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Ticks are important vectors of pathogenic viruses, bacteria, and protozoans to humans, wildlife, and domestic animals. Due to their life cycle, ticks face significant challenges related to water homeostasis. When blood-feeding they must excrete water and ions, but when off-host (for stretches lasting several months) they must conserve water to avoid desiccation. Aquaporins (AQPs), a family of membrane-bound water channels, are key players in osmoregulation in many animals but remain poorly characterized in ticks. Here, we bioinformatically identified AQP-like genes from the deer tick Ixodes scapularis and used phylogenetic approaches to map the evolution of the aquaporin gene family in arthropods. We find that most arachnid AQP-like sequences (including those of I. scapularis) form a monophyletic group clustered within aquaglycerolporins (GLPs) from bacteria to vertebrates. This gene family is absent from insects, revealing divergent evolutionary paths for AQPs in different hematophagous arthropods. We next sequenced the full-length cDNA of I. scapularis aquaporin 1 (IsAQP1) and expressed it heterologously in Xenopus oocytes to functionally characterize its permeability to water and solutes. We additionally examined IsAQP1 expression across different life stages and adult female tissues. We found IsAQP1 to be an efficient water channel with salivary gland-specific expression, implying its function may be closely tied to osmoregulation during blood-feeding. Its functional properties were unique: unlike most GLPs, IsAQP1 has low glycerol permeability, and unlike most AQPs, it is insensitive to mercury. Together, our results suggest IsAQP1 plays an important role in tick water balance during blood feeding and may hold promise as a target for novel vector control efforts.

Native microbiota represent a potential resource for biocontrol of arthropod vectors. Ixodes scapularis is mostly inhabited by the endosymbiotic Rickettsia buchneri, but the composition of bacterial communities varies with life stage, fed status, and/or geographic location. We compared bacterial community diversity among I. scapularis populations sampled within a small geographic range in Central Pennsylvania. We collected and extracted DNA from ticks and sequenced amplicons of the eubacterial 16S rRNA gene from individuals and pooled samples. We then used taxon-specific PCR and/or qPCR to confirm the abundance or infection frequency of select pathogenic and symbiotic bacteria. Bacterial communities were more diverse in pools of males than females and the most abundant taxon was Rickettsia buchneri followed by Coxiellaceae (confirmed by sequencing as an unknown Rickettsiella species). High Rickettsiella titers in pools were likely due to a few heavily infected males. We determined that the infection frequency of Borrelia burgdorferi ranged from 20 to 75%. Titers of Anaplasma phagocytophilum were significantly different between sexes. Amplicon-based bacterial 16S sequencing is a powerful tool for establishing the baseline community diversity and focusing hypotheses for targeted experiments, but care should be taken not to overinterpret data based on too few individuals. We identified intracellular bacterial candidates that may be useful as targets for manipulation.

(1) Background: Native microbiota represent a potential resource for biocontrol of arthropod vectors.Ixodes scapularisare mostly inhabited by the endosymbioticRickettsia buchneri,but the bacterial communty composition varies with life stage, fed status, and/or geographic location. We investigated sex-specific bacterial community diversity fromI. scapulariscollected from central Pennsylvania between populations within a small geographic range. (2) Methods: We sequenced the bacterial 16S rRNA genes from individuals and pooled samples and investigated the abundance or infection frequency of key taxa using taxon-specific PCR and/or qPCR. (3) Results: Bacterial communities were more diverse in pools of males than females. WhenR. buchneriwas not the dominant taxon, Coxiellaceae was dominant. We determined that the infection frequency ofBorrelia burgdorferiranged between 20 to 75%. Titers ofAnaplasma phagocytophilumwere significantly different between sexes. HighRickettsiellatiter in pools were likely due to a few heavily infected males. (4) Conclusion: Bacterial 16S sequencing is useful for establishing the baseline community diversity and focusing hypotheses for targeted experiments. However, care should be taken not to overinterpret data concerning microbial dominance between geographic locations based on a few individuals as this may not accurately represent the bacterial community within tick populations.

