Diversity analyses of bacterial symbionts in four Sclerodermus (Hymenoptera: Bethylidae) parasitic wasps, the dominant biological control agents of wood-boring beetles in China.

Objective: Sclerodermus wasps are important biocontrol agents of a class of wood borers. Bacterial symbionts influence the ecology and biology of their hosts in a variety of ways, including the formation of life-long beneficial or detrimental parasitic infections. However, only a few studies have explored the species and content of the symbionts in the Sclerodermus species. Methods: Here, a high-throughput sequencing study of the V3-V4 region of the 16S ribosomal RNA gene revealed a high level of microbial variety in four Sclerodermus waps, and their diversities and functions were also predicted. Results: The three most prevalent phyla of microorganisms in the sample were Firmicutes, Bacteroides, and Proteus. The KEEG pathways prediction results indicated that the three pathways with the highest relative abundances in the S. sichuanensis species were translation, membrane transport, and nucleotide metabolism. These pathways differed from those observed in S. guani, S. pupariae, and S. alternatusi, which exhibited carbohydrate metabolism, membrane transport, and amino acid metabolism, respectively. Bacteroides were found to be abundant in several species, whereas Wolbachia was the most abundant among S. sichuanensis, with a significant negative correlation between temperature and carriage rate. Conclusions: These results offer insights into the microbial communities associated with the bethylid wasps, which is crucial for understanding how to increase the reproductive capacity of wasps, enhance their parasitic effects, and lower cost in biocontrol.

Mosquito Microbiomes of Rwanda: Characterizing Mosquito Host and Microbial Communities in the Land of a Thousand Hills.

Mosquitoes are a complex nuisance around the world and tropical countries bear the brunt of the burden of mosquito-borne diseases. Rwanda has had success in reducing malaria and some arboviral diseases over the last few years, but still faces challenges to elimination. By building our understanding of in situ mosquito communities in Rwanda at a disturbed, human-occupied site and at a natural, preserved site, we can build our understanding of natural mosquito microbiomes toward the goal of implementing novel microbial control methods. Here, we examined the composition of collected mosquitoes and their microbiomes at two diverse sites using Cytochrome c Oxidase I sequencing and 16S V4 high-throughput sequencing. The majority (36 of 40 species) of mosquitoes captured and characterized in this study are the first-known record of their species for Rwanda but have been characterized in other nations in East Africa. We found significant differences among mosquito genera and among species, but not between mosquito sexes or catch method. Bacteria of interest for arbovirus control, Asaia, Serratia, and Wolbachia, were found in abundance at both sites and varied greatly by species.

Pan-Genome Analysis of Wolbachia, Endosymbiont of Diaphorina citri, Reveals Independent Origin in Asia and North America.

Wolbachia, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus (CLas) that causes citrus greening disease, is naturally infected with Wolbachia (wDi). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 wDi genomes to gain a comprehensive understanding of wDi. Based on average nucleotide identity (ANI) analysis, we classified the wDi strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed wDi genomes. The variation analysis between Asia and North America wDi showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of wDi contributes to our understanding of the geographical population of wDi, the origin of hosts of D. citri, and the interaction between wDi and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB).

Microbiome profile of South Korean vector mosquitoes.

This research offers a comprehensive exploration of the microbial communities associated with vector mosquitoes from South Korea. Aedes albopictus, Anopheles sinensis, and Culex molestus are vectors of pathogens, and understanding the intricacies of their microbiome profile is paramount for unraveling their roles in disease transmission dynamics. In this study, we characterized the microbiome of the midguts of adult female vector mosquitoes collected from different locations in South Korea. After DNA extraction from dissected mosquito midguts, we used the Illumina MiSeq next-generation sequencing to obtain sequences spanning the V4 hypervariable region of the bacteria 16S rRNA. Morphological and molecular characterization using 506-bp mitochondrial 16S rRNA was used to identify the mosquito species before amplicon sequencing. Across the three vector mosquitoes surveyed, 21 bacteria genera belonging to 20 families and 5 phyla were discovered. Proteobacteria and Bacteriodota were the major phyla of bacteria associated with the three mosquito species. There were significant differences in the gut microbiome genera composition between the species and little variation in the gut microbiome between individuals of the same mosquito species. Wolbachia is the most dominant genus in Aedes while Aeromonas, Acinetobacter, and unassigned taxa are the most common in An. sinensis. In addition to that, Chromobacterium, Chryseobacterium, and Aeromonas are dominant in Cx. molestus. This study sheds light on the complex interactions between mosquitoes and their microbiome, revealing potential implications for vector competence, disease transmission, and vector control strategies.

