Microbiome of pear psyllids: A tale about closely related species sharing their endosymbionts.

Psyllids are phloem-feeding insects that can transmit plant pathogens such as phytoplasmas, intracellular bacteria causing numerous plant diseases worldwide. Their microbiomes are essential for insect physiology and may also influence the capacity of vectors to transmit pathogens. Using 16S rRNA gene metabarcoding, we compared the microbiomes of three sympatric psyllid species associated with pear trees in Central Europe. All three species are able to transmit 'Candidatus Phytoplasma pyri', albeit with different efficiencies. Our results revealed potential relationships between insect biology and microbiome composition that varied during psyllid ontogeny and between generations in Cacopsylla pyri and C. pyricola, as well as between localities in C. pyri. In contrast, no variations related to psyllid life cycle and geography were detected in C. pyrisuga. In addition to the primary endosymbiont Carsonella ruddii, we detected another highly abundant endosymbiont (unclassified Enterobacteriaceae). C. pyri and C. pyricola shared the same taxon of Enterobacteriaceae which is related to endosymbionts harboured by other psyllid species from various families. In contrast, C. pyrisuga carried a different Enterobacteriaceae taxon related to the genus Sodalis. Our study provides new insights into host-symbiont interactions in psyllids and highlights the importance of host biology and geography in shaping microbiome structure.

Investigating the microbial community of Cacopsylla spp. as potential factor in vector competence of phytoplasma.

Phytoplasmas are obligatory intracellular bacteria that colonize the phloem of many plant species and cause hundreds of plant diseases worldwide. In nature, phytoplasmas are primarily transmitted by hemipteran vectors. While all phloem-feeding insects could in principle transmit phytoplasmas, only a limited number of species have been confirmed as vectors. Knowledge about factors that might determine the vector capacity is currently scarce. Here, we characterized the microbiomes of vector and non-vector species of apple proliferation (AP) phytoplasma 'Candidatus Phytoplasma mali' to investigate their potential role in the vector capacity of the host. We performed high-throughput 16S rRNA metabarcoding of the two principal AP-vectors Cacopsylla picta and Cacopsylla melanoneura and eight Cacopsylla species, which are not AP-vectors but co-occur in apple orchards. The microbiomes of all species are dominated by Carsonella, the primary endosymbiont of psyllids and a second uncharacterized Enterobacteriaceae endosymbiont. Each Cacopsylla species harboured a species-specific phylotype of both symbionts. Moreover, we investigated differences between the microbiomes of AP-vector versus non-vector species and identified the predominant endosymbionts but also Wolbachia and several minor taxa as potential indicator species. Our study highlights the importance of considering the microbiome in future investigations of potential factors influencing host vector competence. We investigated the potential role of symbiotic bacteria in the acquisition and transmission of phytoplasma. By comparing the two main psyillid vector species of Apple proliferation (AP) phytoplasma and eight co-occurring species, which are not able to vector AP-phytoplasma, we found differences in the microbial communities of AP-vector and non-vector species, which appear to be driven by the predominant symbionts in both vector species and Wolbachia and several minor taxa in the non-vector species. In contrast, infection with AP-phytoplasma did not affect microbiome composition in both vector species. Our study provides new insights into the endosymbiont diversity of Cacopsylla spp. and highlights the importance of considering the microbiome when investigating potential factors influencing host vector competence.

Testing the potential contribution of Wolbachia to speciation when cytoplasmic incompatibility becomes associated with host-related reproductive isolation.

Endosymbiont-induced cytoplasmic incompatibility (CI) may play an important role in arthropod speciation. However, whether CI consistently becomes associated or coupled with other host-related forms of reproductive isolation (RI) to impede the transfer of endosymbionts between hybridizing populations and further the divergence process remains an open question. Here, we show that varying degrees of pre- and postmating RI exist among allopatric populations of two interbreeding cherry-infesting tephritid fruit flies (Rhagoletis cingulata and R. indifferens) across North America. These flies display allochronic and sexual isolation among populations, as well as unidirectional reductions in egg hatch in hybrid crosses involving southwestern USA males. All populations are infected by a Wolbachia strain, wCin2, whereas a second strain, wCin3, only co-infects flies from the southwest USA and Mexico. Strain wCin3 is associated with a unique mitochondrial DNA haplotype and unidirectional postmating RI, implicating the strain as the cause of CI. When coupled with nonendosymbiont RI barriers, we estimate the strength of CI associated with wCin3 would not prevent the strain from introgressing from infected southwestern to uninfected populations elsewhere in the USA if populations were to come into secondary contact and hybridize. In contrast, cytoplasmic-nuclear coupling may impede the transfer of wCin3 if Mexican and USA populations were to come into contact. We discuss our results in the context of the general paucity of examples demonstrating stable Wolbachia hybrid zones and whether the spread of Wolbachia among taxa can be constrained in natural hybrid zones long enough for the endosymbiont to participate in speciation.

