Diversity and evolution of the Wolbachia endosymbionts of Bemisia (Hemiptera: Aleyrodidae) whiteflies

Wolbachia is the most prevalent symbiont described in arthropods to date. Wolbachia can manipulate host reproduction, provide nutrition to insect hosts and protect insect hosts from pathogenic viruses. So far, 13 supergroups of Wolbachia have been identified. The whitefly Bemisia tabaci is a complex containing more than 28 morphologically indistinguishable cryptic species. Some cryptic species of this complex are invasive. In this study, we report a comprehensive survey of Wolbachia in B. tabaci and its relative B. afer from 1658 insects representing 54 populations across 13 provinces of China and one state of Australia. Based on the results of PCR or sequencing of the 16S rRNA gene, the overall rates of Wolbachia infection were 79.6% and 0.96% in the indigenous and invasive Bemisia whiteflies, respectively. We detected a new Wolbachia supergroup by sequencing five molecular marker genes including 16S rRNA, groEL, gltA, hcpA, and fbpA genes. Data showed that many protein‐coding genes have limitations in detecting and classifying newly identified Wolbachia supergroups and thus raise a challenge to the known Wolbachia MLST standard analysis system. Besides, the other Wolbachia strains detected from whiteflies were clustered into supergroup B. Phylogenetic trees of whitefly mitochondrial cytochrome oxidase subunit I and Wolbachia multiple sequencing typing genes were not congruent. In addition, Wolbachia was also detected outside the special bacteriocytes in two cryptic species by fluorescence in situ hybridization, indicating the horizontal transmission of Wolbachia. Our results indicate that members of Wolbachia are far from well explored.     

Genetic distinctness of parthenogenetic forms of European Polydrusus weevils of the subgenus Scythodrusus

Abstract Among eight species of Polydrusus weevils which belong to subgenus Scythodrusus, at least two possess parthenogenetic forms: P. (S.) inustus and P. (S.) pilifer. Both of these species consist of dioecious populations in the Caspian area and of parthenogenetic populations in Eastern Europe (P. (S.) inustus), the Caucasus region (both species) and Middle Asia (P. (S.) pilifer). The origin of parthenogenesis in this subgenus is unresolved; however some data suggest that the parthenogenetic forms are of hybrid ancestry. The genetic distinctness of parthenogenetic Scythodrusus was assessed on the basis of COII, ITS2, EF1‐α and Wolbachiawsp, 16S ribosomal DNA, ftsZ and hcpA sequence comparisons. Both taxa turned out to be monophyletic for all markers, which is an evidence against hybridization of their dioecious ancestors. On the other hand, a high frequency of heterozygous P. (S.) inustus females suggests an origin resulting from hybridization between genetically distinct dioecious representatives of this species. Very similar strains of Wolbachia supergroup A were found in both species, indicating that they have been either inherited from a common ancestor or were transmitted between parthenogenetic Scythodrusus weevils and probably spread randomly across their ranges.     

Discovery of a Novel Wolbachia Supergroup in Isoptera

Wolbachia are one of the most abundant groups of bacterial endosymbionts in the biosphere. Interest in these heritable microbes has expanded with the discovery of wider genetic diversity in undersampled host species. Here, we report on the putative discovery of a new genetic lineage, denoted supergroup H, which infects the Isopteran species Zootermopsis angusticollis and Z. nevadensis. Evidence for this novel supergroup is based on portions of new Wolbachia gene sequences from each species spanning 3.5 kilobases of DNA and the following genes: 16S rDNA, dnaA, gltA, groEL, and ftsZ. Single-gene and concatenated maximum likelihood phylogenies establish this new supergroup and validate the positioning of the other Wolbachia supergroups. This discovery is the first example of a termite Wolbachia that is highly divergent from the Isopteran Wolbachia previously described in supergroup F. This study highlights the importance of multilocus approaches to resolving Wolbachia supergroup relationships. It also suggests that surveys of Wolbachia in more earlier-originating (and undersampled) groups of arthropods are more apt to reveal novel genetic diversity.     

