Kostas D. Mathiopoulos

Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: ascaridoidea) defined by polymerase-chain-reaction-based restriction fragment length polymorphism.

Polymerase-chain-reaction-based restriction fragment length polymorphism analysis was performed to establish genetic markers in rDNA, for the identification of the three sibling species of the Anisakis simplex complex and morphologically differentiated Anisakis species, i.e. Anisakis physeteris, Anisakis schupakovi, Anisakis typica and Anisakis ziphidarum. Different restriction patterns were found between A. simplex sensu stricto and Anisakis pegreffii with two of the restriction endonucleases used (HinfI and TaqI), between A. simplex sensu stricto and A. simplex C with one endonuclease (HhaI), and between A. simplex C and Aniskis pegreffii with three endonucleases (HhaI, HinfI and TaqI), while no variation in patterns was detected among individuals within each species. The species A. physeteris, A. schupakovi, A. typica and A. ziphidarum were found to be different from each other and different from the three sibling species of the A. simplex complex by distinct fragments using 10-12 of the endonucleases tested. The polymorphisms obtained by restriction fragment length polymorphisms have provided a new set of genetic markers for the accurate identification of sibling species and morphospecies.

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.

Spinosad resistance development in wild olive fruit fly Bactrocera oleae (Diptera: Tephritidae) populations in California

BACKGROUND Among target pests of the insecticide spinosad is the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae). In Cyprus, spinosad has been sporadically used since its registration in 2002, whereas in Greece its use has been very limited since its registration in 2004, particularly in biological olive cultivars in Crete. By contrast, in California it has been the only insecticide used against the olive fruit fly since its registration in 2004. This study aimed at examining the resistance status of the olive fruit fly to spinosad. RESULTS Populations from California, Greece and Cyprus, plus a laboratory population, were tested. Bioassays were performed by oral or topical application of different concentrations of the insecticide. Cypriot populations demonstrated no resistance as compared with that of the laboratory population. Among the Greek populations, only one from Crete demonstrated a fourfold increase in resistance, whereas five populations from California demonstrated a 9-13-fold increase. CONCLUSION The observed resistance increase was associated with spinosad applications in the respective areas. These values are relatively low and do not yet pose a serious control problem in the field. However, the observed variation documents that spinosad tolerance has increased in areas where the insecticide has been more extensively used.

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.

A T2504A mutation in the 23S rRNA gene responsible for high-level resistance to linezolid of Staphylococcus epidermidis

Department of Microbiology, Medical School, University of Thessalia, Larissa, Greece; Department of Molecular Microbiology, Institute of BioMedical Research and Technology, Larissa, Greece; Department of Biochemistry and Biotechnology, University of Thessalia, Larissa, Greece; Department of Microbiology, ‘Sismanoglion’ General Hospital of Athens, Athens, Greece; Department of Microbiology, School of Medicine, University of Patras, Patras, Greece

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control. The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A high-quality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT. The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution.

A small deletion in the olive fly acetylcholinesterase gene associated with high levels of organophosphate resistance

Organophosphate resistance in the olive fly was previously shown to associate with two point mutations in the ace gene. The frequency of these mutations was monitored in Bactrocera oleae individuals of increasing resistance. In spite of the difference in resistance among the individuals, there was no correlation between mutation frequencies and resistance level, indicating that other factors may contribute to this variation. The search for additional mutations in the ace gene of highly resistant insects revealed a small deletion at the carboxyl terminal of the protein (termed Delta3Q). Significant correlation was shown between the mutation frequency and resistance level in natural populations. In addition, remaining activity of acetylcholinesterase enzyme (AChE) after dimethoate inhibition was higher in genotypes carrying the mutation. These results strongly suggest a role of Delta3Q in high levels of organophosphate (OP) resistance. Interestingly, the carboxyl terminal of AChE is normally cleaved and substituted by a glycosylphosphatidylinositol (GPI) anchor. We hypothesize that Delta3Q may improve GPI anchoring, thus increasing the amount of AChE that reaches the synaptic cleft. In this way, despite the presence of insecticide, enough enzyme would remain in the cleft for its normal role of acetylcholine hydrolysis, allowing the insect to survive. This provides a previously un-described mechanism of resistance.

Organophosphosphate resistance-related mutations in the acetylcholinesterase gene of Tephritidae

Tephritid insects comprise one of the most devastating families of insect pests worldwide. Organophosphate insecticides have been extensively used for their control during the last 40 years. Inevitably, this has led to the development of resistance. Acetylcholinesterase is the principal target of organophosphates (OP) and, consequently, its gene is the most likely locus where resistance mutations appear. In this review, OP resistance‐associated mutations in Tephritidae are described. Apart from the mutations that lie in the catalytic centre of the enzyme, those that lie at the C‐terminal of the protein are of particular interest as they indicate a possible new mechanism of resistance. Nonetheless, the number of resistance‐associated mutations in the catalytic centre of the Tephritidae ace gene is low, a fact that could be attributed to the fitness cost that any other mutations may have.

Constructing and screening cDNA libraries

The in-depth study of eukaryotic gene structure and expression has been made possible by the ability to clone transcribed DNA. Since RNA per se cannot be cloned (i.e. stably introduced and replicated in a bacterial cell) and RNA-DNA hybrids can only be cloned with very low efficiency, the best choice for the isolation of mRNA is its conversion into double-stranded cDNA. Thus, a good strategy for the isolation of a particular eukaryotic gene is the screening of a cDNA library representing the mRNA population of the appropriate tissue or cell type using an available nucleic acid as a probe. Alternatively, the clone of interest can be identified by virtue of the expression of a specific protein by the target mRNA. In the first case, the hybridization probe could be an existing cloned gene from the same or another species, an oligonucleotide or a pool of oligonucleotides (Suggs et al., 1981), or a PCR product (Lee et al., 1988). In the second approach expressed proteins can be detected by specific antibodies (Young and Davis, 1983b) or by functional assays that determine their biological activity (Yang et al., 1986).

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.