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Microbiota associated with mosquito vector populations impact several traits of mosquitoes, including survival, reproduction, control, and immunity against pathogens. The influence of seasonal variations and mosquito species on mosquito gut microbiota is poorly understood. We sought to determine whether the mosquito microbiota associated with immature stages of two congeners (Culex coronator and Culex nigripalpus) differ temporally and between the two species. Using high throughput 16S rRNA gene sequence analysis, we characterized bacterial and archaeal communities found in the immature stages of the two Culex mosquito species sampled over three seasons to compare the diversity of bacteria between the two species. Beta diversity analyses of the larval microbiota sequences revealed that the two Culex species differed significantly, both temporally within each species and between the two species. Bacteria in Cx. coronator larvae were dominated by Alphaproteobacteria, mainly associated with Roseoccocus and unidentified species of Rhizobiales, and two unidentified species of Cyanobacteria. In contrast, Cx. nigripalpus was dominated by Thorsellia anophelis (Gammaproteobacteria), Clostridium, an unidentified species of Ruminococcacae (Clostridiales), and additional unidentified species associated with Erysipelotrichaceae (Erysipelotrichales), Bacteroidales, and Mollicutes. Results of our study revealed both seasonal and interspecies differences in bacterial community composition associated with the immature stages of Cx. coronator and Cx. nigripalpus vector populations in Florida. These results have important implications for our understanding of the underlying factors of variations in disease transmission among seasons, susceptibility to various pesticides, and other biotic factors, including the role of the microbiota on the spread of invasive species. In addition, our results suggest close associations of certain bacteria species with each of the two Culex species that will be further targeted for their potential in the development of microbial-based control strategies.

Although mosquito microbiota are known to influence reproduction, nutrition, disease transmission, and pesticide resistance, the relationship between host-associated microbial community composition and geographical location is poorly understood. To begin addressing this knowledge gap, we characterized microbiota associated with adult females of Culex nigripalpus mosquito vectors of Saint Louis Encephalitis and West Nile viruses sampled from three locations in Florida (Vero Beach, Palmetto Inland, and Palmetto Coast). High-throughput sequencing of PCR-amplified 16S rRNA genes demonstrated significant differences among microbial communities of mosquitoes sampled from the three locations. Mosquitoes from Vero Beach (east coast Florida) were dominated by uncultivated Asaia sp. (Alphaproteobacteria), whereas microbiota associated with mosquitoes collected from two mosquito populations at Palmetto (west coast Florida) sites were dominated by uncultured Spironema culicis (Spirochaetes), Salinisphaera hydrothermalis (Gammaproteobacteria), Spiroplasma (Mollicutes), uncultured Enterobacteriaceae, Candidatus Megaira (Alphaproteobacteria; Rickettsiae), and Zymobacter (Gammaproteobacteria). The variation in taxonomic profiles of Cx. nigripalpus gut microbial communities, especially with respect to dominating taxa, is a potentially critical factor in understanding disease transmission and mosquito susceptibility to insecticides among different mosquito populations.

Aerobic granules offer enhanced biological nutrient removal and are compact and dense structures resulting in efficient settling properties. Granule instability, however, is still a challenge as understanding of the drivers of instability is poorly understood. In this study, transient instability of aerobic granules, associated with filamentous outgrowth, was observed in laboratory-scale sequencing batch reactors (SBRs). The transient phase was followed by the formation of stable granules. Loosely bound, dispersed, and pinpoint seed flocs gradually turned into granular flocs within 60 days of SBR operation. In stage 1, the granular flocs were compact in structure and typically 0.2 mm in diameter, with excellent settling properties. Filaments appeared and dominated by stage 2, resulting in poor settleability. By stage 3, the SBRs were selected for larger granules and better settling structures, which included filaments that became enmeshed within the granule, eventually forming structures 2-5 mm in diameter. Corresponding changes in sludge volume index were observed that reflected changes in settleability. The protein-to-polysaccharide ratio in the extracted extracellular polymeric substance (EPS) from stage 1 and stage 3 granules was higher (2.8 and 5.7, respectively), as compared to stage 2 filamentous bulking (1.5). Confocal laser scanning microscopic (CLSM) imaging of the biomass samples, coupled with molecule-specific fluorescent staining, confirmed that protein was predominant in stage 1 and stage 3 granules. During stage 2 bulking, there was a decrease in live cells; dead cells predominated. Denaturing gradient gel electrophoresis (DGGE) fingerprint results indicated a shift in bacterial community composition during granulation, which was confirmed by 16S rRNA gene sequencing. In particular, Janthinobacterium (known denitrifier and producer of antimicrobial pigment) and Auxenochlorella protothecoides (mixotrophic green algae) were predominant during stage 2 bulking. The chitinolytic activity of Chitinophaga is likely antagonistic towards Auxenochlorella and may have contributed to stage 3 stable granule formation. Rhodanobacter, known to support complete denitrification, were predominant in stage 1 and stage 3 granules. The relative abundance of Rhodanobacter coincided with high protein concentrations in EPS, suggesting a role in microbial aggregation and granule formation.