Genomically correlated trait combinations and antagonistic selection contributing to counterintuitive genetic patterns of adaptive diapause divergence in Rhagoletis flies.

Adaptation to novel environments can result in unanticipated genomic responses to selection. Here, we illustrate how multifarious, correlational selection helps explain a counterintuitive pattern of genetic divergence between the recently derived apple- and ancestral hawthorn-infesting host races of Rhagoletis pomonella (Diptera: Tephritidae). The apple host race terminates diapause and emerges as adults earlier in the season than the hawthorn host race, to coincide with the earlier fruiting phenology of their apple hosts. However, alleles at many loci associated with later emergence paradoxically occur at higher frequencies in sympatric populations of the apple compared to the hawthorn race. We present genomic evidence that historical selection over geographically varying environmental gradients across North America generated genetic correlations between two life history traits, diapause intensity and diapause termination, in the hawthorn host race. Moreover, the loci associated with these life history traits are concentrated in genomic regions in high linkage disequilibrium (LD). These genetic correlations are antagonistic to contemporary selection on local apple host race populations that favours increased initial diapause depth and earlier, not later, diapause termination. Thus, the paradox of apple flies appears due, in part, to pleiotropy or linkage of alleles associated with later adult emergence and increased initial diapause intensity, the latter trait strongly selected for by the earlier phenology of apples. Our results demonstrate how understanding of multivariate trait combinations and the correlative nature of selective forces acting on them can improve predictions concerning adaptive evolution and help explain seemingly counterintuitive patterns of genetic diversity in nature.

Comparative genome sequencing reveals insights into the dynamics of Wolbachia in native and invasive cherry fruit flies.

Wolbachia is a maternally inherited obligate endosymbiont that can induce a wide spectrum of effects in its host, ranging from mutualism to reproductive parasitism. At the genomic level, recombination within and between strains, transposable elements, and horizontal transfer of strains between host species make Wolbachia an evolutionarily dynamic bacterial system. The invasive cherry fruit fly Rhagoletis cingulata arrived in Europe from North America ~40 years ago, where it now co-occurs with the native cherry pest R. cerasi. This shared distribution has been proposed to have led to the horizontal transfer of different Wolbachia strains between the two species. To better understand transmission dynamics, we performed a comparative genome study of the strain wCin2 in its native United States and invasive European populations of R. cingulata with wCer2 in European R. cerasi. Previous multilocus sequence genotyping (MLST) of six genes implied that the source of wCer2 in R. cerasi was wCin2 from R. cingulata. However, we report genomic evidence discounting the recent horizontal transfer hypothesis for the origin of wCer2. Despite near identical sequences for the MLST markers, substantial sequence differences for other loci were found between wCer2 and wCin2, as well as structural rearrangements, and differences in prophage, repetitive element, gene content, and cytoplasmic incompatibility inducing genes. Our study highlights the need for whole-genome sequencing rather than relying on MLST markers for resolving Wolbachia strains and assessing their evolutionary dynamics.

Population genetics of the brown marmorated stink bug Halyomorpha halys in the early phase of invasion in South Tyrol (Northern Italy).

The brown marmorated stink bug Halyomorpha halys is one of the most harmful invasive species in the world. Native to East Asia, this insect was introduced into North America in the 1990s and into Europe in the 2000s where it subsequently established and spread across the continent. Previous population genetic studies determined the invasion pathways at continental and national levels. However, information on the dynamics on a small-scale is currently scarce. Here we study the genetic diversity and population dynamics of H. halys in South Tyrol, a region in Northern Italy, since its arrival to its widespread establishment over a period of four years. By haplotyping 162 individuals from ten populations (including six previously published individuals) we found a high haplotype diversity in most populations with an increasing diversity across the different years. Most haplotypes were previously found in other regions of Northern Italy, providing evidence for migration from neighboring regions. However, the presence of four previously undescribed haplotypes as well as a haplotype previously found exclusively in Greece highlights additional long-distance dispersal across the continent. Phylogenetic analysis of the haplotypes found in South Tyrol showed that the majority of haplotypes clustered with haplotypes predominantly found in Japan. This suggests a potential recent introduction of H. halys individuals from Japan into Europe, and thus an additional invasion pathway that was previously unidentified.