Aims We aim to determine if Pseudomonas fluorescens is a viable biological control for Erwinia tracheiphila within the insect vector, Acalymma vittatum. Methods and results Pseudomonas fluorescens secreted fluorescein and inhibited growth of E. tracheiphila in disc diffusion assays. To determine if this antagonism was conserved within the insect vector, we performed in vivo assays by orally injecting beetles with bacterial treatments and fluorescent in situ hybridization to determine bacterial presence within the alimentary canal. Conclusions Pseudomonas fluorescens inhibited the growth of E. tracheiphila on a nutrient-limiting medium. In situ experiments demonstrated that P. fluorescens is maintained within the alimentary canal of the beetle for at least 4 days, and co-occurred with E. tracheiphila. When beetles were first presented with Pseudomonas and then challenged with E. tracheiphila, E. tracheiphila was not recovered via FISH after 4 days. These data suggest that P. fluorescens has potential as a biological control agent to limit E. tracheiphila within the insect vector. Significance and impact of the study This is a novel approach for controlling E. tracheiphila that has the potential to decrease reliance on insecticides, providing a safer environment for pollinators and growers.

Bee viral ecology is a fascinating emerging area of research: viruses exert a range of effects on their hosts, exacerbate impacts of other environmental stressors, and, importantly, are readily shared across multiple bee species in a community. However, our understanding of bee viral communities is limited, as it is primarily derived from studies of North American and European Apis mellifera populations. Here, we examined viruses in populations of A. mellifera and 11 other bee species from 9 countries, across 4 continents and Oceania. We developed a novel pipeline to rapidly and inexpensively screen for bee viruses. This pipeline includes purification of encapsulated RNA/DNA viruses, sequence-independent amplification, high throughput sequencing, integrated assembly of contigs, and filtering to identify contigs specifically corresponding to viral sequences. We identified sequences for (+)ssRNA, (−)ssRNA, dsRNA, and ssDNA viruses. Overall, we found 127 contigs corresponding to novel viruses (i.e. previously not observed in bees), with 27 represented by >0.1% of the reads in a given sample, and 7 contained an RdRp or replicase sequence which could be used for robust phylogenetic analysis. This study provides a sequence-independent pipeline for viral metagenomics analysis, and greatly expands our understanding of the diversity of viruses found in bee communities.

BackgroundTick-borne diseases have been increasing at the local, national, and global levels. Researchers studying ticks and tick-borne disease need a thorough knowledge of the pathogens, vectors, and epidemiology of disease spread. Three surveillance approaches are commonly used to provide insight into tick-borne disease risk: human disease case surveillance, active tick surveillance, and passive tick surveillance. Long-term passive surveillance can provide up-to-date data on the spatial variability and temporal dynamics of ectoparasite communities and shed light into the ecology of rarer tick species. We present a retrospective analysis on compiled data of ticks from Pennsylvania over the last 117 years.MethodsWe compiled data from ticks collected during tick surveillance research, and from citizen-based submissions to the Penn State University Department of Entomology (PSUEnt). Specimens were deposited at the PSUEnt arthropod collections that eventually became The Frost Entomological Museum. While most of the specimens were submitted by the public, a subset of the data were collected through active methods (flagging or dragging, or removal of ticks from wildlife). We analyzed all data from 1900-2017 for tick community composition, host associations, and spatio-temporal dynamics.ResultsIn total there were 4,491 submission lots consisting of 7,132 tick specimens. Twenty-four different species were identified, with the large proportion of submissions represented by five tick species. We observed a shift in tick community composition in which the dominant species of tick (Ixodes cookei) was overtaken in abundance byDermacentor variabilisin the early 1990s, and then replaced in abundance byI. scapularis. We analyzed host data and identified overlaps in host range amongst tick species, suggesting potential hubs of pathogen transfer between different tick vectors and their reservoir hosts.ConclusionsWe highlight the importance of long-term passive tick surveillance in investigating the ecology of both common and rare tick species. Information on the geographic distribution, host-association, and seasonality of the tick community can help researchers and health-officials to identify high-risk areas.