Summary Bacillus thuringiensis subsp. israelensis (Bti) is the most widely used biopesticide against mosquitoes and blackflies, with a history of high specificity and efficacy. High doses of Bti have been suggested for extended vector control in some environments; however, the effects of Bti application on the native microfauna in the environment are poorly understood. Two Bti (VectoBac G) treatments (high = 48·1 kg ha−1; low = 0·6 kg ha−1), in addition to an untreated control, were assigned to replicate 300 L microcosms. Culex abundance, phytoplankton biomass, sestonic particulates and nutrients were reduced significantly in the high Bti treatment. These changes affected other physicochemical variables in the water column during the 44-day field study. Bacterial communities present in the water column were assessed by Illumina sequencing of 16S rRNA genes. The most abundant aquatic bacteria in microcosms subject to a low dose of Bti and untreated control, Cyanobacteria, Cytophagales, Cyclobacteriaceae (phylum Bacteroidetes) and Sphingomonas (class Alphaproteobacteria), were suppressed in microcosms subject to a high dose of Bti. Bacteria in the high Bti treatment were dominated by Mucilaginibacter, Sediminibacterium (phylum Sphingobacteria) and Polaromonas (class Betaproteobacteria) and were more diverse than in the other treatments. Synthesis and applications. The biotic and abiotic changes resulting from a biopesticide application that significantly reduced mosquito abundance by more than 50% persisted longer than the period during which larval mosquito numbers were reduced. This warranted further investigation into the ecosystem-level effects of Bti application rates used routinely for mosquito control. Application rates greater than the label rate for a Bti biopesticide can reduce mosquito abundance for an extended period of time and therefore lessen the operational cost of repeated application, but they could exceed manufacturer's recommendations and might violate country-specific regulations governing biopesticide applications in natural habitats. Results of our study suggest that the widespread adoption of Bti application above the recommended label rate should be discouraged in habitats where algal abundance and its effect on primary production, microbial communities and nutrient cycling could affect the functioning of aquatic food webs.

Mosquito microbiota provide important physiological and ecological attributes to mosquitoes, including an impact on their susceptibility to pathogens, fitness, and sensitivity to mosquito control agents. Culex nigripalpus mosquito populations transmit various pathogens, including the Saint Louis and West Nile viruses, and proliferate in nutrient-rich environments, such as in wastewater treatment wetlands. Our study examined whether increases in nutrients within larval mosquito developmental habitats impact microbial communities associated with C. nigripalpus mosquitoes. We characterized the effects of organic enrichments on microbiomes associated with C. nigripalpus mosquitoes and identified potential bacterial microbiota that will be further investigated for whether they alter mosquito life history traits and for their potential role in the development of microbial-based control strategies., Pollution from nutrients in aquatic habitats has been linked to increases in disease vectors, including mosquitoes and other pestiferous insects. One possibility is that changes in mosquito microbiomes are impacted by nutrient enrichments and that these changes affect various traits, including larval development, susceptibility to larval control agents, and susceptibility of the adult mosquitoes to pathogens. We tested this hypothesis using field mesocosms supplemented with low- and high-organic-nutrient regimens and then sampled microbial communities associated with the naturally colonizing Culex nigripalpus mosquito vector. By high-throughput sequencing of 16S rRNA gene sequences, we found no significant differences in overall microbial communities associated with sampled mosquitoes, despite detecting discernible differences in environmental variables, including pH, dissolved oxygen, and nutrient amendments. Nevertheless, indicator species analysis revealed that members of the Clostridiales were significantly associated with mosquitoes that originated from high-nutrient enrichments. In contrast, members of the Burkholderiales were associated with mosquitoes from the low-nutrient enrichment. High bacterial variability associated with the life stages of the C. nigripalpus was largely unaffected by levels of nutrient enrichments that impacted larval microbial resources, including bacteria, ciliates, and flagellates in the larval environments. IMPORTANCE Mosquito microbiota provide important physiological and ecological attributes to mosquitoes, including an impact on their susceptibility to pathogens, fitness, and sensitivity to mosquito control agents. Culex nigripalpus mosquito populations transmit various pathogens, including the Saint Louis and West Nile viruses, and proliferate in nutrient-rich environments, such as in wastewater treatment wetlands. Our study examined whether increases in nutrients within larval mosquito developmental habitats impact microbial communities associated with C. nigripalpus mosquitoes. We characterized the effects of organic enrichments on microbiomes associated with C. nigripalpus mosquitoes and identified potential bacterial microbiota that will be further investigated for whether they alter mosquito life history traits and for their potential role in the development of microbial-based control strategies.