Host plant-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi.

Elucidating the mechanisms and conditions facilitating the formation of biodiversity are central topics in evolutionary biology. A growing number of studies imply that divergent ecological selection may often play a critical role in speciation by counteracting the homogenising effects of gene flow. Several examples involve phytophagous insects, where divergent selection pressures associated with host plant shifts may generate reproductive isolation, promoting speciation. Here, we use ddRADseq to assess the population structure and to test for host-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi (L., 1758) (Diptera: Tephritidae). This tephritid is distributed throughout Europe and western Asia, and has adapted to two different genera of host plants, Prunus spp. (cherries) and Lonicera spp. (honeysuckle). Our data imply that geographic distance and geomorphic barriers serve as the primary factors shaping genetic population structure across the species range. Locally, however, flies genetically cluster according to host plant, with consistent allele frequency differences displayed by a subset of loci between Prunus and Lonicera flies across four sites surveyed in Germany and Norway. These 17 loci display significantly higher FST values between host plants than others. They also showed high levels of linkage disequilibrium within and between Prunus and Lonicera flies, supporting host-related selection and reduced gene flow. Our findings support the existence of sympatric host races in R. cerasi embedded within broader patterns of geographic variation in the fly, similar to the related apple maggot, Rhagoletis pomonella, in North America.

Diversity and distribution of Wolbachia in relation to geography, host plant affiliation and life cycle of a heterogonic gall wasp.

BACKGROUND: The maternally inherited endosymbiont Wolbachia is widespread in arthropods and nematodes and can play an important role in the ecology and evolution of its host through reproductive manipulation. Here, we survey Wolbachia in Belonocnema treatae, a widely distributed North American cynipid gall forming wasp that exhibits regional host specialization on three species of oaks and alternation of sexually and asexually reproducing generations. We investigated whether patterns of Wolbachia infection and diversity in B. treatae are associated with the insect's geographic distribution, host plant association, life cycle, and mitochondrial evolutionary history. RESULTS: Screening of 463 individuals from 23 populations including sexual and asexual generations from all three host plants across the southern U.S. showed an average infection rate of 56% with three common Wolbachia strains: wTre1-3 and an additional rare variant wTre4. Phylogenetic analysis based on wsp showed that these strains are unrelated and likely independently inherited. We found no difference in Wolbachia infection frequency among host plant associated populations or between the asexual and sexual generations, or between males and females of the sexual generation. Partially incomplete Wolbachia transmission rates might explain the occurrence of uninfected individuals. A parallel analysis of the mitochondrial cytochrome oxidase I gene in B. treatae showed high mtDNA haplotype diversity in both infected and uninfected populations suggesting an ancestral infection by Wolbachia as well as a clear split between eastern and western B. treatae mtDNA clades with a sequence divergence of > 6%. The strain wTre1 was present almost exclusively in the western clade while wTre2 and wTre3 occur almost exclusively in eastern populations. In contrast, the same strains co-occur as double-infections in Georgia and triple-infections in two populations in central Florida. CONCLUSIONS: The diversity of Wolbachia across geographically and genetically distinct populations of B. treatae and the co-occurrence of the same strains within three populations highlights the complex infection dynamics in this system. Moreover, the association of distinct Wolbachia strains with mitochondrial haplotypes of its host in populations infected by different Wolbachia strains suggests a potential role of the endosymbiont in reproductive isolation in B. treatae.

Spatial spread of Wolbachia in Rhagoletis cerasi populations.

The bacterial endosymbiont Wolbachia has been used to control insect pests owing to its ability to manipulate their life history and suppress infectious diseases. Therefore, knowledge on Wolbachia dynamics in natural populations is fundamental. The European cherry fruit fly, Rhagoletis cerasi, is infected with the Wolbachia strain wCer2, mainly present in southern and central European populations, and is currently spreading into wCer2-uninfected populations driven by high unidirectional cytoplasmic incompatibility. Here, we describe the distribution of wCer2 along two transition zones where the infection is spreading into wCer2-uninfected R. cerasi populations. Fine-scale sampling of 19 populations in the Czech Republic showed a smooth decrease of wCer2 frequency from south to north within a distance of less than 20 km. Sampling of 12 Hungarian populations, however, showed a sharp decline of wCer2 infection frequency within a few kilometres. We fitted a standard wave equation to our empirical data and estimated a Wolbachia wave speed of 1.9 km yr-1 in the Czech Republic and 1.0 km yr-1 in Hungary. Considering the univoltine life cycle and limited dispersal ability of R. cerasi, our study highlights a rapid Wolbachia spread in natural host populations.