Occurrence of Wolbachia in central European weevils: correlations with host systematics,ecology, and biology

We studied the occurrence of Wolbachia in relation to the systematics, ecology, and biology of 40 weevil species from central Europe. Identification of Wolbachia supergroups and phylogeny was performed on the basis of 16S rDNA, ftsZ, wsp, and hcpA sequences. Sixteen species (40%) were infected by Wolbachia. Six of these possess only supergroup A (15% of all studied species, 37.5% of the infected species), and four harbored only supergroup B (10 and 25%, respectively). Six species were infected by both supergroups A and B or their genomes harbored parts of these two supergroups (15 and 37.5%, respectively). No differences between Wolbachia supergroup frequencies were detected. There was almost no correlation between Wolbachia phylogeny and host systematics and phylogeny at the level of subfamily and tribe, because the representatives of both supergroups were detected in all the studied multi‐species tribes. Wolbachia strains were probably inherited from a common ancestor only in the case of the genus Strophosoma, where two of three analyzed species possessed bacteria which are genetically very close in all the studied genes. There was also only limited congruence between phylogenies obtained from the four studied genes. These results suggest horizontal transmission of Wolbachia strains between species and recombination events between different strains. A significant correlation was detected between infected and uninfected species in relation to mobility (flying species were 2× more frequently infected than non‐flying species), foraging (polyphagous species were 2.5× less frequently infected than mono‐ or oligophagous species), and reproductive mode (parthenogenetic weevils were infected nearly 2× as often as bisexuals). No differences were detected between mesophilous and xerothermophilous species, nor between those inhabiting open areas vs. arboreal species. However, these results might have been influenced by common ancestry among the studied weevils. Because weevils include many plant pests of economic importance, it is possible to use these data in developing alternative, biology‐based strategies for controlling them.     

Wolbachia strains in cryptic species of the Anastrepha fraterculus complex (Diptera,Tephritidae) along the Neotropical Region

Infection by Wolbachia was described previously in eleven species of Anastrepha fruit flies some of which are important pests of fruticulture. One such species is the nominal Anastrepha fraterculus, the South American fruit fly, which actually comprises a complex of cryptic species. The suggestions of using Wolbachia for the control of these pest species, make imperative a more precise characterization of the existing strains of the bacteria. In this study, population samples of the A. fraterculus complex from Brazil, Argentina, Peru, Ecuador, Colombia, Guatemala and Mexico were analyzed for Wolbachia infection. The bacteria were genotyped by the MLST and WSP Typing methodologies. All samples were infected with Wolbachia of supergroup “A”. For each of the five MLST genes, unique as well as already known alleles were detected. Nineteen sequence types for the concatenated sequences of the five MLST genes, and twenty wsp alleles were found in the samples. Host-specific haplotypes, shared strains among distinct hosts, and more than one strain of Wolbachia were found in some population samples. Recombination among the MLST genes and intragenic recombination between wsp haplotypes was rare. Phylogenetic analysis showed a great similarity among the Wolbachia strains in the A. fraterculus complex. However, some strains of Wolbachia are found throughout the Neotropical Region and there are specific strains in determined geographical areas.     

How Diverse Is the Genus Wolbachia? Multiple-Gene Sequencing Reveals a Putatively New Wolbachia Supergroup Recovered from Spider Mites (Acari: Tetranychidae)

