Influences of a Prolific Gut Fungus (Zancudomyces culisetae) on Larval and Adult Mosquito (Aedes aegypti)-Associated Microbiota.

Adult mosquitoes inherit a bacterial community from larvae via transstadial transmission, an understudied process that may influence host-microbe interactions. Microbes contribute to important host life history traits, and analyzing transmitted microbial communities, the interrelationship between larval and adult-associated microbiota, and factors influencing host-microbe relationships provides targets for research. During its larval stage, the yellow fever mosquito (Aedes aegypti) hosts the trichomycete gut fungus Zancudomyces culisetae, and fungal colonization coincides with environmental perturbations in the digestive tract microecosystem. Natural populations are differentially exposed to fungi, thereby potentially harboring distinct microbiota and experiencing disparate host-microbe interactions. This study's objectives were to characterize larval and initial adult microbiomes, investigate variation in diversity and distribution of microbial communities across individuals, and assess whether larval fungal colonization impacted microbiomes at these developmental stages. Laboratory-based fungal infestation assays, sequencing of 16S rRNA gene amplicons, and bacterial load quantification protocols revealed that initial adult microbiomes varied in diversity and distribution. Larval fungal colonization had downstream effects on initial adult microbiomes, significantly reducing microbial community variation, shifting relative abundances of certain bacterial families, and influencing transstadial transmission outcomes of particular genera. Further, abundances of several families consistently decreased in adults relative to levels in larvae, possibly reflecting impacts of host development on specific bacterial taxa. These findings demonstrated that a prolific gut fungus impacted mosquito-associated microbiota at two developmental stages in an insect connected with global human health.IMPORTANCE Mosquitoes are widespread vectors of numerous human pathogens and harbor microbiota known to affect host phenotypic traits. However, little research has directly investigated how bacterial communities associated with larvae and adults are connected. We characterized whole-body bacterial communities in mosquito larvae preceding pupation and in newly emerged adults, and investigated whether a significant biotic factor, fungal colonization of the larval hindgut, impacted these microbiomes. Results showed that fungal colonization reduced microbial community variation across individuals and differentially impacted the outcomes of transstadial transmission for certain bacterial genera, revealing downstream effects of the fungus on initial adult microbiomes. The importance of our research is in providing a thorough comparative analysis of whole-body microbiota harbored in larvae and adults of the yellow fever mosquito (Aedes aegypti) and in demonstrating the important role a widespread gut fungus played in a host-associated microbiome.

Free ammonia offers algal crop protection from predators in dairy wastewater and ammonium-rich media.

Cost-effective methods for protecting crops from grazing organisms like rotifers are needed to reduce the risk of pond crashes in mass algal cultures. We present a novel strategy to optimize the exposure time to free ammonia, via control of media pH, in both defined media and dairy anaerobic digester effluent to suppress rotifers and maintain algal productivity. We tested five different free ammonia exposure times (0, 1, 2, 6, and 12h) and found a significant nonlinear effect of exposure time (p<0.0001) but not pH (p>0.9) on rotifer survival. In both media types, 6-12h of elevated free ammonia significantly reduced Brachionus plicatilis rotifer survival with no negative effects on Nannochloropsis oculata, while shorter exposure times were insufficient to inhibit rotifers, leading to severe algal culture crashes. These results suggest that algal crops can be protected from rotifers, without productivity loss, by elevating free ammonia for 6 or more hours.

Controls on Nitrous Oxide Emissions from the Hyporheic Zones of Streams.

The magnitude and mechanisms of nitrous oxide (N2O) release from rivers and streams are actively debated. The complex interactions of hydrodynamic and biogeochemical controls on emissions of this important greenhouse gas preclude prediction of when and where N2O emissions will be significant. We present observations from column and large-scale flume experiments supporting an integrative model of N2O emissions from stream sediments. Our results show a distinct, replicable, pattern of nitrous oxide generation and consumption dictated by subsurface (hyporheic) residence times and biological nitrogen reduction rates. Within this model, N2O emission from stream sediments requires subsurface residence times (and microbially mediated reduction rates) be sufficiently long (and fast reacting) to produce N2O by nitrate reduction but also sufficiently short (or slow reacting) to limit N2O conversion to dinitrogen gas. Most subsurface exchange will not result in N2O emissions; only specific, intermediate, residence times (reaction rates) will both produce and release N2O to the stream. We also confirm previous observations that elevated nitrate and declining organic carbon reactivity increase N2O production, highlighting the importance of associated reaction rates in controlling N2O accumulation. Combined, these observations help constrain when N2O release will occur, providing a predictive link between stream geomorphology, hydrodynamics, and N2O emissions.