Vector-borne diseases have increased worldwide, facilitated by globalization and variations in climate. Tick and tick-borne disease researchers, veterinarians, medical practitioners, and public health specialists are working to share their expertise on tick ecology, disease transmission, diagnostics, and treatment in order to control tick-borne epidemics and potential pandemics. This review will be a brief overview of the current status of tick-borne diseases, challenges on the scientific and public fronts, and the role of public engagement in improving citizen education within the context of ticks and tick-borne disease research.

Bee viral ecology is a fascinating emerging area of research: viruses exert a range of effects on their hosts, exacerbate the impacts of other environmental stressors, and, importantly, are readily shared across multiple bee species in a community. However, our understanding of bee viral communities is limited, as it is primarily derived from studies of North American and European Apis mellifera populations. Here, we examined viruses in populations of A. mellifera and 11 other bee species from 9 countries, across 5 continents and Oceania. We developed a novel pipeline to rapidly, inexpensively, and robustly screen for bee viruses. This pipeline includes purification of encapsulated RNA/DNA viruses, sequence-independent amplification, high throughput sequencing, integrated assembly of contigs, and filtering to identify contigs specifically corresponding to viral sequences. We identified sequences corresponding to (+)ssRNA, (-)ssRNA, dsRNA, and ssDNA viruses. Overall, we found 127 contigs corresponding to novel viruses (i.e. previously not observed in bees), with 29 represented by >0.1 % of the reads in a given sample. These viruses and viral families were distributed across multiple regions and species. This study provides a robust pipeline for metagenomics analysis of viruses, and greatly expands our understanding of the diversity of viruses found in bee communities.

The bed bug Cimex lectularius is a blood-feeding re-emerging annoyance pest insect that has the ability to transmit Trypanosoma cruzi under experimental laboratory conditions. Aquaporins (AQPs) are water channel proteins that are essential in biological organisms. C. lectularius are constantly exposed to water-related stress, suggesting that AQPs may offer novel control avenues. We identified and cloned four AQPs from C. lectularius, assessed tissue and lifestage-specific expression, and characterized biochemical functions in vitro and in vivo. We identified an efficient water-specific AQP (ClAQP1), two aquaglyceroporins (ClGlp1 and ClGlp2) and a homolog of Drosophila melanogaster big brain (ClBib). ClGlp1 was only functional when co-expressed with the water-specific AQP. Simultaneous RNAi gene silencing of ClAQP1 and ClGlp1 significantly reduced water and urea excretion post blood feeding. The Bib homologue was enriched in embryos, exclusively expressed in ovaries, and when silenced, dramatically increased bug fecundity. Our data demonstrate that AQPs have critical roles in excretion, water homeostasis and reproduction in C. lectularius, and could be potential targets for control in this notorious pest.

The blacklegged tickIxodes scapularisis widely distributed in the United States and transmits multiple pathogens to humans, wildlife and domestic animals. Recently, several novel viruses in the family Bunyaviridae (South Bay virus (SBV) and Blacklegged tick phlebovirus (BTPV)) were identified infecting femaleI. scapularisticks collected in New York State. We used metagenomic sequencing to investigate the distribution of viruses infecting male and femaleI. scapularisticks collected in Centre County, Pennsylvania. We identified both SBV and BTPV in both male and female ticks from all collection locations. The role of maleI. scapularisin pathogen epidemiology has been overlooked because they rarely bite and are not considered important pathogen vectors. However, males may act as reservoirs for pathogens that can then be transmitted to females during mating. Our data highlight the importance of examining all potential avenues of pathogen maintenance and transmission throughout the vector-pathogen life cycle in order to understand the epidemiology of tick-borne pathogens.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing’, prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent., This project has been funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (NIAID, NIH, DHHS) under contract numbers N01-AI30071, HHSN272200900007C, HHSN266200400001C and 5R01GM77117-5. Its contents are solely the responsibility of the authors and do not represent the official views of the NIH. Additional grants and contracts supporting work described in this manuscript were from the NIH-NIAID (HHSN266200400039C and HHSN272200900039C) to F.H.C., and a subcontract under HHSN272200900039C to C.A.H. and J.M.M., the Australian Research Council Discovery Project (DP120100240) to S.C.B. and R.S., the Ministerio de Ciencia e Innovación of Spain (BFU2007–6292; BFU2010–15484) to J.R., BIO2009–07990 and BIO2012–37926 to J.V. NIH-1R01AI090062 to Y.P., L.S., and J.K., NIH 1R21AI096268 and NSF IOS-0949194 to R.M.R., the Xunta de Galicia of Spain (10PXIB918057PR) to J.M.C.T. and M.T., BFU2011–23896 and EU FP7 ANTIGONE (278976) to J.F., the USDA-NRI/CREES (2008-35302-18820) and Texas AgriLife Research Vector Biology grant to P.V.P. and European Research Council Starting Independent Researcher Grant (205202) to R.B., J.M.R was supported by the intramural program of the NIAID, R.M.W. by a Marie Curie International Outgoing Fellowship PIOF-GA-2011–303312, E.M.Z. by Swiss National Science Foundation awards 31003A-125350 and 31003A-143936, J.M.G. by an NIH-NCATS award TL1 TR000162 and NSF Graduate Research Fellowship (DGE 1333468), V.C. by a Boehringer Ingelheim Ph.D. Fellowship, F.G.V. by a Fundação para a Ciência e a Tecnologia, Portugal fellowship (SFRH/BD/22360/2005), C.J.P.G. and F.H. by The Lundbeck Foundation (Denmark), and J.J.G. by NIH awards HHSN272200900040C, R01AI017828 and R01AI043006. Support from the Broad Genomics Platform is gratefully acknowledged.