At least 20% of all arthropods and some nematode species are infected with intracellular bacteria of the genus Wolbachia. This highly diverse genus has been subdivided into eight “supergroups” (A to H) on the basis of nucleotide sequence data. Here, we report the discovery of a new Wolbachia supergroup recovered from the spider mite species Bryobia species V (Acari: Tetranychidae), based on the sequences of three protein-coding genes (ftsZ, gltA, and groEL) and the 16S rRNA gene. Other tetranychid mites possess supergroup B Wolbachia strains. The discovery of another Wolbachia supergroup expands the known diversity of Wolbachia and emphasizes the high variability of the genus. Our data also clarify the existing supergroup structure and highlight the use of multiple gene sequences for robust phylogenetic analysis. In addition to previous reports of recombination between the arthropod-infecting supergroups A and B, we provide evidence for recombination between the nematode-infecting supergroups C and D. Robust delineation of supergroups is essential for understanding the origin and spread of this common reproductive parasite and for unraveling mechanisms of host adaptation and manipulation across a wide range of hosts.     

Wolbachia affect behavior and possibly reproductive compatibility but not thermoresistance,fecundity, and morphology in a novel transinfected host,Drosophila nigrosparsa

Wolbachia, intracellular endosymbionts, are estimated to infect about half of all arthropod species. These bacteria manipulate their hosts in various ways for their maximum benefits. The rising global temperature may accelerate species migration, and thus, horizontal transfer of Wolbachia may occur across species previously not in contact. We transinfected and then cured the alpine fly Drosophila nigrosparsa with Wolbachia strain wMel to study its effects on this species. We found low Wolbachia titer, possibly cytoplasmic incompatibility, and an increase in locomotion of both infected larvae and adults compared with cured ones. However, no change in fecundity, no impact on heat and cold tolerance, and no change in wing morphology were observed. Although Wolbachia increased locomotor activities in this species, we conclude that D. nigrosparsa may not benefit from the infection. Still, D. nigrosparsa can serve as a host for Wolbachia because vertical transmission is possible but may not be as high as in the native host of wMel, Drosophila melanogaster.     

Wolbachia infection in six species of gall wasps and their parasitoids

Wolbachia are endosymbiotic bacteria that are widely present in nematodes and arthropods and sometimes have a significant impact on the evolution, ecology, and biology of their hosts. The co-occurrence of Wolbachia within both Cynipid gall wasps and their parasitoids has rarely been studied. In this study, we report the occurrence of six species of gall wasps and 10 species of their parasitoids in central China. Wolbachia detection using the wsp gene showed that Wolbachia infected two species of gall wasps as well as their parasitoids, indicating that horizontal transmission of Wolbachia occurs between gall wasps and their parasitoids. Given that parasitoids will kill their hosts, Wolbachia may be horizontally transferred from gall wasps to their parasitoids. Using multilocus sequence typing (MLST) analysis, five new strains of Wolbachia were identified, all of which belonged to supergroup A. The strains of Wolbachia that infected gall wasps were not the same as those that infected their parasitoids. This result indicated that Wolbachia may evolve independently in parasitoids after they have been transferred from the host gall wasps.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Strains Typing in Different Geographic Population Spider, <Emphasis Type="Italic">Hylyphantes Graminicola</Emphasis> (Linyphiidae)

The Wolbachia endosymbiont of spiders has not been extensively examined. In order to investigate the distribution, evolutionary history, and reproductive phenotype of Wolbachia in spiders in China, we tested 11 geographic populations of Hylyphantes graminicola. Wolbachia infection has been detected in each population. 10 Wolbachia strains have been characterized by multilocus sequence typing (MLST). Phylogenetic analyses indicated that eight Wolbachia strains in H. graminicola belonged to supergroup B, and two belonged to supergroup A. No correlation existed between Wolbachia diversity and host’s geographic distance. The significant correlation was observed between pairwise distance of H. graminicola COI and genetic divergence of associated Wolbachia strains. We also found that Wolbachia infection frequencies in hosts varied over geographic space.     