Symbiotic Wolbachia bacteria in coccinellid parasitoids: genetic diversity, horizontal transfer, and recombination / Bacterias simbióticas Wolbachia en parasitoides coccinélidos: diversidad genética, transferencia horizontal y recombinación

Parasitoids, which constitute about 25% of all insects and attack arthropods of virtually all taxa, are considered the most suitable vectors for horizontal transmission of the symbiotic bacterium Wolbachia among insects. The parasitoids studied in this article develop in the larvae and pupae of ladybirds. For the first time, Wolbachia was found in parasitic wasp species of the genus Homalotylus (Hymenoptera: Encyrtidae) and from the subfamily Tetrastichinae (Hymenoptera: Eulophidae). To characterize the Wolbachia strains, six bacterial housekeeping genes were examined and compared with previously published Wolbachia gene sequences. The same bacterial strains were found in all individuals of each species of parasitic wasps collected in different places and at different times, which indicates the absence of contamination and testifies to the heritability of the symbionts in the studied chalcids. No evidence was found that the parasitic wasps were infected with Wolbachia, identical to the symbionts of their ladybirds hosts. We found one Wolbachia strain, wHom-2, which is a product of bacterial recombination from unrelated insects, including ladybirds. The lack of correspondence between the molecular phylogenies of Wolbachia strains and mitochondrial DNA of their hosts indicates horizontal transfers of Wolbachia among parasitic wasps of the genus Homalotylus and from the subfamily Tetrastichinae.(AU)

Lab-scale characterization and semi-field trials of Wolbachia Strain wAlbB in a Taiwan Wolbachia introgressed Ae. aegypti strain.

Dengue fever is one of the most severe viral diseases transmitted by Aedes mosquitoes, with traditional approaches of disease control proving insufficient to prevent significant disease burden. Release of Wolbachia-transinfected mosquitoes offers a promising alternative control methodologies; Wolbachia-transinfected female Aedes aegypti demonstrate reduced dengue virus transmission, whilst Wolbachia-transinfected males cause zygotic lethality when crossed with uninfected females, providing a method for suppressing mosquito populations. Although highly promising, the delicate nature of population control strategies and differences between local species populations means that controlled releases of Wolbachia-transinfected mosquitoes cannot be performed without extensive testing on specific local Ae. aegypti populations. In order to investigate the potential for using Wolbachia to suppress local Ae. aegypti populations in Taiwan, we performed lab-based and semi-field fitness trials. We first transinfected the Wolbachia strain wAlbB into a local Ae. aegypti population (wAlbB-Tw) and found no significant changes in lifespan, fecundity and fertility when compared to controls. In the laboratory, we found that as the proportion of released male mosquitoes carrying Wolbachia was increased, population suppression could reach up to 100%. Equivalent experiments in semi-field experiments found suppression rates of up to 70%. The release of different ratios of wAlbB-Tw males in the semi-field system provided an estimate of the optimal size of male releases. Our results indicate that wAlbB-Tw has significant potential for use in vector control strategies aimed at Ae. aegypti population suppression in Taiwan. Open field release trials are now necessary to confirm that wAlbB-Tw mediated suppression is feasible in natural environments.

Microbiome analyses of 12 psyllid species of the family Psyllidae identified various bacteria including Fukatsuia and Serratia symbiotica, known as secondary symbionts of aphids.