The bark gnawing beetle, Nemosoma attenuatum Van Dyke (Coleoptera: Trogossitidae), has been previously described as an associate of twig beetles in the genus Pityophthorus Eichhoff (Coleoptera: Curculionidae) and other bark beetles. Presented here are post hoc observations on the seasonal occurrence of N. attenuatum, collected and identified as part of an ecological study of Pityophthorus species inhabiting native Monterey pines and their potential role as vectors of a fungal pathogen Fusarium circinatum. Adult N. attenuatum were recovered from flight traps between May 2001 and July 2003. Additionally, adult and larval N. attenuatum were discovered in cut P. radiata branches suspended in canopies of P. radiata in native stands. The highest flight trap counts were recorded during May 2001, suggesting a mass emergence. The highest numbers of N. attenuatum were observed from branches suspended between January to March. Two N. attenuatum larvae were discovered within the galleries of Pityophthorus setosus, al...

Endosymbiotic Wolbachia bacteria have been previously shown to infect laboratory colonies of the human bed bug, Cimex lectularius L. (Heteroptera: Cimicidae), but little information exists regarding the extent of infection in natural populations. We assayed C. lectularius populations from five North American regions (California, Connecticut, Florida, New York, and Toronto, Canada) and one African region (Macha, Zambia) for Wolbachia infection by the polymerase chain reaction (PCR). Wolbachia infections were prevalent in all populations assayed (83-100%). There were no significant differences in infection frequency between geographic regions, between sexes, or between life stages (adult versus nymph). The potential utility of Wolbachia for alternative bed bug control strategies is discussed.

Pitch canker, caused by the fungus Fusarium circinatum, is a disease affecting many pine tree species. In California, Pinus radiata (Monterey pine) is the principal pine host affected by pitch canker. This investigation into factors affecting infection frequency by F. circinatum of P. radiata examined the influence of: (i) wound size; (ii) relative humidity; (iii) time of inoculation; (iv) inoculum density; and (v) wound age. Wounded branches sustained significantly more infections when large-diameter (1·6 mm) rather than small-diameter (0·5 mm) wounds were made. Infection frequencies tended to be higher at 100% RH than at ambient humidity, although these differences were not statistically significant. Infection frequencies were significantly higher on branches inoculated after 17·00 h than on branches inoculated before noon. Infection frequencies were significantly higher for wounded branches spray-inoculated with 5 × 107 rather than 1 × 107 spores mL−1. Infection frequencies of pruning wounds declined as wounds aged.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

