Kevin A. Kuehn

Algal regulation of extracellular enzyme activity in stream microbial communities associated with inert substrata and detritus

Abstract We tested the hypothesis that algae influence the activities of extracellular enzymes involved in mineralization processes within microbial assemblages in streams. We tested the prediction that the factors that influence algal biomass and photosynthesis (i.e., diel fluctuations in photosynthetically active radiation [PAR], long-term variations in light regime, and community development stage) would have a corresponding effect on extracellular enzyme activities. We also tested the prediction that algae would influence enzyme activities on inorganic substrata and in detrital communities where they ultimately would influence plant litter decomposition rates. We allowed microbial communities to develop on inert substrata (glass-fiber filters) or on leaf litter in artificial streamside channels. For each community type, we examined the effects of long-term light manipulations, community development stage, and diel periodicity on the activities of β-glucosidase, alkaline phosphatase, leucine-aminopeptidase, and phenol oxidase. In addition, we measured the decomposition rates of the leaf litter substrata in the low- and high-light treatments. Our results support the prediction that factors that influence algal photosynthesis and biomass in the short (diel fluctuations in PAR) and long (shading, community development stage) term ultimately influence enzyme activities in microbial communities associated with both inorganic substrata and detritus. Furthermore, decomposition rates of organic detritus probably are enhanced by algal colonization and activity. Algal photosynthesis might enhance redox and pH conditions within microbial communities, and in turn, might increase the activities of oxidative and hydrolytic enzymes. As a consequence, photoautotrophic activities might stimulate heterotrophic pathways in stream ecosystems by creating conditions favorable for decomposition of both dissolved and particulate organic detritus.

Priming in the microbial landscape: periphytic algal stimulation of litter‐associated microbial decomposers

Microbial communities associated with submerged detritus in aquatic ecosystems often comprise a diverse mixture of autotrophic and heterotrophic microbes, including algae, bacteria, protozoa, and fungi. Recent studies have documented increased rates of plant litter mass loss when periphytic algae are present. We conducted laboratory and field experiments to assess potential metabolic interactions between natural autotrophic and heterotrophic microbial communities inhabiting submerged decaying plant litter of Typha angustifolia and Schoenoplectus acutus. In the field, submerged plant litter was either exposed to natural sunlight or placed under experimental canopies that manipulated light availability and growth of periphytic algae. Litter was collected and returned to the laboratory, where algal photosynthesis was manipulated (light/dark incubation), while rates of bacterial and fungal growth and productivity were simultaneously quantified. Bacteria and fungi were rapidly stimulated by exposure to light, thus establishing the potential for algal priming of microbial heterotrophic decay activities. Experimental incubations of decaying litter with 14C- and 13C-bicarbonate established that inorganic C fixed by algal photosynthesis was rapidly transferred to and assimilated by heterotrophic microbial decomposers. Periphytic algal stimulation of microbial heterotrophs, especially fungal decomposers, is an important and largely unrecognized interaction within the detrital microbial landscape, which may transform our current conceptual understanding of microbial secondary production and organic matter decomposition in aquatic ecosystems.

Constitutive Differences between Natural and Artificial Container Mosquito Habitats: Vector Communities, Resources, Microorganisms, and Habitat Parameters

ABSTRACT Aquatic containers, including tree holes and vehicle tires, harbor a diverse assemblage of mosquitoes capable of vectoring important diseases. Many studies have examined containers as a mosquito breeding site, although no data exist that have simultaneously compared mosquito communities between tree holes and tires, and few have quantified differences in environmental factors or food resources that may be important for explaining population or community differences. At two times (early and late summer 2009) we sampled two tire and two tree hole sites in south-central Mississippi, and for each container we enumerated mosquito larvae and measured several environmental parameters (canopy cover, water volume, and detritus), and biomass and productivity of fungi and bacteria, and species richness and abundance of protozoans. Tree holes held less water but were more shaded compared with tires; however, after correcting for volume differences, tree holes contained more detritus and were higher in some microorganism measures (protozoan richness, bacterial productivity in the water column). Based on community dissimilarity analysis of mosquitoes, strong differences existed between container types and sampling period; Aedes albopictus (Skuse) and Culex quinquefasciatus (Say) were dominant in tires, whereas Ae. triseriatus (Say) and Orthopodomyia signifera (Coquillett) were dominant in tree holes. This study also reports the use of tires by the invasive mosquito Cx. coronator (Dyar and Knab). Tree holes supported a higher density of larvae but fewer species than tires, though there was variation across time. Our work illustrates that detrital inputs and some microorganisms differ in fundamental ways between tires and tree holes, and because of compositional differences in mosquito communities, these small aquatic habitats cannot be considered to be homogeneous mosquito habitats.

