Old residents and new arrivals of Rhagoletis species in Europe.

The genus Rhagoletis (Diptera: Tephritidae) comprises more than 65 species distributed throughout Europe, Asia and America, including many species of high economic importance. Currently, there are three Rhagoletis species that infest fruits and nuts in Europe. The European cherry fruit fly, Rhagoletis cerasi (may have invaded Europe a long time ago from the Caucasian area of West Asia), and two invasive species (recently introduced from North America): the eastern American cherry fruit fly, R. cingulata, and the walnut husk fly, R. completa. The presence of different Rhagoletis species may enhance population dynamics and establish an unpredictable economic risk for several fruit and nut crops in Europe. Despite their excessive economic importance, little is known on population dynamics, genetics and symbiotic associations for making sound pest control decisions in terms of species-specific, environmental friendly pest control methods. To this end, the current paper (a) summarizes recently accumulated genetic and population data for the European Rhagoletis species and their association with the endosymbiont Wolbachia pipientis, and (b) explores the possibility of using the current knowledge for implementing the innovative biological control methods of sterile insect technique and incompatible insect technique.

Tetracycline-suppressible female lethality and sterility in the Mexican fruit fly, Anastrepha ludens.

The sterile insect technique (SIT) involves the mass release of sterile males to suppress insect pest populations. SIT has been improved for larval pests by the development of strains for female-specific tetracycline-suppressible (Tet-off) embryonic lethal systems for male-only populations. Here we describe the extension of this approach to the Mexican fruit fly, Anastrepha ludens, using a Tet-off driver construct with the Tet-transactivator (tTA) under embryo-specific Anastrepha suspensa serendipity α (As-sry-α) promoter regulation. In the absence of tetracycline, tTA acts upon a Tet-response element linked to the pro-apoptotic cell death gene lethal effector, head involuation defective (hid), from A. ludens (Alhid(Ala2) ) that contains a sex-specific intron splicing cassette, resulting in female-specific expression of the lethal effector. Parental adults double-homozygous for the driver/effector vectors were expected to yield male-only progeny when reared on Tet-free diet, but a complete lack of oviposited eggs resulted for each of the three strains tested. Ovary dissection revealed nonvitellogenic oocytes in all strains that was reversible by feeding females tetracycline for 5 days after eclosion, resulting in male-only adults in one strain. Presumably the sry-α promoter exhibits prezygotic maternal expression as well as zygotic embryonic expression in A. ludens, resulting in a Tet-off sterility effect in addition to female-specific lethality.

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans.

Wolbachia manipulate insect host biology through a variety of means that result in increased production of infected females, enhancing its own transmission. A Wolbachia strain (wInn) naturally infecting Drosophila innubila induces male killing, while native strains of D. melanogaster and D. simulans usually induce cytoplasmic incompatibility (CI). In this study, we transferred wInn to D. melanogaster and D. simulans by embryonic microinjection, expecting conservation of the male-killing phenotype to the novel hosts, which are more suitable for genetic analysis. In contrast to our expectations, there was no effect on offspring sex ratio. Furthermore, no CI was observed in the transinfected flies. Overall, transinfected D. melanogaster lines displayed lower transmission rate and lower densities of Wolbachia than transinfected D. simulans lines, in which established infections were transmitted with near-perfect fidelity. In D. simulans, strain wInn had no effect on fecundity and egg-to-adult development. Surprisingly, one of the two transinfected lines tested showed increased longevity. We discuss our results in the context of host-symbiont co-evolution and the potential of symbionts to invade novel host species.

Population genetic structure and secondary endosymbionts of Q Bemisia tabaci (Hemiptera: Aleyrodidae) from Greece.