BACKGROUND: Psyllids (Hemiptera: Psylloidea) comprise a group of plant sap-sucking insects that includes important agricultural pests. They have close associations not only with plant pathogens, but also with various microbes, including obligate mutualists and facultative symbionts. Recent studies are revealing that interactions among such bacterial populations are important for psyllid biology and host plant pathology. In the present study, to obtain further insight into the ecological and evolutionary behaviors of bacteria in Psylloidea, we analyzed the microbiomes of 12 psyllid species belonging to the family Psyllidae (11 from Psyllinae and one from Macrocorsinae), using high-throughput amplicon sequencing of the 16S rRNA gene. RESULTS: The analysis showed that all 12 psyllids have the primary symbiont, Candidatus Carsonella ruddii (Gammaproteobacteria: Oceanospirillales), and at least one secondary symbiont. The majority of the secondary symbionts were gammaproteobacteria, especially those of the family Enterobacteriaceae (order: Enterobacteriales). Among them, symbionts belonging to "endosymbionts3", which is a genus-level monophyletic group assigned by the SILVA rRNA database, were the most prevalent and were found in 9 of 11 Psyllinae species. Ca. Fukatsuia symbiotica and Serratia symbiotica, which were recognized only as secondary symbionts of aphids, were also identified. In addition to other Enterobacteriaceae bacteria, including Arsenophonus, Sodalis, and "endosymbionts2", which is another genus-level clade, Pseudomonas (Pseudomonadales: Pseudomonadaceae) and Diplorickettsia (Diplorickettsiales: Diplorickettsiaceae) were identified. Regarding Alphaproteobacteria, the potential plant pathogen Ca. Liberibacter europaeus (Rhizobiales: Rhizobiaceae) was detected for the first time in Anomoneura mori (Psyllinae), a mulberry pest. Wolbachia (Rickettsiales: Anaplasmataceae) and Rickettsia (Rickettsiales: Rickettsiaceae), plausible host reproduction manipulators that are potential tools to control pest insects, were also detected. CONCLUSIONS: The present study identified various bacterial symbionts including previously unexpected lineages in psyllids, suggesting considerable interspecific transfer of arthropod symbionts. The findings provide deeper insights into the evolution of interactions among insects, bacteria, and plants, which may be exploited to facilitate the control of pest psyllids in the future.

Narrow Genetic Diversity of Wolbachia Symbionts in Acrididae Grasshopper Hosts (Insecta, Orthoptera).

Bacteria of the Wolbachia genus are maternally inherited symbionts of Nematoda and numerous Arthropoda hosts. There are approximately 20 lineages of Wolbachia, which are called supergroups, and they are designated alphabetically. Wolbachia strains of the supergroups A and B are predominant in arthropods, especially in insects, and supergroup F seems to rank third. Host taxa have been studied very unevenly for Wolbachia symbionts, and here, we turn to one of largely unexplored insect families: Acrididae. On the basis of five genes subject to multilocus sequence typing, we investigated the incidence and genetic diversity of Wolbachia in 41 species belonging three subfamilies (Gomphocerinae, Oedipodinae, and Podisminae) collected in Turkey, Kazakhstan, Tajikistan, Russia, and Japan, making 501 specimens in total. Our results revealed a high incidence and very narrow genetic diversity of Wolbachia. Although only the strains belonging to supergroups A and B are commonly present in present, the Acrididae hosts here proved to be infected with supergroups B and F without A-supergroup variants. The only trace of an A-supergroup lineage was noted in one case of an inter-supergroup recombinant haplotype, where the ftsZ gene came from supergroup A, and the others from supergroup B. Variation in the Wolbachia haplotypes in Acrididae hosts within supergroups B and F was extremely low. A comprehensive genetic analysis of Wolbachia diversity confirmed specific features of the Wolbachia allelic set in Acrididae hosts. This result can help to elucidate the crucial issue of Wolbachia biology: the route(s) and mechanism(s) of Wolbachia horizontal transmission.

Cytoplasmic incompatibility in the semivoltine longicorn beetle Acalolepta fraudatrix (Coleoptera: Cerambycidae) double infected with Wolbachia.

Wolbachia are obligatory endosymbiotic α-proteobacteria found in many arthropods. They are maternally inherited, and can induce reproductive alterations in the hosts. Despite considerable recent progress in studies on the associations between Wolbachia and various taxonomic groups of insects, none of the researches have revealed the effects of Wolbachia on longicorn beetles as the host insect. Acalolepta fraudatrix is a forest longicorn beetle that is distributed in East Asia. In this study, the relationship between Wolbachia and A. fraudatrix was investigated. Out of two populations of A. fraudatrix screened for Wolbachia using the genes ftsZ, wsp, and 16S rRNA, only one of the populations showed detection of all three genes indicating the presence of Wolbachia. Electron microscopy and fluorescent in situ hybridization also confirmed that the A. fraudatrix population was infected with Wolbachia. Sequencing the wsp genes derived from single insects revealed that two strains of Wolbachia coexisted in the insects based on the detection of two different sequences of the wsp gene. We designated these strains as wFra1 and wFra2. The bacterial titers of wFra1 were nearly 2-fold and 3-fold higher than wFra2 in the testes and ovaries, respectively. The two strains of Wolbachia in the insects were completely eliminated by rearing the insects on artificial diets containing 1% concentration of tetracycline for 1 generation. Reciprocal crosses between Wolbachia-infected and Wolbachia-uninfected A. fraudatrix demonstrated that only eggs produced by the crosses between Wolbachia-infected males and Wolbachia-uninfected females did not hatch, indicating that Wolbachia infecting A. fraudatrix causes cytoplasmic incompatibility in the host insect. This is the first report showing the effect of Wolbachia on reproductive function in a longicorn beetle, A. fraudatrix.

