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.     

Wolbachia infections of tephritid fruit flies: molecular evidence for five distinct strains in a single host species

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and can induce cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing or feminization in their hosts. Here, we report phylogenetic relationships of Wolbachia in tephritid fruit flies based on wsp gene sequences. We also report, for the first time, five distinct strains of Wolbachia in Bactrocera ascita sp. B. Four of the five Wolbachia strains found in this species were in the same groups as those found in other tephritid fruit flies, suggesting possible horizontal transmission of Wolbachia from other fruit flies into B. ascita sp. B. The unreliability of wsp-specific group primers demonstrated in this study suggests that these primers might be useful only for preliminary identification of Wolbachia. Final determination of group affiliation needs to be verified with wsp sequence data. Received: 12 December 2001 / Accepted: 11 January 2002     

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.     

Molecular Characterization of Wolbachia Strains Associated with the Invasive Asian Citrus Psyllid Diaphorina citri in Brazil

Wolbachia is a symbiont intensively studied due to its ability to interfere with their host’s reproduction, and it has been recently proposed as an alternative tool to control insect pests or vectors of diseases. The Asian citrus psyllid Diaphorina citri is an important pest of citrus since it vectors the bacterium that causes the "Huanglongbing" disease in citrus. The frequency and diversity of Wolbachia associated with D. citri is unknown, limiting the utilization of Wolbachia as an alternative strategy for insect management. Thus, we aimed to determine the natural rate of infection, to characterize the Wolbachia strains associated with this psyllid by "multilocus sequencing typing” (MLST) and wsp analysis, and to verify the association of the symbiont to particular genotypes of the host. Analysis indicated Wolbachia infects 100 % of all specimens tested from all 15 sampled populations. MLST revealed the occurrence of five new sequence types (STs) of Wolbachia, while analysis based on the wsp sequences indicated only four different types of Wolbachia. ST-173 was predominant, while the remaining STs were population specific. Analysis of the host–symbiont relationship did not reveal any particular association of Wolbachia and haplotypes or a decrease in nucleotide diversity of D. citri in populations in which more than one ST was recorded. The consequences of the diversity of STs reported are still unknown, but the fact that Wolbachia infection is fixed and that there is one ST with a broad distribution highlights the use of this symbiont as an alternative strategy to control D. citri.     

Male-killing Wolbachia in two species of insect

The inherited bacterium Wolbachia spreads through the manipulation of host reproduction, and has been suggested to be an important factor in arthropod evolution, from host speciation to the evolution of sex-determination systems. Past work has shown that members of this group may produce cytoplasmic incompatibility, feminize genetically male hosts, and induce host parthenogenesis. Here, we report an expansion of the range of reproductive manipulations produced by members of this clade, recording Wolbachia strains that kill male hosts during embryogenesis in two host species, the ladybird Adalia bipunctata, and the butterfly Acraea encedon. Both male-killing bacteria belong to the B group of Wolbachia. However, phylogenetic analyses were unable to resolve whether the bacteria in the two species are monophyletic, or represent independent origins of male-killing among the B-group Wolbachia. We also found significant divergence within the wsp gene of Wolbachia strains found in different A. bipunctata individuals, suggesting this host species contains two Wolbachia strains, diverged in wsp sequence but monophyletic. Our observations reinforce the notion that Wolbachia may be an important agent driving arthropod evolution, and corroborates previous suggestions that male-killing behaviour is easily evolved by invertebrate symbionts.     

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus,Anopheles sinensis,Armigeres subalbatus,Culex pipiens and Cx. tritaeniorhynchus in China

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.     

The diversity,recombination and horizontal transmission of Wolbachia in spiders in China

Wolbachia are maternally inherited endosymbiotic bacteria. These intracellular bacteria are common in arthropods and could manipulate host reproduction in diverse ways, such as feminization, parthenogenesis, male killing and cytoplasmic incompatibility. In spiders, infection by Wolbachia has been found in a total of 99 species belonging to 62 genera and 17 families. Furthermore, recent studies analyzed the phylogeny of Wolbachia in Hylyphantes graminicola, 2 cave spiders and Agelenopsis species using multilocus sequence typing (MLST) approach. However, the diversity of Wolbachia strains determined by MLST in spiders from China is still largely unknown.In this study, we collected 1153 spider individuals from Mangshan in China and screened for Wolbachia in 975 individuals representing 68 spider species belonging to 45 genera of 16 families. We analyzed the phylogenetic relationship between Wolbachia and their host spiders by MLST approach. We found novel infections of Wolbachia in 1 family, 9 genera and 20 species of spiders. We found 13 new Wolbachia strains and suggest that group A is more common than group B in Wolbachia that infect spiders. Our results revealed three recombination events of the concatenated multilocus sequences in Wolbachia that infect spiders. Furthermore, our results demonstrated the phylogenetic incongruence between Wolbachia and spiders, suggesting the horizontal transmission of Wolbachia in spiders.We suggest that recombination and horizontal transmission may play an important role in the diversity and evolution of Wolbachia in spiders.     