We investigated the molecular diversity of the major agricultural pest Bemisia tabaci and of its associated secondary endosymbionts in Greece. Analyzing mitochondrial DNA, we found that the Q1 (=Q west) is predominant. We used eight microsatellite polymorphic markers to study the genetic structure of 37 populations from mainland and insular Greece, collected on different host species from outdoor and protected crops as well as from non-cultivated plants. In some cases, gene flow was found to be low even between populations separated by just a few kilometres. Bayesian analysis identified two main genetic groups, the first encompassing populations from south Crete and the second composed of populations from north Crete, two other Aegean islands and mainland Greece. Genetic differentiation was not correlated with different host plant species or habitat, or greenhouse versus open environment populations. Gene flow significantly decreased with geographic distance, but no isolation by distance existed when only the samples from mainland Greece or only the samples from Crete were considered. The secondary symbionts Wolbachia and Hamiltonella were present at high frequencies while Arsenophonus, Cardinium and Rickettsia were absent from Greek populations. Multilocus sequence typing of Wolbachia identified two Wolbachia strains. These two strains were found together in most of the populations studied but never in the same host individual. Their role on the observed population structure is discussed.

Cytogenetic analysis of the Ethiopian fruit fly Dacus ciliatus (Diptera: Tephritidae).

The Ethiopian fruit fly, Dacus ciliatus, is an important pest of cucurbits, which recently invaded the Middle East. The genetics and cytogenetics of D. ciliatus have been scarcely studied. Such information is, however, an essential basis for understanding the biology of insect pests, as well as for the design of modern control strategies. We report here the mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this species. The mitotic metaphase complement consists of six pairs of chromosomes, including one pair of heteromorphic sex (XX/XY) chromosomes. The heterogametic sex is ascribed to the male. 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 a heterochromatic mass corresponding to the sex chromosomes. Banding patterns, as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between D. ciliatus and Bactrocera oleae are proposed by comparing chromosome banding patterns and by in situ hybridization of the hsp70 gene.

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.

Combining sterile and incompatible insect techniques for the population suppression of Drosophila suzukii.

The spotted wing Drosophila, Drosophila suzukii, has recently invaded Europe and the Americas, and it is a major threat for a wide variety of commercial soft fruits both in open field and greenhouse production systems. D. suzukii infests a wide range of ripening fruits, leading to substantial yield and revenue losses. As the application of insecticides close to the harvest period poses great concerns, the development of an efficient environment-friendly control approach to fight D. suzukii is necessary. In this study, we exploited the sterile insect technique (SIT) in combination with Wolbachia symbiosis as a population suppression approach that can constitute a potential component of an area-wide integrated pest management program. We aimed to establish a combined SIT/incompatible insect technique (IIT) protocol that would require lower irradiation doses as a complementary tool for D. suzukii management. Two D. suzukii lines trans-infected with the Wolbachia wHa and wTei strains were irradiated at doses four times less than usual (e.g., 45 Gy), and the egg hatching and adult emergence were determined. Our results indicated that wHa and wTei females as well as wHa males were sterile at this low dose. The longevity, adult emergence and flight ability of adults were evaluated, and no major effect caused by irradiation was detected. Our data indicate that a SIT/IIT protocol can be a competent approach for D. suzukii management.

Challenging the Wigglesworthia, Sodalis, Wolbachia symbiosis dogma in tsetse flies: Spiroplasma is present in both laboratory and natural populations.

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the "Wigglesworthia-Sodalis-Wolbachia dogma" operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G. tachinoides, members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density in G. f. fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G. f. fuscipes.

Wolbachia infection and cytoplasmic incompatibility in Drosophila species.

Forty-one stocks from 30 Drosophila species were surveyed for Wolbachia infection using PCR technology. D. sechellia and two strains of D. auraria were found to be infected and were tested for the expression of cytoplasmic incompatibility, along with D. ananassae and D. melanogaster strains, which are already known to be infected. D. ananassae and D. melanogaster show levels of incompatibility up to 25%, while D. auraria and D. sechellia exhibit levels of egg mortality approximately 60%. A dot-blot assay using the dnaA sequence as probe was developed to assess the infection levels in individual males that were used in incompatibility crosses. A positive correlation between bacterial density and cytoplasmic incompatibility was observed. The stocks examined can be clustered into at least two groups, depending on the levels of infection relative to the degree of cytoplasmic incompatibility exhibited. One group, containing D. simulans Hawaii, D. sechellia, and D. auraria, exhibits high levels of cytoplasmic incompatibility relative to levels of infection; all the other species and D. simulans Riverside exhibit significantly lower levels of cytoplasmic incompatibility relative to levels of infection. These data show that, in addition to bacterial density, bacterial and/ or host factors also affect the expression of cytoplasmic incompatibility.