Molecular detection and characterization of the endosymbiont Wolbachia in the European hedgehog flea, Archaeopsylla erinacei.

Wolbachia, the endosymbiont of arthropods and onchocercid nematodes is present in many medically important insect species, being also considered for the indirect control of parasitic ones. Archaeopsylla erinacei is a flea species infesting hedgehogs acting as vector of Rickettsia felis, Bartonella henselae, and Rickettsia helvetica, thus having public health relevance. The Wolbachia surface protein (wsp) and 16S rRNA genes were used to determine the presence, prevalence and molecular typing of Wolbachia in this flea species collected in two regions of southern Italy. Of the 45 fleas tested (n = 16 males, 35.6%; n = 29 females, 64.4%), 43 (95.6%; 95% CI: 84.8-99.2) scored positive for Wolbachia, of which 15 (33.3%) and 28 (62.2%) were males and females, respectively. The sex-wise prevalence of this endosymbiont was almost equal in both sexes (males 93.8%; 95% CI: 69.5-99.7; females 96.7%; 95% CI: 83.1-99.8). Single locus sequence analysis (SLST) of Wolbachia revealed two sequence types for 16S rRNA gene, named as wAr_15227 and wAr_15234, which came from two different areas, equally distributed in male and female fleas, whilst only one sequence type was identified for wsp gene. The phylogenetic analysis placed the two 16S rRNA sequence types in paraphyletic clades belonging to the supergroup A and B, respectively. Whilst, the tree of wsp gene clustered the corresponding sequence in the same clade including those of Wolbachia supergroup A. In MLST analyses, both Wolbachia sequence types clustered in a monophyletic clade with Drosophila nikananu (wNik) and Drosophila sturtevanti (wStv) from supergroup A. ClonalFrame analysis revealed a recombination event in the wAr_15234 strain which came from Apulia region. Scientific knowledge of the presence/prevalence of Wolbachia among medically important fleas, may contribute to develop an alternative biological method for the vector control.

Genetic innovations in animal-microbe symbioses.

Animal hosts have initiated myriad symbiotic associations with microorganisms and often have maintained these symbioses for millions of years, spanning drastic changes in ecological conditions and lifestyles. The establishment and persistence of these relationships require genetic innovations on the parts of both symbionts and hosts. The nature of symbiont innovations depends on their genetic population structure, categorized here as open, closed or mixed. These categories reflect modes of inter-host transmission that result in distinct genomic features, or genomic syndromes, in symbionts. Although less studied, hosts also innovate in order to preserve and control symbiotic partnerships. New capabilities to sequence host-associated microbial communities and to experimentally manipulate both hosts and symbionts are providing unprecedented insights into how genetic innovations arise under different symbiont population structures and how these innovations function to support symbiotic relationships.

Molecular phylogeny of heritable symbionts and microbiota diversity analysis in phlebotominae sand flies and Culex nigripalpus from Colombia.