Japanese barberry seed predation by Rhagoletis meigenii fruit flies harboring Wolbachia endosymbionts

The phytophagous fruit fly Rhagoletis meigenii harbors the bacterium Wolbachia pipientis and, together with Japanese barberry, form a tri-partite symbiosis. R. meigenii is a seed predator of invasive Japanese barberry plants and is dependent on this insect-plant interaction for reproductive success. The endosymbiotic bacterium W. pipientis is a reproductive parasite known to alter the sex ratios of offspring and the fitness of infected host insects. We investigated Japanese barberry fruit for the degree of infestation by R. meigenii and characterized the Wolbachia strain infecting R. meigenii. Densities of R. meigenii in four naturalized stands of Japanese barberry revealed low numbers of fruit flies with high variability in the population densities observed among individual plants. Overall, R. meigenii infested roughly 10–20 % of the Japanese barberry fruits analyzed; fruit with two seeds (vs. one seed) were the most frequently infested. Approximately, 90 % of the R. meigenii tested positive for Wolbachia infection via PCR amplification of the Wolbachia surface protein (wsp) gene. No bacterial strain diversity was observed when comparing multi-locus sequence typing (MLST) profiles within or among five R. meigenii populations in Maine, although the MLST profile obtained from R. meigenii differed from three co-occurring Rhagoletis. The Wolbachia endosymbiont of R. meigenii is a member of the Wolbachia supergroup A and the ST-13 cluster complex.     

Wolbachia in Anastrepha Fruit Flies (Diptera: Tephritidae)

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and cause a variety of reproductive abnormalities, such as cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing, and host feminization. In this study, we used three sets of Wolbachia-specific primers (16S rDNA, ftsZ, and wsp) in conjunction with the polymerase chain reaction (PCR), cloning and sequencing to study the infection of fruit flies (Anastrepha spp. and Ceratitis capitata) by Wolbachia. The flies were collected at several localities in Brazil and at Guayaquil, Ecuador. All of the fruit flies studied were infected with Wolbachia supergroup A, in agreement with the high prevalence of this group in South America. Phylogenetic analysis showed that the wsp gene was the most sensitive gene for studying the relationships among Wolbachia strains. The Wolbachia sequences detected in these fruit flies were similar to those such as wMel reported for other fruit flies. These results show that the infection of Anastrepha fruit flies by Wolbachia is much more widespread than previously thought.     

Clarithromycin and Amoxicillin Susceptibility of Helicobacter pylori Strains Isolated from Adult Patients with Gastric or Duodenal Ulcer in Italy

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences. Received: 10 January 2000 / Accepted: 22 February 2000     

Chaos of Wolbachia Sequences Inside the Compact Fig Syconia of Ficus benjamina (Ficus: Moraceae)

Figs and fig wasps form a peculiar closed community in which the Ficus tree provides a compact syconium (inflorescence) habitat for the lives of a complex assemblage of Chalcidoid insects. These diverse fig wasp species have intimate ecological relationships within the closed world of the fig syconia. Previous surveys of Wolbachia, maternally inherited endosymbiotic bacteria that infect vast numbers of arthropod hosts, showed that fig wasps have some of the highest known incidences of Wolbachia amongst all insects. We ask whether the evolutionary patterns of Wolbachia sequences in this closed syconium community are different from those in the outside world. In the present study, we sampled all 17 fig wasp species living on Ficus benjamina, covering 4 families, 6 subfamilies, and 8 genera of wasps. We made a thorough survey of Wolbachia infection patterns and studied evolutionary patterns in wsp (Wolbachia Surface Protein) sequences. We find evidence for high infection incidences, frequent recombination between Wolbachia strains, and considerable horizontal transfer, suggesting rapid evolution of Wolbachia sequences within the syconium community. Though the fig wasps have relatively limited contact with outside world, Wolbachia may be introduced to the syconium community via horizontal transmission by fig wasps species that have winged males and visit the syconia earlier.     

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.     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct ‘supergroups’ and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species’ sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering ∼6.2 kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.     