Fungi in Biogeochemical Cycles: The role of fungi in carbon and nitrogen cycles in freshwater ecosystems

Introduction Fungi are adapted to a diverse array of freshwater ecosystems. In streams and rivers, flowing water provides a mechanism for downstream dispersal of fungal propagules. The dominant group of fungi in these habitats, aquatic hyphomycetes, have conidia that are morphologically adapted (tetraradiate and sigmoid) for attachment to their substrates (leaf litter andwoody debris from riparian vegetation) in flowing water (Webster, 1959; Webster & Davey, 1984). In freshwater wetlands and lake littoral zones, production of emergent aquatic macrophytes is often extremely high, resulting in an abundance of plantmaterial that eventually enters the detrital pool. The dead shootmaterial of thesemacrophytes (leaf blades, leaf sheaths and culms) often remains standing for long periods of time before collapsing to the sediments or water. This plant matter is colonized by fungi that are adapted for surviving the harsh conditions that prevail in the standing-dead environment (Kuehn et al., 1998). There are a number of other freshwater ecosystems where fungi are present and exhibit interesting adaptations, e.g. aero-aquatic fungi in woodland ponds, zoosporic organisms (Chytridiomycota and Oomycota) in a variety of habitats including the pelagic zones of lakes, and Trichomycetes that inhabit the guts of a variety of aquatic insects. Despite the well-known occurrence of these fungal groups in aquatic habitats, virtually nothing is known concerning their roles in biogeochemical processes. Overall, the contributions of fungi to biogeochemical cycles have been understudied in most freshwater ecosystems. Most studies examining fungal participation in biogeochemical cycles in freshwater ecosystems focused on the role of fungi in the decomposition of plant litter.Historically, the lackof appropriatemethods toaccuratelyquantify

Application of the [3H]Leucine Incorporation Technique for Quantification of Bacterial Secondary Production Associated with Decaying Wetland Plant Litter

ABSTRACT The radiolabeled leucine incorporation technique for quantifying rates of bacterial production has increased in popularity since its original description for bacterioplankton communities. Prior studies addressing incorporation conditions (e.g., substrate saturation) for bacterial communities in other habitats, such as decaying plant litter, have reported a wide range of final leucine concentrations (400 nM to 50 μM) required to achieve saturation-level uptake. We assessed the application of the [3H]leucine incorporation procedure for measuring bacterial production on decaying wetland plant litter. Substrate saturation experiments (nine concentrations, 10 nM to 50 μM final leucine concentration) were conducted on three dates for microbial communities colonizing the submerged litter of three emergent plant species (Typha angustifolia, Schoenoplectus validus, and Phragmites australis). A modified [3H]leucine protocol was developed by coupling previously described incubation and alkaline extraction protocols with microdialysis (500 molecular weight cutoff membrane) of the final radiolabeled protein extract. The incorporation of [3H]leucine into protein exhibited a biphasic saturation curve, with lower apparent Km values ranging from 400 nM to 4.2 μM depending on the plant species studied. Upper apparent Km values ranged from 1.3 to 59 μM. These results suggest differential uptake by litter-associated microbial assemblages, with the lower apparent Km values possibly representing bacterial uptake and higher apparent Km values representing a combination of both bacterial and nonbacterial (e.g., eukaryotic) uptake.

Evaluation of the efficacy of the photosystem II inhibitor DCMU in periphyton and its effects on nontarget microorganisms and extracellular enzymatic reactions

Abstract We examined the efficacy of the photosystem II inhibitor 3-(3,4-diclorophenyl)-1,1-dimethyl urea (DCMU) for inhibition of algal photosynthesis in periphyton associated with submerged decomposing litter of Typha angustifolia. We also investigated the possible nontarget effects of DCMU exposure on heterotrophic microorganisms (i.e., bacteria and fungi) and extracellular enzyme activity associated with decaying litter. Standing-dead Typha leaf litter was submerged for 34 and 73 d, returned to the laboratory, and used for controlled laboratory experiments that examined the effect of DCMU on algal ([14C]bicarbonate, pulse-amplitude modulated fluorometry), bacterial ([3H]leucine), and fungal ([14C]acetate) production. Simultaneous assays also were conducted to examine the effect of DCMU on the activities of 4 extracellular enzymes (β-glucosidase, β-xylosidase, leucine-aminopeptidase, and phosphatase). DCMU significantly inhibited algal photosynthesis in light-exposed periphyton (p always < 0.0003), with strong inhibitory effects occurring within 5 min after exposure to DCMU. In contrast, DCMU had no significant direct effect on bacterial (p > 0.5) or fungal production (p > 0.3). Extracellular enzyme activities also were not significantly affected by exposure to DCMU. Heterotrophic microbial and enzyme activity assays were conducted in darkness to avoid any indirect effects of DCMU (i.e., heterotrophic responses to the inhibition of photosynthesis, rather than to DCMU itself). The apparent lack of nontarget effects of DCMU on heterotrophic microbial processes, combined with good efficacy against algal photosynthesis, suggest that DCMU may a useful selective inhibitor for investigations of interactions among litter-inhabiting microbiota.