Reproductive Manipulators in the Bark Beetle Pityogenes chalcographus (Coleoptera: Curculionidae)-The Role of Cardinium, Rickettsia, Spiroplasma, and Wolbachia.

Heritable bacterial endosymbionts can alter the biology of numerous arthropods. They can influence the reproductive outcome of infected hosts, thus affecting the ecology and evolution of various arthropod species. The spruce bark beetle Pityogenes chalcographus (L.) (Coleoptera: Curculionidae: Scolytinae) was reported to express partial, unidirectional crossing incompatibilities among certain European populations. Knowledge on the background of these findings is lacking; however, bacterial endosymbionts have been assumed to manipulate the reproduction of this beetle. Previous work reported low-density and low-frequency Wolbachia infections of P. chalcographus but found it unlikely that this infection results in reproductive alterations. The aim of this study was to test the hypothesis of an endosymbiont-driven incompatibility, other than Wolbachia, reflected by an infection pattern on a wide geographic scale. We performed a polymerase chain reaction (PCR) screening of 226 individuals from 18 European populations for the presence of the endosymbionts Cardinium, Rickettsia, and Spiroplasma, and additionally screened these individuals for Wolbachia. Positive PCR products were sequenced to characterize these bacteria. Our study shows a low prevalence of these four endosymbionts in P. chalcographus. We detected a yet undescribed Spiroplasma strain in a single individual from Greece. This is the first time that this endosymbiont has been found in a bark beetle. Further, Wolbachia was detected in three beetles from two Scandinavian populations and two new Wolbachia strains were described. None of the individuals analyzed were infected with Cardinium and Rickettsia. The low prevalence of bacteria found here does not support the hypothesis of an endosymbiont-driven reproductive incompatibility in P. chalcographus.

The hitchhiker's guide to Europe: the infection dynamics of an ongoing Wolbachia invasion and mitochondrial selective sweep in Rhagoletis cerasi.

Wolbachia is a maternally inherited and ubiquitous endosymbiont of insects. It can hijack host reproduction by manipulations such as cytoplasmic incompatibility (CI) to enhance vertical transmission. Horizontal transmission of Wolbachia can also result in the colonization of new mitochondrial lineages. In this study, we present a 15-year-long survey of Wolbachia in the cherry fruit fly Rhagoletis cerasi across Europe and the spatiotemporal distribution of two prevalent strains, wCer1 and wCer2, and associated mitochondrial haplotypes in Germany. Across most of Europe, populations consisted of either 100% singly (wCer1) infected individuals with haplotype HT1, or 100% doubly (wCer1&2) infected individuals with haplotype HT2, differentiated only by a single nucleotide polymorphism. In central Germany, singly infected populations were surrounded by transitional populations, consisting of both singly and doubly infected individuals, sandwiched between populations fixed for wCer1&2. Populations with fixed infection status showed perfect association of infection and mitochondria, suggesting a recent CI-driven selective sweep of wCer2 linked with HT2. Spatial analysis revealed a range expansion for wCer2 and a large transition zone in which wCer2 splashes appeared to coalesce into doubly infected populations. Unexpectedly, the transition zone contained a large proportion (22%) of wCer1&2 individuals with HT1, suggesting frequent intraspecific horizontal transmission. However, this horizontal transmission did not break the strict association between infection types and haplotypes in populations outside the transition zone, suggesting that this horizontally acquired Wolbachia infection may be transient. Our study provides new insights into the rarely studied Wolbachia invasion dynamics in field populations.

Identification and characterization of specific motifs in effector proteins of plant parasites using MOnSTER.