Introduction The most significant vector of tick-borne pathogens in the United States is Ixodes scapularis Say (the blacklegged tick). Previous studies have identified significant genetic, behavioral and morphological differences between northern vs. southern populations of this tick. Because tick-borne pathogens are dependent on their vectors for transmission, a baseline understanding of the vector population structure is crucial to determining the risks and epidemiology of pathogen transmission. Methods We investigated population genetic variation of I. scapularis populations in the eastern United States using a multilocus approach. We sequenced and analyzed the mitochondrial COI and 16S genes and three nuclear genes (serpin2, ixoderin B and lysozyme) from wild specimens. Results We identified a deep divergence (3–7%) in I. scapularis COI gene sequences from some southern specimens, suggesting we had sampled a different Ixodes species. Analysis of mitochondrial 16S rRNA sequences did not support this hypothesis and indicated that all specimens were I. scapularis. Phylogenetic analysis and analysis of molecular variance (AMOVA) supported significant differences between northern vs. southern populations. Demographic analysis suggested that northern populations had experienced a bottleneck/expansion event sometime in the past, possibly associated with Pleistocene glaciation events. Conclusions Similar to other studies, our data support the division of northern vs. southern I. scapularis genetic lineages, likely due to differences in the demographic histories between these geographic regions. The deep divergence identified in some COI gene sequences highlights a potential hazard of relying solely on COI for species identification (“barcoding”) and population genetics in this important vector arthropod.

Background Bacterial vaginosis (BV) is a common gynecologic diagnosis characterized by dysbiosis of the vaginal microbiota. It is often accompanied by vaginal symptoms such as odor and discharge, but can be asymptomatic. Despite over 50 years of research, the etiology of BV is not well understood, which is a major impediment to treatment and prevention of BV. Results Here we report on the temporal dynamics of 25 vaginal communities over a 10 week period using samples collected daily from women who were diagnosed with symptomatic BV (15 women), asymptomatic BV (6 women), and women who did not have BV (4 women). Conclusion This unique resource of samples and data will contribute to a better understanding of the role that the vaginal microbes have in the natural history of BV and lead to improved diagnosis and treatment.

Vaginal HIV microbicides offer great promise in preventing HIV transmission, but failures of phase 3 clinical trials, in which microbicide-treated subjects had an increased risk of HIV transmission, raised concerns about endpoints used to evaluate microbicide safety. A possible explanation for the increased transmission risk is that the agents shifted the vaginal bacterial community, resulting in loss of natural protection and enhanced HIV transmission susceptibility. We characterized vaginal microbiota, using pyrosequencing of bar-coded 16S rRNA gene fragments, in samples from 35 healthy, sexually abstinent female volunteer subjects (ages 18 to 50 years) with regular menses in a repeat phase 1 study of twice-daily application over 13.5 days of 1 of 3 gel products: a hydroxyethylcellulose (HEC)-based “universal” placebo (10 subjects), 6% cellulose sulfate (CS; 13 subjects), and 4% nonoxynol-9 (N-9; 12 subjects). We used mixed effects models inferred using Bayesian Markov chain Monte Carlo methods, which showed that treatment with active agents shifted the microbiota toward a community type lacking significant numbers of Lactobacillus spp. and dominated by strict anaerobes. This state of the vaginal microbiota was associated with a low or intermediate Nugent score and was not identical to bacterial vaginosis, an HIV transmission risk factor. The placebo arm contained a higher proportion of communities dominated by Lactobacillus spp., particularly L. crispatus, throughout treatment. The data suggest that molecular evaluation of microbicide effects on vaginal microbiota may be a critical endpoint that should be incorporated in early clinical assessment of microbicide candidates., IMPORTANCE Despite large prevention efforts, HIV transmission and acquisition rates remain unacceptably high. In developing countries, transmission mainly occurs through heterosexual intercourse, where women are significantly more vulnerable to infection than men. Vaginal microbicides are considered to be one of the most promising female-controlled products, in that women themselves insert the microbicides into the vagina to prevent HIV transmission during sexual intercourse. The failure of several microbicides in clinical trials has raised questions concerning the low in vivo efficacy of such anti-HIV molecules. This study was designed to gain insights into the failures of two microbicides by testing the hypothesis that the microbicides negatively affect a critical line of defense against HIV, the vaginal microbiota. The results suggest that in the early assessment of candidate microbicides, culture-independent evaluation of their effect on the vaginal microbiota should be considered and may constitute a critical endpoint.