Presence of the endosymbiont Wolbachia among some fruit flies (Diptera: Tephritidae) from Iran: A multilocus sequence typing approach

Wolbachia is a widespread endosymbiont of insects with a diverse range of biological effects on its hosts. We studied the prevalence of Wolbachia in some important species of tephritids in Iran. Among different populations of five fruit fly species, Dacus ciliatus (cucurbit fly), Rhagoletis cerasi (cherry fruit fly), Ceratitis capitata (Mediterranean fruit fly), Myiopardalis pardalina (melon fly) and Carypomya vesuviana (jujube fly), two species, R. cerasi and C. vesuviana, showed infection with separate Wolbachia strains, namely wCer6 and wVes1, respectively. C. vesuviana is introduced here as a novel host for Wolbachia. Genotyping of Wolbachia strains in 12 populations of five fruit fly species, using multilocus sequence typing (MLST) and the wsp gene sequence showed the occurrence of two new strains as well as a new strain type (ST) belonging to the A supergroup. On the basis of the results of this study, 12 barcodes under five species of Iranian tephritids have been added to the database of DNA barcodes. Inter- and intra-specific differences among COI sequences showed a clear gap in barcoding among most fruit flies.     

Comparative Genomics of Wolbachia and the Bacterial Species Concept

The importance of host-specialization to speciation processes in obligate host-associated bacteria is well known, as is also the ability of recombination to generate cohesion in bacterial populations. However, whether divergent strains of highly recombining intracellular bacteria, such as Wolbachia, can maintain their genetic distinctness when infecting the same host is not known. We first developed a protocol for the genome sequencing of uncultivable endosymbionts. Using this method, we have sequenced the complete genomes of the Wolbachia strains wHa and wNo, which occur as natural double infections in Drosophila simulans populations on the Seychelles and in New Caledonia. Taxonomically, wHa belong to supergroup A and wNo to supergroup B. A comparative genomics study including additional strains supported the supergroup classification scheme and revealed 24 and 33 group-specific genes, putatively involved in host-adaptation processes. Recombination frequencies were high for strains of the same supergroup despite different host-preference patterns, leading to genomic cohesion. The inferred recombination fragments for strains of different supergroups were of short sizes, and the genomes of the co-infecting Wolbachia strains wHa and wNo were not more similar to each other and did not share more genes than other A- and B-group strains that infect different hosts. We conclude that Wolbachia strains of supergroup A and B represent genetically distinct clades, and that strains of different supergroups can co-exist in the same arthropod host without converging into the same species. This suggests that the supergroups are irreversibly separated and that barriers other than host-specialization are able to maintain distinct clades in recombining endosymbiont populations. Acquiring a good knowledge of the barriers to genetic exchange in Wolbachia will advance our understanding of how endosymbiont communities are constructed from vertically and horizontally transmitted genes.     

Various infection status and molecular evidence for horizontal transmission and recombination of Wolbachia and Cardinium among rice planthoppers and related species

Wolbachia and Cardinium are widely distributed and are considered important for their ability to disturb reproduction and affect other fitness‐related traits of their hosts. By using multilocus sequence typing (MLST), RFLP (restriction fragment length polymorphism) and 16S ribosomal DNA gene sequencing methods, we extensively surveyed Wolbachia and Cardinium infection status of four predominant rice planthoppers and one kind of leafhopper in different rice fields. The results demonstrated that Sogatella furcifera (Horváth) and Laodelphax striatellus (Fallén) were infected with the same Wolbachia strain (wStri), while Nilaparvata lugens (Stål) and its closely related species Nilaparvata muiri China were infected with two phylogeneticlly distant strains, wLug and wMui, respectively. Three new Wolbachia strains (provisionally named wMfas1, wMfas2 and wMfas3) were detected in the leafhopper Macrosteles fascifrons (Stål). Only S. furcifera was co‐infected with Cardinium, which indicated that the distribution of Cardinium in these rice planthoppers was narrower than that of Wolbachia. Unambiguous intragenic recombination events among these Wolbachia strains and incongruent phylogenetic relationships show that the connections between different Wolbachia strains and hosts were more complex than we expected. These results suggest that horizontal transmission and host associated specialization are two factors affecting Wolbachia and Cardinium infections among planthoppers and their related species.     

