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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., (© 2022. The Author(s).)

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., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Intracellular bacteria are ubiquitous in the insect world, with perhaps the best-studied example being the alphaproteobacterium, Wolbachia. Like most endosymbionts, Wolbachia cannot be cultivated outside of its host cells, hindering traditional microbial characterization techniques. Furthermore, multiple Wolbachia strains can be present within a single host, and certain strains can be present in densities below the detection limit of current methods. To date, Wolbachia has most commonly been studied using polymerase chain reaction (PCR) amplification and Sanger DNA sequencing by targeting specific genes in the bacterium's genome. PCR amplification and Sanger sequencing of multiple Wolbachia strains requires analysis of individually cloned sequences, which is resource and labor intensive. To help mitigate these difficulties, we present a modified double digest restriction site associated DNA sequencing (ddRADseq) approach to target and sequence in parallel multiple genes by adding restriction enzyme recognition sites to gene-specific PCR primers. Adopting this strategy allows us to uniquely tag and sequence amplicons from multiple hosts simultaneously on an Illumina MiSeq platform. Our approach represents an efficient and cost-effective method to characterize multiple target genes in population surveys.

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 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 w Cer2, mainly present in southern and central European populations, and is currently spreading into w Cer2-uninfected populations driven by high unidirectional cytoplasmic incompatibility. Here, we describe the distribution of w Cer2 along two transition zones where the infection is spreading into w Cer2-uninfected R. cerasi populations. Fine-scale sampling of 19 populations in the Czech Republic showed a smooth decrease of w Cer2 frequency from south to north within a distance of less than 20 km. Sampling of 12 Hungarian populations, however, showed a sharp decline of w Cer2 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., (© 2018 The Authors.)

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.

The eastern cherry fruit fly, Rhagoletis cingulata Loew (Diptera: Tephritidae), is an economically important pest of cherries in North America. In 1983 it was first reported in Europe where it shares its ecological niche with the native European cherry fruit fly, Rhagoletis cerasi L. (Diptera: Tephritidae). Their coexistence in Europe led to the recent horizontal transmission of the Wolbachia strain wCer1 from R. cerasi to R. cingulata Horizontal Wolbachia transmission is mediated by either sharing of ecological niches or by interacting species such as parasitoids. Here we describe for the first time that two braconid wasps, Psyttalia rhagoleticola Sachtleben (Hymenoptera: Braconidae) and Utetes magnus Fischer (Hymenoptera: Braconidae), naturally parasitizing R. cerasi, use the invasive R. cingulata in Europe as a new host. In contrast, no parasitoids that parasitize R. cingulata in its native American range were detected in the introduced European range. Diagnostic Wolbachia PCR screening and sequence analyses demonstrated that all P. rhagoleticola individuals were infected with the newly described Wolbachia strain wRha while all U. magnus individuals were uninfected. wRha is different from wCer1 but had an Wolbachia surface protein (wsp) gene sequence that was identical to wCer2 of R. cerasi and wCin2 of R. cingulata. However, multi locus sequence typing revealed differences in all loci between wRha and the tephritid's strains. The horizontal transmission of wCer1 between the two tephritid species did not result in fixed heritable infections in the parasitoids. However, the parasitoids may have acted as a transient wCer1 vector., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.)

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., (© 2016 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

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., (© 2013 John Wiley & Sons Ltd.)

Wolbachia are wide-spread, endogenous α-Proteobacteria of arthropods and filarial nematodes. 15-75% of all insect species are infected with these endosymbionts that alter their host's reproduction to facilitate their spread. In recent years, many insect species infected with multiple Wolbachia strains have been identified. As the endosymbionts are not cultivable outside living cells, strain typing relies on molecular methods. A Multi Locus Sequence Typing (MLST) system was established for standardizing Wolbachia strain identification. However, MLST requires hosts to harbour individual and not multiple strains of supergroups without recombination. This study revisits the applicability of the current MLST protocols and introduces Allele Intersection Analysis (AIA) as a novel approach. AIA utilizes natural variations in infection patterns and allows correct strain assignment of MLST alleles in multiply infected host species without the need of artificial strain segregation. AIA identifies pairs of multiply infected individuals that share Wolbachia and differ in only one strain. In such pairs, the shared MLST sequences can be used to assign alleles to distinct strains. Furthermore, AIA is a powerful tool to detect recombination events. The underlying principle of AIA may easily be adopted for MLST approaches in other uncultivable bacterial genera that occur as multiple strain infections and the concept may find application in metagenomic high-throughput parallel sequencing projects.