Effects of crowding and temperature on Wolbachia infection density among life cycle stages of Aedes albopictus

Species of the genus Wolbachia are a group of Rickettsia-like, maternally-inherited bacteria (gram negative), which cause various reproductive alterations in their arthropod and nematode hosts including cytoplasmic incompatibility (CI), male-killing, parthenogenesis and feminization. They can be divided into supergroups such as A and B based on phylogenetic analysis of 16S rDNA sequences. In this study, we examined the relative infection densities of Wolbachia strains among life cycle stages in the mosquito, Aedes albopictus in terms of crowding effect and temperature effect. A. albopictus is known to be superinfected with both A- and B-supergroup Wolbachia which cause CI. The relative Wolbachia densities within each individual mosquito were determined and quantified by using real-time quantitative PCR assay based on the wsp gene. We found that B-supergroup Wolbachia strain densities in this host species were consistently and significantly higher than in the A-supergroup. Larval crowding also reduced adult size of mosquitoes. Our results show clearly that the higher densities of mosquito larvae cause lower densities of Wolbachia strains. Examination of the effect of temperature on Wolbachia density in each stage of the mosquito clearly revealed a significant decrease in bacterial density following exposure to elevated temperature (37 °C) in both males and females.     

Two New Strains of Wolbachia Affecting Natural Avocado Thrips

Wolbachia is an obligate intracellular bacterium with a high frequency of infection and a continental distribution in arthropods and nematodes. This endosymbiont can induce various reproductive phenotypes in their hosts and has been previously found naturally in several pests including thrips (Thripidae). These insects cause physical fruit damage and economic losses in avocado. The presence of Wolbachia was evaluated for the first time in avocado thrips populations of Frankliniella sp. and Scirtothrips hansoni sp.n. from eastern Antioquia. DNA from adult thrips individuals was used to assess the detection of Wolbachia by amplifying a fragment (600 bp) of the Wolbachia major surface protein (wsp) gene. Results confirmed the presence of two new Wolbachia strains in these two thrips species, with a higher percentage of natural infection in S. hansoni sp.n. The first Wolbachia species was found in Frankliniella sp. and belongs to supergroup A and the second was detected in S. hansoni sp.n. and is part of supergroup B. Wolbachia was more frequently found in females (32.73%), and only found in one male. Analysis of phylogenetic relationships, suggests that the two new Wolbachia sequences (wFran: Frankliniella and wShan: Scirtothrips hansoni) detected here represent two new groups for this endosymbiont. The haplotype network shows the presence of two possible haplotypes for each strain. Future studies to evaluate the possible use of Wolbachia as a control agent in avocado thrips are necessary.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00951-5.     

High incidences and similar patterns of Wolbachia infection in fig wasp communities from three different continents

Abstract Wolbachia are endosymbiotic bacteria that infect numerous arthropod species. Previous studies in Panama and Australia revealed that the majority of fig wasp species harbor Wolbachia infections, but that similar patterns of incidence have evolved independently with different wasp species and Wolbachia strains on the two continents. We found Wolbachia infections in 25/47 species (53%) of fig wasp associated with 25 species of Chinese figs. Phylogenetic analyses of Wolbachia wsp sequences indicated that very similar strains are not obviously found in either closely related or ecologically linked fig wasps species. The extremely high prevalence of Wolbachia in fig wasps (over 50% of species infected) is not constrained by geographical origin and is a recurrent theme of fig wasp/Wolbachia interactions.     

Multi-Infections of Feminizing Wolbachia Strains in Natural Populations of the Terrestrial Isopod Armadillidium Vulgare

Maternally inherited Wolbachia (α-Proteobacteria) are widespread parasitic reproductive manipulators. A growing number of studies have described the presence of different Wolbachia strains within a same host. To date, no naturally occurring multiple infections have been recorded in terrestrial isopods. This is true for Armadillidium vulgare which is known to harbor non simultaneously three Wolbachia strains. Traditionally, such Wolbachia are detected by PCR amplification of the wsp gene and strains are characterized by sequencing. The presence of nucleotide deletions or insertions within the wsp gene, among these three different strains, provides the opportunity to test a novel genotyping method. Herein, we designed a new primer pair able to amplify products whose lengths are specific to each Wolbachia strain so as to detect the presence of multi-infections in A. vulgare. Experimental injections of Wolbachia strains in Wolbachia-free females were used to validate the methodology. We re-investigated, using this novel method, the infection status of 40 females sampled in 2003 and previously described as mono-infected based on the classical sequencing method. Among these females, 29 were identified as bi-infected. It is the first time that naturally occuring multiple infections of Wolbachia are detected within an individual A. vulgare host. Additionally, we resampled 6 of these populations in 2010 to check the infection status of females.     

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.     

Evolutionary history of <Emphasis Type="Italic">Wolbachia</Emphasis> infections in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Background  

Wolbachia are endosymbiotic bacteria that commonly infect numerous arthropods. Despite their broad taxonomic distribution, the transmission patterns of these bacteria within and among host species are not well understood. We sequenced a portion of the wsp gene from the Wolbachia genome infecting 138 individuals from eleven geographically distributed native populations of the fire ant Solenopsis invicta. We then compared these wsp sequence data to patterns of mitochondrial DNA (mtDNA) variation of both infected and uninfected host individuals to infer the transmission patterns of Wolbachia in S. invicta.