Plant pathogens cause billions of dollars of crop loss every year and are a major threat to global food security. Identifying and characterizing pathogens effectors is crucial towards their improved control. Because of their poor sequence conservation, effector identification is challenging, and current methods generate too many candidates without indication for prioritizing experimental studies. In most phyla, effectors contain specific sequence motifs which influence their localization and targets in the plant. Therefore, there is an urgent need to develop bioinformatics tools tailored for pathogen effectors. To circumvent these limitations, we have developed MOnSTER a specific tool that identifies clusters of motifs of protein sequences (CLUMPs). MOnSTER can be fed with motifs identified by de novo tools or from databases such as Pfam and InterProScan. The advantage of MOnSTER is the reduction of motif redundancy by clustering them and associating a score. This score encompasses the physicochemical properties of AAs and the motif occurrences. We built up our method to identify discriminant CLUMPs in oomycetes effectors. Consequently, we applied MOnSTER on plant parasitic nematodes and identified six CLUMPs in about 60% of the known nematode candidate parasitism proteins. Furthermore, we found co-occurrences of CLUMPs with protein domains important for invasion and pathogenicity. The potentiality of this tool goes beyond the effector characterization and can be used to easily cluster motifs and calculate the CLUMP-score on any set of protein sequences.

Divergent evolutionary histories of two sympatric spruce bark beetle species.

Ips typographus and Pityogenes chalcographus are two sympatric Palearctic bark beetle species with wide distribution ranges. As both species are comparable in biology, life history, and habitat, including sharing the same host, Picea abies, they provide excellent models for applying a comparative approach in which to identify common historical patterns of population differentiation and the influence of species-specific ecological characteristics. We analysed patterns of genetic diversity, genetic structure and demographic history of ten I. typographus and P. chalcographus populations co-distributed across Europe using both COI and ITS2 markers. Rather than similarities, our results revealed striking differences. Ips typographus was characterised by low genetic diversity, shallow population structure and strong evidence that all extant haplogroups arose via a single Holocene population expansion event. In contrast, genetic variation and structuring were high in P. chalcographus indicating a longer and more complex evolutionary history. This was estimated to be five times older than I. typographus, beginning during the last Pleistocene glacial maximum over 100 000 years ago. Although the expansions of P. chalcographus haplogroups also date to the Holocene or just prior to its onset, we show that these occurred from at least three geographically separated glacial refugia. Overall, these results suggest that the much longer evolutionary history of P. chalcographus greatly influenced the levels of phylogeographic subdivision among lineages and may have led to the evolution of different life-history traits which in turn have affected genetic structure and resulted in an advantage over the more aggressive I. typographus.

Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe.

The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species boundaries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well-characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolbachia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in several European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some populations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hungary. In summary, our study provides strong evidence for a very recent inter-specific Wolbachia transmission with a subsequent spatial spread in field populations.

Division of labor within psyllids: metagenomics reveals an ancient dual endosymbiosis with metabolic complementarity in the genus Cacopsylla.

IMPORTANCE: Heritable beneficial bacterial endosymbionts have been crucial for the evolutionary success of numerous insects by enabling the exploitation of nutritionally limited food sources. Herein, we describe a previously unknown dual endosymbiosis in the psyllid genus Cacopsylla, consisting of the primary endosymbiont "Candidatus Carsonella ruddii" and a co-occurring Enterobacteriaceae bacterium for which we propose the name "Candidatus Psyllophila symbiotica." Its localization within the bacteriome and its small genome size confirm that Psyllophila is a co-primary endosymbiont widespread within the genus Cacopsylla. Despite its highly eroded genome, Psyllophila perfectly complements the tryptophan biosynthesis pathway that is incomplete in the co-occurring Carsonella. Moreover, the genome of Psyllophila is almost as small as Carsonella's, suggesting an ancient dual endosymbiosis that has now reached a precarious stage where any additional gene loss would make the system collapse. Hence, our results shed light on the dynamic interactions of psyllids and their endosymbionts over evolutionary time.

Seasonal wild dance of dual endosymbionts in the pear psyllid Cacopsylla pyricola (Hemiptera: Psylloidea).

Most sap-feeding insects maintain obligate relationships with endosymbiotic bacteria that provide their hosts with essential nutrients. However, knowledge about the dynamics of endosymbiont titers across seasons in natural host populations is scarce. Here, we used quantitative PCR to investigate the seasonal dynamics of the dual endosymbionts "Candidatus Carsonella ruddii" and "Ca. Psyllophila symbiotica" in a natural population of the pear psyllid Cacopsylla pyricola (Hemiptera: Psylloidea: Psyllidae). Psyllid individuals were collected across an entire year, covering both summer and overwintering generations. Immatures harboured the highest titers of both endosymbionts, while the lowest endosymbiont density was observed in males. The density of Carsonella remained high and relatively stable across the vegetative period of the pear trees, but significantly dropped during the non-vegetative period, overlapping with C. pyricola's reproductive diapause. In contrast, the titer of Psyllophila was consistently higher than Carsonella's and exhibited fluctuations throughout the sampling year, which might be related to host age. Despite a tightly integrated metabolic complementarity between Carsonella and Psyllophila, our findings highlight differences in their density dynamics throughout the year, that might be linked to their metabolic roles at different life stages of the host.