Haplodiploidy is a sex determination system in which fertilized diploid eggs develop into females and unfertilized haploid eggs develop into males. The evolutionary explanations for this phenomenon include the possibility that haplodiploidy can be reinforced by infection with endosymbiotic bacteria, such as Wolbachia. The subfamily Scolytinae contains species with haplodiploid and diploid sex determination systems. Thus, we studied the association with Wolbachia in 12 diploid and 11 haplodiploid scolytine beetles by analyzing wsp and multilocus sequence typing (MLST) of five loci in this endosymbiont. Wolbachia genotypes were compared with mitochondrial (COI) and nuclear (EF) genotypes in the scolytines. Eight of the 23 scolytine species were infected with Wolbachia, with haplodiploids at significantly higher rates than diploid species. Cloning and sequencing detected multiple infections with up to six Wolbachia strains in individual species. Phylogenetic analyses of wsp and five MLST genes revealed different Wolbachia strains in scolytines. Comparisons between the beetle and Wolbachia phylogenies revealed that closely related beetles were infected with genetically different Wolbachia strains. These results suggest the horizontal transmission of multiple Wolbachia strains between scolytines. We discuss these results in terms of the evolution of different sex determination systems in scolytine beetles. [ABSTRACT FROM AUTHOR]

Rhagoletis pomonella Walsh (Diptera: Tephritidae) is a model species for sympatric speciation through host race formation on apple and hawthorn. The bacterial endosymbiont Wolbachia, a manipulator of arthropod reproduction, has been considered to contribute to speciation in several species. A potential role of Wolbachia in sympatric speciation of R. pomonella remains to be tested despite an earlier detection by PCR. In this study, we isolated Wolbachia from R. pomonella individuals from both host species using multi-locus sequence typing (MLST) and the surface protein wsp. By cloning and sequencing of 311 plasmids, we found sequence types of at least four wPom strains. A complete MLST profile was obtained only for wPom1, whereas MLST loci of the other putative strains were difficult to assign because of multiple infections and low sample numbers. wPom1 occurs in both host races, whereas different sequence types were found at low frequencies only in apple-infesting R. pomonella. This warrants further investigation as it cannot be excluded that Wolbachia plays a part in this model of sympatric speciation. [ABSTRACT FROM AUTHOR]

Numerous terrestrial arthropods are infected with the alphaproteobacterium Wolbachia. This endosymbiont is usually transmitted vertically from infected females to their offspring and can alter the reproduction of hosts through various manipulations, like cytoplasmic incompatibility (CI), enhancing its spread in new host populations. Studies on the spatial and temporal dynamics of Wolbachia under natural conditions are scarce. Here, we analyzed Wolbachia infection frequencies in populations of the European cherry fruit fly, Rhagoletis cerasi (L.), in central Germany—an area of an ongoing spread of the CI-inducing strain wCer2. In total, 295 individuals from 19 populations were PCR-screened for the presence of wCer2 and their mitochondrial haplotype. Results were compared with historic data to understand the infection dynamics of the ongoing wCer2 invasion. An overall wCer2 infection frequency of about 30% was found, ranging from 0% to 100% per population. In contrast to an expected smooth transition from wCer2-infected to completely wCer2-uninfected populations, a relatively scattered infection pattern across geography was observed. Moreover, a strong Wolbachia-haplotype association was detected, with only a few rare misassociations. Our results show a complex dynamic of an ongoing Wolbachia spread in natural field populations of R. cerasi. [ABSTRACT FROM AUTHOR]