Elucidating the factors that impinge on the stability of bacterial communities in the vagina may help in predicting the risk of diseases that affect women's health. Here, we describe the temporal dynamics of the composition of vaginal bacterial communities in 32 reproductive-age women over a 16-week period. The analysis revealed the dynamics of five major classes of bacterial communities and showed that some communities change markedly over short time periods, whereas others are relatively stable. Modeling community stability using new quantitative measures indicates that deviation from stability correlates with time in the menstrual cycle, bacterial community composition, and sexual activity. The women studied are healthy; thus, it appears that neither variation in community composition per se nor higher levels of observed diversity (co-dominance) are necessarily indicative of dysbiosis.

Celiac disease (CD) is a unique autoimmune disorder in which the genetic factors (DQ2/DQ8) and the environmental trigger (gluten) are known and necessary but not sufficient for its development. Other environmental components contributing to CD are poorly understood. Studies suggest that aspects of gluten intake might influence the risk of CD occurrence and timing of its onset, i.e., the amount and quality of ingested gluten, together with the pattern of infant feeding and the age at which gluten is introduced in the diet. In this study, we hypothesize that the intestinal microbiota as a whole rather than specific infections dictates the switch from tolerance to immune response in genetically susceptible individuals. Using a sample of infants genetically at risk of CD, we characterized the longitudinal changes in the microbial communities that colonize infants from birth to 24 months and the impact of two patterns of gluten introduction (early vs. late) on the gut microbiota and metabolome, and the switch from gluten tolerance to immune response, including onset of CD autoimmunity. We show that infants genetically susceptible to CD who are exposed to gluten early mount an immune response against gluten and develop CD autoimmunity more frequently than at-risk infants in which gluten exposure is delayed until 12 months of age. The data, while derived from a relatively small number of subjects, suggest differences between the developing microbiota of infants with genetic predisposition for CD and the microbiota from infants with a non-selected genetic background, with an overall lack of bacteria of the phylum Bacteriodetes along with a high abundance of Firmicutes and microbiota that do not resemble that of adults even at 2 years of age. Furthermore, metabolomics analysis reveals potential biomarkers for the prediction of CD. This study constitutes a definite proof-of-principle that these combined genomic and metabolomic approaches will be key to deciphering the role of the gut microbiota on CD onset.

The endosymbiotic bacterium Wolbachia is being investigated as a potential control agent in several important vector insect species. Recent studies have shown that Wolbachia can protect the insect host against a wide variety of pathogens, resulting in reduced transmission of parasites and viruses. It has been proposed that compromised vector competence of Wolbachia-infected insects is due to up-regulation of the host innate immune system or metabolic competition. Anopheles mosquitoes, which transmit human malaria parasites, have never been found to harbor Wolbachia in nature. While transient somatic infections can be established in Anopheles, no stable artificially-transinfected Anopheles line has been developed despite numerous attempts. However, cultured Anopheles cells can be stably infected with multiple Wolbachia strains such as wAlbB from Aedes albopictus, wRi from Drosophila simulans and wMelPop from Drosophila melanogaster. Infected cell lines provide an amenable system to investigate Wolbachia-Anopheles interactions in the absence of an infected mosquito strain. We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes. Wolbachia infection had a dramatic strain-specific effect on gene expression in this cell line, with almost 700 genes in total regulated representing a diverse array of functional classes. Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts. This is in stark contrast to the induction of immune genes observed in other insect hosts. We also identified genes that may be potentially involved in Wolbachia-induced reproductive and pathogenic phenotypes. Somatically-infected mosquitoes had similar responses to cultured cells. The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria., Author Summary Wolbachia are bacteria that infect many insect species, but do not infect Anopheles mosquitoes. These mosquitoes transmit Plasmodium parasites, which cause malaria in humans. Wolbachia infection in Aedes aegypti mosquitoes reduces their ability to transmit diverse pathogens including viruses, nematodes and bird malaria parasites. Wolbachia-induced stimulation of the mosquito's innate immune system has been suggested as a mechanism conferring this pathogen interference. Since no Wolbachia-infected Anopheles mosquito strain exists, we used infected cultured Anopheles cells to examine the effect of infection on Anopheles gene expression. Wolbachia had a profound influence on Anopheles gene expression. Many of the genes regulated by Wolbachia have been seen in other studies to influence Plasmodium levels in mosquitoes, but interestingly and in contrast to other mosquitoes, many of the host genes were suppressed rather than induced.