Wolbachia infection dynamics in a natural population of the pear psyllid Cacopsylla pyri (Hemiptera: Psylloidea) across its seasonal generations.

Wolbachia is one of the most abundant intracellular symbionts of arthropods and has profound effects on host biology. Wolbachia transmission and host phenotypes often depend on its density within the host, which can be affected by multiple biotic and abiotic factors. However, very few studies measured Wolbachia density in natural host populations. Here, we describe Wolbachia in the pear psyllid Cacopsylla pyri from three populations in the Czech Republic. Using phylogenetic analyses based on wsp and multilocus sequence typing genes, we demonstrate that C. pyri harbours three new Wolbachia strains from supergroup B. A fourth Wolbachia strain from supergroup A was also detected in parasitised immatures of C. pyri, but likely came from a hymenopteran parasitoid. To obtain insights into natural Wolbachia infection dynamics, we quantified Wolbachia in psyllid individuals from the locality with the highest prevalence across an entire year, spanning several seasonal generations of the host. All tested females were infected and Wolbachia density remained stable across the entire period, suggesting a highly efficient vertical transmission and little influence from the environment and different host generations. In contrast, we observed a tendency towards reduced Wolbachia density in males which may suggest sex-related differences in Wolbachia-psyllid interactions.

Wolbachia-Mitochondrial DNA Associations in Transitional Populations of Rhagoletis cerasi.

The endosymbiont Wolbachia can manipulate arthropod host reproduction by inducing cytoplasmic incompatibility (CI), which results in embryonic mortality when infected males mate with uninfected females. A CI-driven invasion of Wolbachia can result in a selective sweep of associated mitochondrial haplotype. The co-inheritance of Wolbachia and host mitochondrial DNA can therefore provide significant information on the dynamics of an ongoing Wolbachia invasion. Therefore, transition zones (i.e., regions where a Wolbachia strain is currently spreading from infected to uninfected populations) represent an ideal area to investigate the relationship between Wolbachia and host mitochondrial haplotype. Here, we studied Wolbachia-mitochondrial haplotype associations in the European cherry fruit fly, Rhagoletis cerasi, in two transition zones in the Czech Republic and Hungary, where the CI-inducing strain wCer2 is currently spreading. The wCer2-infection status of 881 individuals was compared with the two known R. cerasi mitochondrial haplotypes, HT1 and HT2. In accordance with previous studies, wCer2-uninfected individuals were associated with HT1, and wCer2-infected individuals were mainly associated with HT2. We found misassociations only within the transition zones, where HT2 flies were wCer2-uninfected, suggesting the occurrence of imperfect maternal transmission. We did not find any HT1 flies that were wCer2-infected, suggesting that Wolbachia was not acquired horizontally. Our study provides new insights into the dynamics of the early phase of a Wolbachia invasion.

Identification of Plant DNA in Adults of the Phytoplasma Vector Cacopsylla picta Helps Understanding Its Feeding Behavior.

Apple proliferation is an economically important disease and a threat for commercial apple cultivation. The causative pathogen, the bacterium 'Candidatus Phytoplasma mali', is mainly transmitted by Cacopsylla picta, a phloem-feeding insect that develops on the apple tree (Malus spp.). To investigate the feeding behavior of adults of the phytoplasma vector Cacopsylla picta in more detail, we used deep sequencing technology to identify plant-specific DNA ingested by the insect. Adult psyllids were collected in different apple orchards in the Trentino-South Tyrol region of northern Italy. DNA from the whole body of the insect was extracted and analyzed for the presence of plant DNA by performing PCR with two plant-specific primers that target the chloroplast regions trnH-psbA and rbcLa. DNA from 23 plant genera (trnH) and four plant families (rbcLa) of woody and herbaceous plant taxa was detected. Up to six and three plant genera and families, respectively, could be determined in single specimens. The results of this study contribute to a better understanding of the feeding behavior of adult Cacopsylla picta.