Impacts of an ethanol-blended fuel release on groundwater and fate of produced methane: Simulation of field observations.

In a field experiment at Vandenberg Air Force Base (VAFB) designed to mimic the impact of a small-volume release of E10 (10% ethanol and 90% conventional gasoline), two plumes were created by injecting extracted groundwater spiked with benzene, toluene, and o-xylene, abbreviated BToX (No-Ethanol Lane) and BToX plus ethanol (With-Ethanol Lane) for 283 days. We developed a reactive transport model to understand processes controlling the fate of ethanol and BToX. The model was calibrated to the extensive field dataset and accounted for concentrations of sulfate, iron, acetate, and methane along with iron-reducing bacteria, sulfate-reducing bacteria, fermentative bacteria, and methanogenic archaea. The benzene plume was about 4.5 times longer in the With-Ethanol Lane than in the No-Ethanol Lane. Matching this different behavior in the two lanes required inhibiting benzene degradation in the presence of ethanol. Inclusion of iron reduction with negligible growth of iron-reducers was required to reproduce the observed constant degradation rate of benzene. Modeling suggested that vertical dispersion and diffusion of sulfate from an adjacent aquitard were important sources of sulfate in the aquifer. Matching of methane data required incorporating initial fermentation of ethanol to acetate, methane loss by outgassing, and methane oxidation coupled to sulfate and iron reduction. Simulation of microbial growth using dual Monod kinetics, and including inhibition by more favorable electron acceptors, generally resulted in reasonable yields for microbial growth of 0.01-0.05.

Hyporheic microbial community development is a sensitive indicator of metal contamination.

Accurate natural resource damage assessment necessitates monitoring organisms or communities that respond most sensitively to contaminants. Observational studies have demonstrated a correlation between fluvial heavy metal deposition and hyporheic microbial community structure. To establish a causal relationship between sediment metal content and the structure of colonizing bacterial communities, we performed a controlled field experiment River sediments of 1.75-2.36 mm in diameter with five different contaminant concentrations were collected from an environmental metal contamination gradient. Sediments were sterilized and then recolonized by incubation in the hyporheic zone of an uncontaminated river (i.e., a common garden experiment was performed). A significant correlation between hyporheic microbial community structure and heavy metal contamination (R2 = 0.81) was observed. The abundance of two phylogenetic groups was highly correlated with the level of heavy metal contamination (Group I, R2 = 0.96; Group III, R2 = 0.96, most closely affiliated with the alpha- and gamma-proteobacteria, respectively). Microbial community structural responses were detected at metal concentrations an order of magnitude lower than those previously reported to impact benthic macroinvertebrate communities. We conclude that hyporheic microbial communities could offer the most sensitive method for assessing natural resource damage in lotic ecosystems in response to fluvial heavy metal deposition.

Impact of ethanol on the natural attenuation of benzene, toluene, and o-xylene in a normally sulfate-reducing aquifer.