F supergroup <Emphasis Type="Italic">Wolbachia</Emphasis> in bush crickets: what do patterns of sequence variation reveal about this supergroup and horizontal transfer between nematodes and arthropods?

Wolbachia pipientis, an intracellular, α-proteobacterium, is commonly found in arthropods and filarial nematodes. Most infected insects are known to harbor strains of Wolbachia from supergroups A or B, whereas supergroups C and D occur only in filarial nematodes. Here, we present molecular evidence from two genes (ftsZ and 16S rDNA) that 2 Orthopterans (the bush cricket species Orocharis saltator and Hapithus agitator; Gryllidae: Eneopterinae) are infected with Wolbachia from the F supergroup. Additionally, a series of PCR tests revealed that these bush cricket specimens did not harbor nematodes, thus indicating that our positive results were not a by-product of nematodes being present in these cricket samples. Patterns of molecular variation suggest that (1) strains of F supergroup Wolbachia exhibit less genetic variation than the nematode-specific C and D supergroups but more than the A and B supergroups found in arthropods and (2) that there is no evidence of recombination within F supergroup strains. The above data support previous findings that F supergroup Wolbachia is not only harbored in both nematodes and arthropods, but that horizontal transfer has likely occurred recently between these diverse taxonomic groups (although the exact details of such horizontal transmissions remain unclear). Moreover, the limited genetic variation and lack of recombination in the F supergroup suggest that this clade of Wolbachia has radiated relatively rapidly with either (1) little time for recombination to occur or (2) selection against recombination as occurs in the mutualistic C and D strains of Wolbachia – both of which remain to be explored further.     

Wolbachia have made it twice: Hybrid introgression between two sister species of Eurema butterflies

Wolbachia, cytoplasmically inherited endosymbionts of arthropods, are known to hijack their host reproduction in various ways to increase their own vertical transmission. This may lead to the selective sweep of associated mitochondria, which can have a large impact on the evolution of mitochondrial lineages. In Japan, two different Wolbacahia strains (wCI and wFem) are found in two sister species of pierid butterflies, Eurema mandarina and Eurema hecabe. In both species, females infected with wCI (C females) produce offspring with a nearly 1:1 sex ratio, while females infected with both wCI and wFem (CF females) produce all‐female offspring. Previous studies have suggested the historical occurrence of hybrid introgression in C individuals between the two species. Furthermore, hybrid introgression in CF individuals is suggested by the distinct mitochondrial lineages between C females and CF females of E. mandarina. In this study, we performed phylogenetic analyses based on nuclear DNA and mitochondrial DNA markers of E. hecabe with previously published data on E. mandarina. We found that the nuclear DNA of this species significantly diverged from that of E. mandarina. By contrast, mitochondrial DNA haplotypes comprised two clades, mostly reflecting Wolbachia infection status rather than the individual species. Collectively, our results support the previously suggested occurrence of two independent historical events wherein the cytoplasms of CF females and C females moved between E. hecabe and E. mandarina through hybrid introgression.     

Frequency of infection with A and B supergroup <Emphasis Type="Italic">Wolbachia</Emphasis> in insects and pests associated with mulberry and silkworm

Wolbachia is a ubiquitous, Gram-negative, vertically transmitted, alpha-proteobacterium that causes an array of reproductive abnormalities including cytoplasmic incompatibility, feminization of genetic males, parthenogenesis in a number of insect species, among others. Wolbachia is now being exploited as an agent for pest and vector control. Previous surveys indicated that it is commonly seen in 16–76% of arthropods. In this paper, using polymerase chain reaction assay based on specific amplification of the ftsZ-A and-B supergroup Wolbachia gene fragments, we found that 30% of insects and pests screened were positive for Wolbachia. Among them 66.7% harbour double Wolbachia infection, while 33.3% harbour single Wolbachia infection. These results indicate widespread infection with both double and single Wolbachia, and provide a wealth of information to exploit this endobacterium for the management of pests and vectors.     