Wolbachia transfer from Drosophila melanogaster into D. simulans: Host effect and cytoplasmic incompatibility relationships.

Wolbachia are maternally transmitted endocellular bacteria causing a reproductive incompatibility called cytoplasmic incompatibility (CI) in several arthropod species, including Drosophila. CI results in embryonic mortality in incompatible crosses. The only bacterial strain known to infect Drosophila melanogaster (wDm) was transferred from a D. melanogaster isofemale line into uninfected D. simulans isofemale lines by embryo microinjections. Males from the resulting transinfected lines induce >98% embryonic mortality when crossed with uninfected D. simulans females. In contrast, males from the donor D. melanogaster line induce only 18-32% CI on average when crossed with uninfected D. melanogaster females. Transinfected D. simulans lines do not differ from the D. melanogaster donor line in the Wolbachia load found in the embryo or in the total bacterial load of young males. However, >80% of cysts are infected by Wolbachia in the testes of young transinfected males, whereas only 8% of cysts are infected in young males from the D. melanogaster donor isofemale line. This difference might be caused by physiological differences between hosts, but it might also involve tissue-specific control of Wolbachia density by D. melanogaster. The wDm-transinfected D. simulans lines are unidirectionally incompatible with strains infected by the non-CI expressor Wolbachia strains wKi, wMau, or wAu, and they are bidirectionally incompatible with strains infected by the CI-expressor Wolbachia strains wHa or wNo. However, wDm-infected males do not induce CI toward females infected by the CI-expressor strain wRi, which is found in D. simulans continental populations, while wRi-infected males induce partial CI toward wDm-infected females. This peculiar asymmetrical pattern could reflect an ongoing divergence between the CI mechanisms of wRi and wDm. It would also confirm other results indicating that the factor responsible for CI induction in males is distinct from the factor responsible for CI rescue in females.

Wolbachia infections are distributed throughout insect somatic and germ line tissues.

Wolbachia are intracellular microorganisms that form maternally-inherited infections within numerous arthropod species. These bacteria have drawn much attention, due in part to the reproductive alterations that they induce in their hosts including cytoplasmic incompatibility (CI), feminization and parthenogenesis. Although Wolbachia's presence within insect reproductive tissues has been well described, relatively few studies have examined the extent to which Wolbachia infects other tissues. We have examined Wolbachia tissue tropism in a number of representative insect hosts by western blot, dot blot hybridization and diagnostic PCR. Results from these studies indicate that Wolbachia are much more widely distributed in host tissues than previously appreciated. Furthermore, the distribution of Wolbachia in somatic tissues varied between different Wolbachia/host associations. Some associations showed Wolbachia disseminated throughout most tissues while others appeared to be much more restricted, being predominantly limited to the reproductive tissues. We discuss the relevance of these infection patterns to the evolution of Wolbachia/host symbioses and to potential applied uses of Wolbachia.

Taxonomic status of the intracellular bacterium Wolbachia pipientis.

Wolbachia pipientis is a maternally inherited, intracellular bacterium found in more than 20 % of all insects, as well as numerous other arthropods and filarial nematodes. It has been the subject of a growing number of studies in recent decades, because of the remarkable effects it has on its arthropod hosts, its potential as a tool for biological control of arthropods of agricultural and medical importance and its use as a target for treatment of filariasis. W. pipientis was originally discovered in cells of the mosquito Culex pipiens and is the only formally described member of the genus. Molecular sequence-based studies have revealed a number of phylogenetically diverse strains of W. pipientis. Owing to uncertainty about whether W. pipientis comprises more than one species, researchers in the field now commonly refer to W. pipientis simply as Wolbachia. In this note, we briefly review higher-level phylogenetic and recombination studies of W. pipientis and propose that all the intracellular symbionts known to cluster closely with the type strain of W. pipientis, including those in the currently recognized supergroups (A-H), are officially given this name.