BACKGROUND: Secondary symbionts of insects include a range of bacteria and fungi that perform various functional roles on their hosts, such as fitness, tolerance to heat stress, susceptibility to insecticides and effects on reproduction. These endosymbionts could have the potential to shape microbial communites and high potential to develop strategies for mosquito-borne disease control. METHODOLOGY/PRINCIPAL FINDINGS: The relative frequency and molecular phylogeny of Wolbachia, Microsporidia and Cardinium were determined of phlebotomine sand flies and mosquitoes in two regions from Colombia. Illumina Miseq using the 16S rRNA gene as a biomarker was conducted to examine the microbiota. Different percentages of natural infection by Wolbachia, Cardinium, and Microsporidia in phlebotomines and mosquitoes were detected. Phylogenetic analysis of Wolbachia shows putative new strains of Lutzomyia gomezi (wLgom), Brumptomyia hamata (wBrham), and a putative new group associated with Culex nigripalpus (Cnig) from the Andean region, located in Supergroup A and Supergroup B, respectively. The sequences of Microsporidia were obtained of Pi. pia and Cx. nigripalpus, which are located on phylogeny in the IV clade (terrestrial origin). The Cardinium of Tr. triramula and Ps. shannoni were located in group C next to Culicoides sequences while Cardinium of Mi. cayennensis formed two putative new subgroups of Cardinium in group A. In total were obtained 550 bacterial amplicon sequence variants (ASVs) and 189 taxa to the genus level. The microbiota profiles of Sand flies and mosquitoes showed mainly at the phylum level to Proteobacteria (67.6%), Firmicutes (17.9%) and Actinobacteria (7.4%). High percentages of relative abundance for Wolbachia (30%-83%) in Lu. gomezi, Ev. dubitans, Mi. micropyga, Br. hamata, and Cx. nigripalpus were found. ASVs assigned as Microsporidia were found in greater abundance in Pi. pia (23%) and Cx. nigripalpus (11%). An important finding is the detection of Rickettsia in Pi. pia (58,8%) and Bartonella sp. in Cx. nigripalpus. CONCLUSIONS/SIGNIFICANCE: We found that Wolbachia infection significantly decreased the alpha diversity and negatively impacts the number of taxa on sand flies and Culex nigripalpus. The Principal Coordinate Analysis (PCoA) is consistent, which showed statistically significant differences (PERMANOVA, F = 2.4744; R2 = 0.18363; p-value = 0.007) between the microbiota of sand flies and mosquitoes depending on its origin, host and possibly for the abundance of some endosymbionts (Wolbachia, Rickettsia).

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.

Greenhead (Tabanus nigrovittatus) Wolbachia and Its Microbiome: A Preliminary Study.

Endosymbiotic Wolbachia bacteria are known to influence the host physiology, microbiota composition, and dissemination of pathogens. We surveyed a population of Tabanus nigrovittatus, commonly referred to as "greenheads," from Crane Beach (Ipswich, MA, USA) for the presence of the alphaproteobacterial symbiont Wolbachia. We studied the COI (mitochondrial cytochrome oxidase) marker gene to evaluate the phylogenetic diversity of the studied specimens. The DNA sequences show strong similarity (between 99.9 and 98%) among the collected specimens but lower similarity to closely related entries in the NCBI database (only between 96.3 and 94.7%), suggesting a more distant relatedness. Low levels of Wolbachia presence necessitated a nested PCR approach, and using 5 markers (ftsZ, fbpA, dnaA, coxA, and gatB), we determined that two recognized "supergroups" of Wolbachia species were represented in the studied specimens, members of clades A and B. Using next-generation sequencing, we also surveyed the insect gut microbiomes of a subset of flies, using Illumina and PacBio 16S rRNA gene sequencing with barcoded primers. The composition of Proteobacteria also varied from fly to fly, with components belonging to Gammaproteobacteria making up the largest percentage of organisms (30 to 70%) among the microbiome samples. Most of the samples showed the presence of Spiroplasma, a member of the phylum Mollicutes, although the frequency of its presence was variable, ranging from 2 to 57%. Another noteworthy bacterial phylum consistently identified was Firmicutes, though the read abundances were typically below 10%. Of interest is an association between Wolbachia presence and higher Alphaproteobacteria representation in the microbiomes, suggesting that the presence of Wolbachia affects the host microbiome. IMPORTANCE Tabanus nigrovittatus greenhead populations contain two supergroups of Wolbachia endosymbionts, members of supergroups A and B. Analysis of the greenhead microbiome using next-generation sequencing revealed that the majority of bacterial species detected belonged to Gammaproteobacteria, with most of the samples also showing the presence of Spiroplasma, a member of the Mollicutes phylum also known to infect insects. An association between Wolbachia presence and higher Alphaproteobacteria representation in the microbiomes suggests that Wolbachia presence affects the host microbiome composition.

Two Newly Introduced Wolbachia Endosymbionts Induce Cell Host Differences in Competitiveness and Metabolic Responses.