Multiple Wolbachia infections in Rhagoletis pomonella

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.     

Evolutionary Dynamics of wAu-Like Wolbachia Variants in Neotropical Drosophila spp.

Wolbachia bacteria are common intracellular symbionts of arthropods and have been extensively studied in Drosophila. Most research focuses on two Old Word hosts, Drosophila melanogaster and Drosophila simulans, and does not take into account that some of the Wolbachia associations in these species may have evolved only after their fast global expansion and after the exposure to Wolbachia of previously isolated habitats. Here we looked at Wolbachia of Neotropical Drosophila species. Seventy-one lines of 16 Neotropical Drosophila species sampled in different regions and at different time points were analyzed. Wolbachia is absent in lines of Drosophila willistoni collected before the 1970s, but more recent samples are infected with a strain designated wWil. Wolbachia is absent in all other species of the willistoni group. Polymorphic wWil-related strains were detected in some saltans group species, with D. septentriosaltans being coinfected with at least four variants. Based on wsp and ftsZ sequence data, wWil of D. willistoni is identical to wAu, a strain isolated from D. simulans, but can be discriminated when using a polymorphic minisatellite marker. In contrast to wAu, which infects both germ line and somatic tissues of D. simulans, wWil is found exclusively in the primordial germ line cells of D. willistoni embryos. We report on a pool of closely related Wolbachia strains in Neotropical Drosophila species as a potential source for the wAu strain in D. simulans. Possible evolutionary scenarios reconstructing the infection history of wAu-like Wolbachia in Neotropical Drosophila species and the Old World species D. simulans are discussed.     

Toward a Wolbachia Multilocus Sequence Typing System: Discrimination of Wolbachia Strains Present in Drosophila Species

Among the diverse maternally inherited symbionts in arthropods, Wolbachia are the most common and infect over 20% of all species. In a departure from traditional genotyping or phylogenetic methods relying on single Wolbachia genes, the present study represents an initial Multilocus Sequence Typing (MLST) analysis to discriminate closely related Wolbachia pipientis strains, and additional data on sequence diversity in Wolbachia. We report a new phylogenetic characterization of four genes (aspC, atpD, sucB, and pdhB), and provide an expanded analysis of markers described in previous studies (16S rDNA, ftsZ, groEL, dnaA, and gltA). MLST analysis of the bacterial strains present in 16 different Drosophila–Wolbachia associations detected four distinct clonal complexes that also corresponded to maximum-likelihood identified phylogenetic clades. Among the 16 associations analyzed, six could not be assigned to MLST clonal complexes and were also shown to be in conflict with relationships predicted by maximum-likelihood phylogenetic inferences. The results demonstrate the discriminatory power of MLST for identifying strains and clonal lineages of Wolbachia and provide a robust foundation for studying the ecology and evolution of this widespread endosymbiont.     

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

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

First record of the bacterial endosymbiont Wolbachia for phytophagous hoverflies from genus Merodon (Diptera: Syrphidae)

Wolbachia is a widespread bacterial endosymbiont among arthropod species. It influences the reproduction of the host species and also mitochondrial DNA diversity. Until now there were only a few studies that detected Wolbachia infections in hoverflies (Diptera: Syrphidae), and this is the first broader study with the aim of examining the incidence of Wolbachia in the hoverfly genus Merodon. The obtained results indicate an infection rate of 96% and the presence of both Wolbachia supergroup A and B, which are characteristic for most of the infected arthropod species. Additionally, the presence of multiple Wolbachia strains in the Merodon aureus group species was detected and the mitochondrial DNA COI‐based relationships of the group are discussed in the light of infection. Finally, we discuss plant‐mediated horizontal transmission of Wolbachia strains among the studied hoverfly species.