Side-by-side experiments were conducted in a sulfate-reducing aquifer at a former fuel station to evaluate the effect of ethanol on biodegradation of other gasoline constituents. On one side, for approximately 9 months we injected groundwater amended with 1-3 mg/L benzene, toluene, and o-xylene (BToX). On the other side, we injected the same, adding approximately 500 mg/L ethanol. Initially the BToX plumes on both sides ("lanes") extended approximately the same distance. Thereafter, the plumes in the "No Ethanol Lane" retracted significantly, which we hypothesize to be due to an initial acclimation period followed by improvement in efficiency of biodegradation under sulfate-reducing conditions. In the "With Ethanol Lane", the BToX plumes also retracted, but more slowly and not as far. The preferential biodegradation of ethanol depleted dissolved sulfate, leading to methanogenic/acetogenic conditions. We hypothesize that BToX in the ethanol-impacted lane were biodegraded in part within the methanogenic/acetogenic zone and, in part, within sulfate-reducing zones developing along the plume fringes due to mixing with sulfate-containing groundwater surrounding the plumes due to dispersion and/or shifts in flow direction. Overall, this research confirms that ethanol may reduce rates of biodegradation of aromatic fuel components in the subsurface, in both transient and near steady-state conditions.

Relationship between communities and processes; new insights from a field study of a contaminated ecosystem.

We used a 93-year-old mine waste contamination gradient in alluvial soil to explore the relationship between ecosystem level functioning and community structure in a chronically stressed ecosystem. The sensitivity of broad functional parameters (in situ soil respiration, microbial biomass, above and below ground plant biomass) and microbial diversity [phospholipid fatty acid (PLFA) abundance and richness] were compared. Functional responses were linear with respect to contaminants while thresholds were detected in the community structural response to contamination along the gradient. For example, in situ soil respiration was negatively and linearly correlated to contamination concentration (R = -0.783, P < 0.01), but changes in microbial community structure only became evident where contaminant concentrations were greater than 28 times above background levels. Our results suggest that functional redundancy does not prevent depression of ecosystem function in the long-term.

Determining rates of change and evaluating group-level resiliency differences in hyporheic microbial communities in response to fluvial heavy-metal deposition.

Prior field studies by our group have demonstrated a relationship between fluvial deposition of heavy metals and hyporheic-zone microbial community structure. Here, we determined the rates of change in hyporheic microbial communities in response to heavy-metal contamination and assessed group-level differences in resiliency in response to heavy metals. A controlled laboratory study was performed using 20 flowthrough river mesocosms and a repeated-measurement factorial design. A single hyporheic microbial community was exposed to five different levels of an environmentally relevant metal treatment (0, 4, 8, 16, and 30% sterilized contaminated sediments). Community-level responses were monitored at 1, 2, 4, 8, and 12 weeks via denaturing gradient gel electrophoresis and quantitative PCR using group-specific primer sets for indigenous populations most closely related to the alpha-, beta-, and gamma-proteobacteria. There was a consistent, strong curvilinear relationship between community composition and heavy-metal contamination (R(2) = 0.83; P < 0.001), which was evident after only 7 days of metal exposure (i.e., short-term response). The abundance of each phylogenetic group was negatively affected by the heavy-metal treatments; however, each group recovered from the metal treatments to a different extent and at a unique rate during the course of the experiment. The structure of hyporheic microbial communities responded rapidly and at contamination levels an order of magnitude lower than those shown to elicit a response in aquatic macroinvertebrate assemblages. These studies indicate that hyporheic microbial communities are a sensitive and useful indicator of heavy-metal contamination in streams.

GC fractionation enhances microbial community diversity assessment and detection of minority populations of bacteria by denaturing gradient gel electrophoresis.

Effectively and accurately assessing total microbial community diversity is one of the primary challenges in modern microbial ecology. This is particularly true with regard to the detection and characterization of unculturable populations and those present only in low abundance. We report a novel strategy, GC fractionation combined with denaturing gradient gel electrophoresis (GC-DGGE), which combines mechanistically different community analysis approaches to enhance assessment of microbial community diversity and detection of minority populations of microbes. This approach employs GC fractionation as an initial step to reduce the complexity of the community in each fraction. This reduced complexity facilitates subsequent detection of diversity in individual fractions. DGGE analysis of individual fractions revealed bands that were undetected or only poorly represented when total bacterial community DNA was analyzed. Also, directed cloning and sequencing of individual bands from DGGE lanes corresponding to individual G+C fractions allowed detection of numerous phylotypes that were not recovered using a traditional random cloning and sequencing approach.

Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient.

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R(2) = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R(2) = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the alpha, beta, and gamma-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.