Wolbachia endosymbionts can induce multiple reproductive manipulations in their hosts, with cytoplasmic incompatibility (CI) being one of the most common manipulations. Two important agricultural pests, the white-backed planthopper (Sogatella furcifera) and the brown planthopper (Nilaparvata lugens), are usually infected with CI-inducing Wolbachia strain wFur and non-CI-inducing Wolbachia strain wLug, respectively. The biological effects of these infections when present in a host cell are unknown. Here, we introduced the two Wolbachia strains into an Aedes albopictus cell line to stably establish a wFur-infected cell line (WFI) and a wLug-infected cell line (WLI). In a mixed culture, WFI cells were completely replaced by WLI cells, pointing to a stronger competitiveness of the WLI cell line. We found that infection by both Wolbachia strains reduced cell growth rates, but WLI had a higher cell growth rate than WFI, and this difference in cell growth rate combined with possible Wolbachia differences in diffusivity may have affected cell competitiveness. By examining gene expression and metabolites in the two lines, we found that some genes and key metabolites responded to differences in cell competitiveness. These results point to potential mechanisms that could contribute to the relative performance of hosts infected by these strains and also highlight the substantial impact of a non-CI Wolbachia on metabolism, which may in turn influence the fitness of its native host. IMPORTANCEWolbachia transinfection in insects can be used to suppress pests and block virus transmission. We stably introduced two Wolbachia strains from rice planthoppers into cell lines of an important arbovirus mosquito vector, Aedes albopictus. The levels of competitiveness of host cells from the lines infected by the two Wolbachia strains were different, as were metabolic responses of the cell lines. These results suggest potential metabolic effects of Wolbachia on native hosts that could be exploited when they are transinfected into novel hosts for pest control.

Persistent Spodoptera frugiperda rhabdovirus infection in Sf9 cells is not restricted by Wolbachia wMelPop-CLA and wAlbB strains and is targeted by the RNAi machinery.

The endosymbiotic bacterium Wolbachia pipientis confers RNA virus refractoriness in Drosophila and Aedes mosquitoes. Questions remain about the Wolbachia-virus restriction phenotype and how extensive this phenomenon may be within other arthropods. Here, we generated two Spodoptera frugiperda cell lines stably transinfected with two strains of Wolbachia, wAlbB and wMelPop-CLA. Despite the high density of Wolbachia in stably infected Sf9 cells, RT-PCR indicated the presence of the negative-sense RNA virus Spodoptera frugiperda rhabdovirus (SfRV) in Wolbachia-infected and uninfected cell lines. No differences in the replication of SfRV between Sf9 and Wolbachia-infected cells was found. RNA-Seq analysis of the parental Sf9 cells supported SfRV's presence in these cells with abundant 20 nt virus-derived small RNAs indicating active replication of SfRV in these cells. Overall, this study supports a growing body of evidence that Wolbachia does not restrict negative-sense RNA viruses and generates an in vitro model to examine Lepidoptera-Wolbachia virus interactions.

Wolbachia prevalence in the vector species Culex pipiens and Culex torrentium in a Sindbis virus-endemic region of Sweden.

BACKGROUND: Wolbachia pipientis are endosymbiotic bacteria present in a large proportion of terrestrial arthropods. The species is known to sometimes affect the ability of its host to transmit vector-borne pathogens. Central Sweden is endemic for Sindbis virus (SINV), where it is mainly transmitted by the vector species Culex pipiens and Culex torrentium, with the latter established as the main vector. In this study we investigated the Wolbachia prevalence in these two vector species in a region highly endemic for SINV. METHODS: Culex mosquitoes were collected using CDC light traps baited with carbon dioxide over 9 years at 50 collection sites across the River Dalälven floodplains in central Sweden. Mosquito genus was determined morphologically, while a molecular method was used for reliable species determination. The presence of Wolbachia was determined through PCR using general primers targeting the wsp gene and sequencing of selected samples. RESULTS: In total, 676 Cx. pipiens and 293 Cx. torrentium were tested for Wolbachia. The prevalence of Wolbachia in Cx. pipiens was 97% (95% CI 94.8-97.6%), while only 0.7% (95% CI 0.19-2.45%) in Cx. torrentium. The two Cx. torrentium mosquitoes that were infected with Wolbachia carried different types of the bacteria. CONCLUSIONS: The main vector of SINV in the investigated endemic region, Cx. torrentium, was seldom infected with Wolbachia, while it was highly prevalent in the secondary vector, Cx. pipiens. The presence of Wolbachia could potentially have an impact on the vector competence of these two species. Furthermore, the detection of Wolbachia in Cx. torrentium could indicate horizontal transmission of the endosymbiont between arthropods of different species.

