polytene chromosome map from larval salivary glands. The mitotic complement consists of six chromosome pairs including a heteromorphic sex (XX/XY) chromosome pair. Analysis of the polytene complement has shown a total of five polytene chromosomes (10 polytene arms) that correspond to the five autosomes. The most important landmarks of each polytene chromosome and characteristic asynapsis at a specific chromosomal region are presented and discussed. Chromosomal homology between B. dorsalis and Ceratitis capitata has been determined by comparing chromosome banding patterns. The detection of chromosome inversions in both B. dorsalis strains is shown and discussed. Our results show that the polytene maps presented here are suitable for cytogenetic analysis of this species and can be used for comparative studies among species of the Tephritidae family. They also provide a diagnostic tool that could accelerate species identification within the B. dorsalis complex and could shed light on the ongoing speciation in this complex. Polytene chromosome maps can facilitate the development of biological control methods and support the genome mapping project of the species that is currently in progress.

Genetic changes during laboratory domestication of an olive fly SIT strain

Early attempts to apply the SIT on the olive fly, Bactrocera oleae, were unsuccessful, mainly due to the low competitiveness of the sterile mass‐reared males compared with the wild ones. Recently, new efforts are underway in Israel to develop a vigorous and efficient mass‐reared olive fly laboratory strain. To understand the genetics of the adaptation process and possibly link the corresponding loss of competitiveness to genetic markers, we followed the fluctuation of genotypic frequencies of ten microsatellite markers during the course of 22 generations of the colonization of a wild Israeli population in laboratory conditions. Effective and observed allele number is halved after 11 generations, so is mean heterozygosity. Practically, there is very little change between F0 and F1, there are substantial changes between F1 and F2–F5, and there is a virtual complete adaptation to the new laboratory environment by F11, because no more changes are observed between F11 and F22. If we assume that the loss of allele number and heterozygosity also reflects the loss of the ‘wild’ character of the colonized strain and, possibly, the loss of a substantial part of its natural vigour, our results indicate that there is an apparent need to refresh a mass‐reared colony with wild material at about every five to eight generations. Furthermore, simulation models indicated that while most of the observed allele frequency fluctuations were due to random drift, some alleles were probably under selection.

Genetic and cytogenetic analysis of the American cherry fruit fly, Rhagoletis cingulata (Diptera: Tephritidae)

The American eastern cherry fruit fly, Rhagoletis cingulata, a pest of cherries in the western hemisphere, invaded Europe in 1983, and since then dispersed to several European countries. Information on the genetics and cytogenetics of this pest is very scarce. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of R. cingulata are presented here. The mitotic metaphase complement consists of six pairs of chromosomes with the sex chromosomes being very small and similar in size. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei and an extrachromosomal heterochromatic mass, which corresponds to the sex chromosomes. The banding patterns and the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between R. cingulata, R. completa and R. cerasi are also proposed, based on the comparison of chromosome banding patterns. Furthermore, the detection and characterization of Wolbachia pipientis in the R. cingulata population studied is presented and the potential correlation with the asynaptic phenomena found in its polytene complement is discussed. In addition, 10 out of 24 microsatellite markers developed for other Rhagoletis species are cross-amplified, evaluated and proposed as useful markers for population and genetic studies in R. cingulata.

Cross-amplified microsatellites in the European cherry fly, Rhagoletis cerasi : medium polymorphic–highly informative markers

The European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), is a major pest of cherries in Europe and parts of Asia. Despite its big economic significance, there is a lack of studies on the genetic structure of its natural populations. Knowledge about an insect pest on molecular, genetic and population levels facilitates the development of environmentally friendly control methods. In this study, we present the development of 13 microsatellite markers for R. cerasi, through cross-species amplification. These markers have been used for the genotyping of 130 individuals from five different sampling sites in Greece. Our results indicate that (i) cross-species amplification is a versatile and rapid tool for developing microsatellite markers in Rhagoletis spp., (ii) the microsatellite markers presented here constitute an important tool for population studies on this pest, and (iii) there is clear structuring of natural European cherry fly populations.

Microsatellite and Wolbachia analysis in Rhagoletis cerasi natural populations: population structuring and multiple infections

Rhagoletis cerasi (Diptera: Tephritidae) is a major pest of sweet and sour cherries in Europe and parts of Asia. Despite its economic significance, there is a lack of studies on the genetic structure of R. cerasi populations. Elucidating the genetic structure of insects of economic importance is crucial for developing phenological-predictive models and environmental friendly control methods. All natural populations of R. cerasi have been found to harbor the endosymbiont Wolbachia pipientis, which widely affects multiple biological traits contributing to the evolution of its hosts, and has been suggested as a tool for the biological control of insect pests and disease vectors. In the current study, the analysis of 18 R. cerasi populations collected in Greece, Germany, and Russia using 13 microsatellite markers revealed structuring of R. cerasi natural populations, even at close geographic range. We also analyzed the Wolbachia infection status of these populations using 16S rRNA-, MLST- and wsp-based approaches. All 244 individuals screened were positive for Wolbachia. Our results suggest the fixation of the wCer1 strain in Greece while wCer2, wCer4, wCer5, and probably other uncharacterized strains were also detected in multiply infected individuals. The role of Wolbachia and its potential extended phenotypes needs a thorough investigation in R. cerasi. Our data suggest an involvement of this symbiont in the observed restriction in the gene flow in addition to a number of different ecological factors.