Oral microbes form a complex and dynamic biofilm community, which is subjected to daily host and environmental challenges. Dysbiosis of the oral biofilm is correlated with local and distal infections and postulating a baseline for the healthy core oral microbiota provides an opportunity to examine such shifts during the onset and recurrence of disease. Here we quantified the daily, weekly, and monthly variability of the oral microbiome by sequencing the largest oral microbiota time-series to date, covering multiple oral sites in ten healthy individuals. Temporal dynamics of salivary, dental, and tongue consortia were examined by high-throughput 16S rRNA gene sequencing over 90 days, with four individuals sampled additionally 1 year later. Distinct communities were observed between dental, tongue, and salivary samples, with high levels of similarity observed between the tongue and salivary communities. Twenty-six core OTUs that classified within Streptococcus, Fusobacterium, Haemophilus, Neisseria, Prevotella, and Rothia genera were present in ≥95% samples and accounted for ~65% of the total sequence data. Phylogenetic diversity varied from person to person, but remained relatively stable within individuals over time compared to inter-individual variation. In contrast, the composition of rare microorganisms was highly variable over time, within most individuals. Using machine learning, an individual's oral microbial assemblage could be correctly assigned to them with 88–97% accuracy, depending on the sample site; 83% of samples taken a year after initial sampling could be confidently traced back to the source subject., Peering into the mouth: Bacterial turnover in plaque and saliva A study of bacteria in the mouth reveals insights into their diversity, stability, and variability among people and over time. By tracking daily, weekly, and monthly fluctuations of plaque and salivary bacteria in ten healthy volunteers, Dilani Senadheera at the Faculty of Dentistry, University of Toronto and co-researchers in Canada reveal significant differences in the “microbiome” present in dental, tongue and saliva samples over time. They found considerable variation in these communities between individuals, sufficient to identify a person with “bacterial fingerprints” using plaque or saliva even after 1 year. The researchers reveal a “core community” that spans different persons, oral sites, and time, suggesting some level of stability. This study is useful to understand the diversity and community drifts in different oral sites over time, which is important when plaque and saliva are used for bacterial analysis in diagnostic, risk-prediction, and forensic applications.

The ability of Panulirus ornatus phyllosoma to capture, feed and process diets of different texture and particle size were studied during developmental stages I, III and V. Diets were captured using the spines and dactyl on the second and third pereiopods and brought to the oral cavity assisted by the second and third maxillapeds. Optimal capture was obtained when diets were a gelatinous-muscular consistency, and hard particles were caught when embedded in a muscular carrier. A range of food textures, from gelatinous to hard, were able to be masticated in the oral cavity by stage V phyllosoma. Masticated particles were filtered by phyllosoma through spines and hairs in the proventriculum, during stages I–II, and a filter press in stages III–V. Maximum particle size passing into the midgut gland for digestion was 7, 3 and 0.5 µm in stage I, III and V phyllosoma, respectively. B cells were prominent in the proximal ends of the caece of stage V phyllosoma and were visible in live animals under light microscopy.

Background The native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes. Results Beta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only ~2 % of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82 % of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes. Conclusions Bacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0475-8) contains supplementary material, which is available to authorized users.

Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes., IMPORTANCE The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This is arguably the most powerful application of metagenomics for the recovery of novel genes and a natural partner of the stable-isotope-probing approach for targeting active-yet-uncultured microorganisms. We expanded on previous efforts to combine stable-isotope probing and metagenomics, enriching microorganisms from multiple soils that were active in degrading plant-derived carbohydrates, followed by construction of a cellulose-based metagenomic library and recovery of glycoside hydrolases through functional metagenomics. The major advance of our study was the discovery of active-yet-uncultivated soil microorganisms and enrichment of their glycoside hydrolases. We recovered positive cosmid clones in a higher frequency than would be expected with direct metagenomic analysis of soil DNA. This study has generated an invaluable metagenomic resource that future research will exploit for genetic and enzymatic potential.

Anaerobic ammonia-oxidizing (anammox) bacteria are able to oxidize ammonia and reduce nitrite to produce N2 gas. After being discovered in a wastewater treatment plant (WWTP), anammox bacteria were subsequently characterized in natural environments, including marine, estuary, freshwater, and terrestrial habitats. Anammox bacteria play an important role in removing fixed N from both engineered and natural ecosystems, but broad scale anammox bacterial distributions, based on available data, have not yet been summarized. The objectives of this study were to explore global distributions and diversity of anammox bacteria and to identify factors that influence their biogeography. Over 6,000 anammox 16S rRNA gene sequences from the public database were analyzed in this current study. Data ordinations indicated that salinity was an important factor governing anammox bacterial distributions, with distinct populations inhabiting natural and engineered ecosystems. Gene phylogenies and rarefaction analysis demonstrated that freshwater environments and the marine water column harbored the highest and the lowest diversity of anammox bacteria, respectively. A co-occurrence network analysis indicated that Ca. Scalindua strongly correlated with other Ca. Scalindua taxa, whereas Ca. Brocadia co-occurred with taxa from both known and unknown anammox genera. Our survey provides a better understanding of ecological factors affecting anammox bacterial distributions and provides a comprehensive baseline for understanding the relationships among anammox communities in global environments.

Microbes are important for mosquito nutrition, growth, reproduction and control. In this study, we examined bacterial communities associated with larval mosquitoes and their habitats. Specifically, we characterized bacterial communities associated with late larval instars of the western encephalitis mosquito ( Culex tarsalis ), the submerged portions of two emergent macrophytes (California bulrush, Schoenoplectus californicus and alkali bulrush, Schoenoplectus maritimus ), and the associated water columns to investigate potential differential use of resources by mosquitoes in different wetland habitats. Using next-generation sequence data from 16S rRNA gene hypervariable regions, the alpha diversity of mosquito gut microbial communities did not differ between pond mesocosms containing distinct monotypic plants. Proteobacteria, dominated by the genus Thorsellia (Enterobacteriaceae), was the most abundant phylum recovered from C . tarsalis larvae. Approximately 49% of bacterial OTUs found in larval mosquitoes were identical to OTUs recovered from the water column and submerged portions of the two bulrushes. Plant and water samples were similar to one another, both being dominated by Actinobacteria, Bacteroidetes, Cyanobacteria , Proteobacteria and Verrucomicrobia phyla. Overall, the bacterial communities within C . tarsalis larvae were conserved and did not change across sampling dates and between two distinct plant habitats. Although Thorsellia spp. dominated mosquito gut communities, overlap of mosquito gut, plant and water-column OTUs likely reveal the effects of larval feeding. Future research will investigate the role of the key indicator groups of bacteria across the different developmental stages of this mosquito species.

Background Periodontitis is an infectious and inflammatory disease of polymicrobial etiology that can lead to the destruction of bones and tissues that support the teeth. The management of chronic periodontitis (CP) relies heavily on elimination or at least control of known pathogenic consortia associated with the disease. Until now, microbial plaque obtained from the subgingival (SubG) sites has been the primary focus for bacterial community analysis using deep sequencing. In addition to the use of SubG plaque, here, we investigated whether plaque obtained from supragingival (SupG) and tongue dorsum sites can serve as alternatives for monitoring CP-associated bacterial biomarkers. Results Using SubG, SupG, and tongue plaque DNA from 11 healthy and 13 diseased subjects, we sequenced V3 regions (approximately 200 bases) of the 16S rRNA gene using Illumina sequencing. After quality filtering, approximately 4.1 million sequences were collapsed into operational taxonomic units (OTUs; sequence identity cutoff of >97%) that were classified to a total of 19 phyla spanning 114 genera. Bacterial community diversity and overall composition was not affected by health or disease, and multiresponse permutation procedure (MRPP) on Bray-Curtis distance measures only supported weakly distinct bacterial communities in SubG and tongue plaque depending on health or disease status (P