Kinetics of pure cultures of hydrogen-oxidizing denitrifying bacteria and modeling of the interactions among them in mixed cultures.

In this study we report the isolation of four denitrifying bacteria from a batch reactor, where the progress of hydrogenotrophic denitrification was examined. Only three of the strains had the ability to use hydrogen as electron donor. In the present work, kinetic batch experiments were carried out in order to study the dynamic characteristics of pure and defined mixed cultures of hydrogen-oxidizing denitrifying bacteria, under anoxic conditions, in a defined synthetic medium, in the presence of nitrates. Kinetic models were developed and the kinetic parameters were determined from the batch experiments for each bacterium separately. The behavior of mixed cultures and the interactions between the bacteria were described using kinetic models based on the kinetic models developed for each bacterium separately and their predictions were compared with the results from mixed culture experiments. The mathematical models that were developed and validated in the present work are capable of describing the behavior of the bacteria in pure and mixed cultures, and in particular, the kinetics of nitrate and nitrite reduction and cell growth.

Extrachromosomal DNA of the symbiont Sodalis glossinidius.

The extrachromosomal DNA of Sodalis glossinidius from two tsetse fly species was sequenced and contained four circular elements: three plasmids, pSG1 (82 kb), pSG2 (27 kb), and pSG4 (11 kb), and a bacteriophage-like pSG3 (19 kb) element. The information suggests S. glossinidius is evolving towards an obligate association with tsetse flies.

Cytogenetic analysis of three genetic sexing strains of Ceratitits capitata.

Polytene chromosomes of three genetic sexing strains of Ceratitis capitata were analyzed. The genetic sexing mechanism is based on a pupal color dimorphism (white-brown) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the w locus (white pupal case). The analyzed polytene chromosomes were derived from two different pupal tissues, the orbital bristle and fat body cells. The Y chromosome is visible in both tissues, while the autosomes present a different banding pattern. Based on these features, the autosome breakpoints in the three Y; autosome translocations were mapped, and the homology of the translocated autosome in both tissues was established. In addition, the location of the break-points was compared to the stability of these three strains.

Integumental phosphatase isoenzymes from white puparia of Ceratitis capitata: isolation and characterization.

Two specific alkaline phosphatase forms were identified in the integument of wild-type Ceratitis capitata during transition of larvae to pupae. The separation was achieved by DEAE-cellulose chromatography; alkaline phosphatase 1 and alkaline phosphatase 2 were eluted in 0.1 and 0.4 M KCl, respectively. Both isoenzymes have a molecular weight of approximately 180,000. The pH curve reveals two peaks for both alkaline phosphatases: one at 9.4 and the other at 11.0. The two isoenzymes at both pH optima catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate, but not phosphoserine, phosphothreonine, ATP, or AMP. However, at pH 9.4, alkaline phosphatase 1 is more effective than ALPase 2 and exhibits a preference for phosphotyrosine. The divalent cations Mn2+, Mg2+, and Ba2+ activate the enzymes, while Cu2+ and Zn2+ are inhibitors for both isoenzymes. Both isoenzymes are inactivated by EDTA. The effect of amino acids on enzyme activity was also tested. Alkaline phosphatase 1 is inhibited by L-tyrosine, while alkaline phosphatase 2 is unaffected. L-Phenylalanine has no effect on either isoenzyme. Both isoenzymes are inhibited by urea and 2-mercaptoethanol. Simultaneous addition of urea and 2-mercaptoethanol reveals that ALPase 1 is more sensitive to these inhibitors than ALPase 2.

Wolbachia neither induces nor suppresses transcripts encoding antimicrobial peptides.