Taxonomic classification of the bacterial endosymbiont Wolbachia based on next-generation sequencing: is there molecular evidence for its presence in tardigrades?

We used high-throughput sequencing of 16S rRNA to test whether tardigrade species are infected with Wolbachia parasites. We applied SILVA and Greengenes databases that allowed taxonomic classification of bacterial sequences to OTUs. The results obtained from both databases differed considerably in the number of OTUs, and only the Greengenes database allowed identification of Wolbachia (infection was also supported by comparison of sequences to NCBI database). The putative bacterial endosymbiont Wolbachia was discovered only in adult eutardigrades, while bacteria identified down to the order Rickettsiales were detected in both eutardigrade eggs and adult specimens. Nevertheless, the frequency of Wolbachia in the bacterial communities of the studied eutardigrades was low. Similarly, in our positive control, i.e., a fairy shrimp Streptocephalus cafer, which was found to be infected with Wolbachia in our previous study using Sanger sequencing, only the Rickettsiales were detected. We also carried out phylogenetic reconstruction using Wolbachia sequences from the SILVA and Greengenes databases, Alphaproteobacteria putative endosymbionts and Rickettsiales OTUs obtained in previous studies on the microbial community of tardigrades, and Rickettsiales and Wolbachia OTUs obtained in the current study. Our discovery of Wolbachia in tardigrades can fuel new research to uncover the specifics of this interaction.

Wolbachia supergroup E found in Hypochthonius rufulus (Acari: Oribatida) in Poland.

Data on the spread of intracellular bacteria in oribatid mites (Acari: Oribatida) are scarce. Our work fills a gap in the research on endosymbionts in this group of invertebrates and provides information on Wolbachia infection in Hypochthonius rufulus (Acari: Oribatida) from soil, litter and moss sample collected in south-eastern Poland. This is the first report of Wolbachia in H. rufulus. Phylogeny based on the analysis of the 16S rRNA, gatB, fbpA, gltA, ftsZ and hcpA gene sequences revealed that Wolbachia from H. rufulus represented supergroup E and was related to bacterial endosymbionts of Collembola. The unique sequence within Wolbachia supergroup E was detected for the 16S rRNA gene of the bacteria from H. rufulus. The sequences of Wolbachia 16S rRNA and housekeeping genes have been deposited in publicly available databases and are an important source of molecular data for comparative studies.

DNA recombination and repair in Wolbachia: RecA and related proteins.

Wolbachia is an obligate intracellular bacterium that has undergone extensive genomic streamlining in its arthropod and nematode hosts. Because the gene encoding the bacterial DNA recombination/repair protein RecA is not essential in Escherichia coli, abundant expression of this protein in a mosquito cell line persistently infected with Wolbachia strain wStri was unexpected. However, RecA's role in the lytic cycle of bacteriophage lambda provides an explanation for retention of recA in strains known to encode lambda-like WO prophages. To examine DNA recombination/repair capacities in Wolbachia, a systematic examination of RecA and related proteins in complete or nearly complete Wolbachia genomes from supergroups A, B, C, D, E, F, J and S was undertaken. Genes encoding proteins including RecA, RecF, RecO, RecR, RecG and Holliday junction resolvases RuvA, RuvB and RuvC are uniformly absent from Wolbachia in supergroup C and have reduced representation in supergroups D and J, suggesting that recombination and repair activities are compromised in nematode-associated Wolbachia, relative to strains that infect arthropods. An exception is filarial Wolbachia strain wMhie, assigned to supergroup F, which occurs in a nematode host from a poikilothermic lizard. Genes encoding LexA and error-prone polymerases are absent from all Wolbachia genomes, suggesting that the SOS functions induced by RecA-mediated activation of LexA do not occur, despite retention of genes encoding a few proteins that respond to LexA induction in E. coli. Three independent E. coli accessions converge on a single Wolbachia UvrD helicase, which interacts with mismatch repair proteins MutS and MutL, encoded in nearly all Wolbachia genomes. With the exception of MutL, which has been mapped to a eukaryotic association module in Phage WO, proteins involved in recombination/repair are uniformly represented by single protein annotations. Putative phage-encoded MutL proteins are restricted to Wolbachia supergroups A and B and show higher amino acid identity than chromosomally encoded MutL orthologs. This analysis underscores differences between nematode and arthropod-associated Wolbachia and describes aspects of DNA metabolism that potentially impact development of procedures for transformation and genetic manipulation of Wolbachia.