Using microarray-based comparative genome hybridization (mCGH), the genomic content ofWolbachia pipientis wMel fromDrosophila melanogasterwas compared to the closely relatedWolbachiafromD. innubila(wInn),D. santomea(wSan), and three strains fromD. simulans(wAu,wRi,wSim). A large number of auxiliary genes are identified in these five strains, with most absent/divergent genes being unique to a given strain. Each strain caused an average of ∼60 genes to be removed from the core genome. As such, these organisms do not appear to have the streamlined genomes expected of obligate intracellular bacteria. Prophage, hypothetical and ankyrin repeat genes are over-represented in the absent/divergent genes, with 21–87 % of absent/divergent genes coming from prophage regions. The onlywMel region absent/divergent in all five query strains is that containing WD_0509 to WD_0511, including a DNA mismatch repair protein MutL-2, a degenerate RNase, and a conserved hypothetical protein. A region flanked by the two portions of the WO-B prophage inwMel is found in four of the fiveWolbachiastrains as well as on a plasmid of a rickettsial endosymbiont ofIxodes scapularis, suggesting lateral gene transfer between these two obligate intracellular species. Overall, these insect-associatedWolbachiahave highly mosaic genomes, with lateral gene transfer playing an important role in their diversity and evolution.

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia -permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod- Rickettsia interactions. We used two cell lines ( Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species ( Rickettsia felis ) and two spotted-fever-group rickettsial species ( R. montanensis and R. peacockii ) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA , sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii . The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia -arthropod interactions.

Endosymbiotic Wolbachia bacteria have been previously shown to infect laboratory colonies of the human bed bug, Cimex lectularius L. (Heteroptera: Cimicidae), but little information exists regarding the extent of infection in natural populations. We assayed C. lectularius populations from five North American regions (California, Connecticut, Florida, New York, and Toronto, Canada) and one African region (Macha, Zambia) for Wolbachia infection by the polymerase chain reaction (PCR). Wolbachia infections were prevalent in all populations assayed (83-100%). There were no significant differences in infection frequency between geographic regions, between sexes, or between life stages (adult versus nymph). The potential utility of Wolbachia for alternative bed bug control strategies is discussed.

Wolbachia spp. are obligate maternally inherited endosymbiotic bacteria that infect diverse arthropods and filarial nematodes. Previous microscopic and molecular studies have identified Wolbachia in several bed bug species (Cimicidae), but little is known about how widespread Wolbachia infections are among the Cimicidae. Because cimicids of non-medical importance are not commonly collected, we hypothesized that preserved museum specimens could be assayed for Wolbachia infections. For the screening of museum specimens, we designed a set of primers that specifically amplify small diagnostic fragments (130 to 240 bp) of the Wolbachia 16S rRNA gene. Using these and other previously published primers, we screened 39 cimicid species (spanning 16 genera and all 6 recognized subfamilies) and 2 species of the sister family Polyctenidae for Wolbachia infections using museum and wild-caught material. Amplified fragments were sequenced to confirm that our primers were amplifying Wolbachia DNA. We identified 10 infections, 8 of which were previously undescribed. Infections in the F supergroup were common in the subfamily Cimicinae, while infections in the A supergroup were identified in the subfamilies Afrocimicinae and Haematosiphoninae. Even though specimens were degraded, we detected infections in over 23% of cimicid species. Our results indicate that Wolbachia infections may be common among cimicids and that archived museum material is a useful untapped resource for invertebrate endosymbiont surveys. The new screening primers listed in this report will be useful for other researchers conducting Wolbachia surveys with specimens with less-than-optimum DNA quality.