Wolbachia are intracellular maternally inherited microorganisms that are associated with reproductive abnormalities such as cytoplasmic incompatibility (CI), feminization and parthenogenesis in the various arthropod species they infect. Surveys indicate that these bacteria infect more than 16% of all insect species as well as isopods, mites and nematodes, making Wolbachia one of the most ubiquitous parasites yet described. However, nothing is known about the interactions of this bacterium with the host's immune system. We studied the expression of inducible antimicrobial markers in the adults of two Wolbachia infected insect species, Drosophila simulans and Aedes albopictus. The lack of available immune markers in the mosquito species led us to clone part of the defensin gene from this species, which was found to be very similar to the other mosquito defensins cloned from Anopheles gambiae and Aedes aegypti. Comparisons of the expression pattern of the antibacterial markers between Wolbachia-infected and cured lines, and also between bacteria-challenged and unchallenged adults indicated that Wolbachia does not either constitutively induce or suppress the transcription of these antibacterial genes. In addition, no difference in the transcription of these genes was found between double and single Wolbachia-infected strains or between strains in which Wolbachia has different tissue tropisms.

Biochemical and genetic studies on alkaline phosphatase of Ceratitis capitata.

Two forms of alkaline phosphatase exist in the integument of the "white pupae" (wp) and dark pupae (dp) mutant strains of Ceratitis capitata, during transition from larvae to pupae. They were separated by DEAE-cellulose chromatography. Both isoenzymes have a molecular weight of approximately 180,000 and two pH optima, at 9.4 and at 11.0. The isoenzymes of the "dark pupae" mutant catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate but not phosphoserine, phosphothreonine, ATP, and AMP. In contrast, the isoenzymes of the white pupae mutant hydrolyze all the substrates tested. The ALPase 1 of the dark pupae mutant was inhibited by L-tyrosine, but L-phenylalanine had no effect on either isoenzyme. The effects of divalent cations, EDTA, temperature, urea, and 2-mercaptoethanol were also investigated. Electrophoretic analysis did not reveal any variants of the larval and pupal isoenzymes, but ALPase A, an adult stage-specific isoenzyme, was found to be polymorphic. The electrophoretic variants were shown to be controlled by three codominant alleles located on the third chromosome of Ceratitis capitata. Since we found no hybrid enzyme, we conclude that ALPase A is monomeric.

A prokaryotic dnaA sequence in Drosophila melanogaster: Wolbachia infection and cytoplasmic incompatibility among laboratory strains.

Using oligonucleotide primers derived from the aligned polypeptide sequences of several prokaryotic dnaA genes, we amplified from Drosophila melanogaster DNA a 557 bp fragment containing a single open reading frame. The predicted peptide sequence shows a significant similarity to previously characterized protein sequences that are encoded by the dnaA genes of several prokaryotes. The dnaA sequences are also detectable by PCR in DNA from Drosophila simulans and Nasonia vitripennis flies which are infected by a symbiotic bacterium assigned to the type species Wolbachia pipientis. A tetracycline treatment that eradicates bacterial parasites from insects, abolishes the dnaA sequences from Drosophila and Nasonia DNA. In addition, dnaA-positive Drosophila melanogaster contain numerous rod-shaped bacteria in embryos, which are abolished in subsequent generations after treatment with tetracycline. Combined with phylogenetic analysis of DnaA and 16S rRNA sequences, these results show that the dnaA cognate comes from Wolbachia. A survey of Drosophila stocks using PCR amplification of dnaA and 16S rRNA sequences showed that Wolbachia is widely spread among D. melanogaster laboratory strains but absent from several established strains of the Mediterranean fruit fly Ceratitis capitata. Evidence is also presented that presence of the bacterium can cause partial cytoplasmic incompatibility between infected and non-infected D. melanogaster strains.

Gene organization of the dnaA region of Wolbachia.

The dnaA region of Wolbachia, an intracellular bacterial parasite of insects, is unique. A glnA cognate was found upstream of the dnaA gene, while neither of the two open reading frames detected downstream of dnaA has any homologue in the database. This unusual gene arrangement may reflect requirements associated with the unique ecological niche this agent occupies.