Mosquito Larvae in Tires from Mississippi, United States: The Efficacy of Abiotic and Biotic Parameters in Predicting Spatial and Temporal Patterns of Mosquito Populations and Communities

ABSTRACT Container systems, including discarded vehicle tires, which support populations of mosquitoes, have been of interest for understanding the variables that produce biting adults that serve as both nuisances and as public health threats. We sampled tires in six sites at three times in 2012 across the state of Mississippi to understand the biotic and abiotic variables responsible for explaining patterns of larvae of common species, species richness, and total abundance of mosquitoes. From 498 tires sampled, we collected >58,000 immatures representing 16 species, with the most common species including Aedes albopictus (Skuse), Culex quinquefasciatus (L.), Orthopodomyia signifera (Coquillett), Aedes triseriatus (Say), Toxorhynchites rutilus septentrionalis (Coquillett), and Culex territans (Walker) accounting for ∼97% of all larvae. We also documented 32 new county records for resident species and recent arrivals in the state, including Aedes japonicus japonicus (Theobald) and Culex coronator (Dyar & Knab). Cluster analysis, which was used to associate sites and time periods based on similar mosquito composition, did reveal patterns across the state; however, there also were more general patterns between species and genera and environmental factors. Broadly, Aedes was often associated with factors related to detritus, whereas Culex was frequently associated with habitat variables (e.g., tire size and water volume) and microorganisms. Some Culex did lack factors connecting variation in early and late instars, suggesting differences between environmental determinants of oviposition and survival. General patterns between the tire environment and mosquito larvae do appear to exist, especially at the generic level, and point to inherent differences between genera that may aid in predicting vector locations and populations.

Changes in nutrient stoichiometry, elemental homeostasis and growth rate of aquatic litter-associated fungi in response to inorganic nutrient supply

Aquatic fungi mediate important energy and nutrient transfers in freshwater ecosystems, a role potentially altered by widespread eutrophication. We studied the effects of dissolved nitrogen (N) and phosphorus (P) concentrations and ratios on fungal stoichiometry, elemental homeostasis, nutrient uptake and growth rate in two experiments that used (1) liquid media and a relatively recalcitrant carbon (C) source and (2) fungi grown on leaf litter in microcosms. Two monospecific fungal cultures and a multi-species assemblage were assessed in each experiment. Combining a radioactive tracer to estimate fungal production (C accrual) with N and P uptake measurements provided an ecologically relevant estimate of mean fungal C:N:P of 107:9:1 in litter-associated fungi, similar to the 92:9:1 obtained from liquid cultures. Aquatic fungi were found to be relatively homeostatic with respect to their C:N ratio (~11:1), but non-homeostatic with respect to C:P and N:P. Dissolved N greatly affected fungal growth rate and production, with little effect on C:nutrient stoichiometry. Conversely, dissolved P did not affect fungal growth and production but controlled biomass C:P and N:P, probably via luxury P uptake and storage. The ability of fungi to immobilize and store excess P may alter nutrient flow through aquatic food webs and affect ecosystem functioning.

Effects of UV Radiation on Wetland Periphyton: Algae, Bacteria, and Extracellular Polysaccharides

ABSTRACT To study the effects of UV radiation on wetland periphyton, glass substrata were placed under acrylic mesocosms in a small freshwater marsh in Michigan, USA. One treatment excluded light in the UV range (<400nm), and another allowed the passage of all light. Substrata were sampled on four dates during August and September 2004. Chlorophyll a, algal community composition, ash-free dry mass, bacterial density and viability, and total extracellular polysaccharide (EPS) content were measured. Algal biomass, algal community composition, bacterial abundance, and ash-free dry mass were relatively insensitive to UV treatment; however, the proportion of non-viable bacteria (p = 0.006) and area-specific EPS content (p = 0.025) were significantly greater in the UV-exposed treatment. These results suggest that ambient levels of UV can damage periphytic bacteria and may cause corresponding functional changes within naturally occurring periphytic communities.

Contribution of surface leaf-litter breakdown and forest composition to benthic oxygen demand and ecosystem respiration in a South Georgia blackwater river

Abstract Many North American blackwater rivers exhibit low dissolved O2 (DO) that may be the result of benthic respiration. We examined how tree species affected O2 demand via the quantity and quality of litter produced. In addition, we compared areal estimates of surface leaf-litter microbial respiration to sediment O2 demand (SOD) and ecosystem respiration (ER) in stream and swamp reaches of a blackwater river to quantify contributions of surface litter decomposition to O2 demand. Litter inputs averaged 917 and 678 g m-2 y-1 in the swamp and stream, respectively. Tree species differentially affected O2 demand via the quantity and quality of litter produced. Bald cypress (Taxodium distichum) contributed most litter inputs because of its dominance and because it produced more litter per tree, thereby making greater relative contributions to O2 demand in the swamp. In the stream, water oak (Quercus nigra) produced litter supporting lower fungal biomass and O2 uptake rates, but produced more litter than red maple (Acer rubrum). Breakdown rates in the swamp were faster, whereas standing stock decreases were lower than in the stream, indicating greater organic matter retention. Surface litter microbial respiration accounted for 89% of SOD (6.37 g O2 m-2 d-1), and 57 to 89% of ER in the swamp. Our findings suggest that surface litter drives the majority of O2 demand in some blackwater swamps, and tree species with higher rates of litterfall may make larger contributions to ER. Forested swamps may be hotspots of O2 demand in blackwater rivers because low water velocities enhance retention.