Aerobic anoxygenic phototrophs play important roles in nutrient cycling within cyanobacterial Microcystis bloom microbiomes.

BACKGROUND: During the bloom season, the colonial cyanobacterium Microcystis forms complex aggregates which include a diverse microbiome within an exopolymer matrix. Early research postulated a simple mutualism existing with bacteria benefitting from the rich source of fixed carbon and Microcystis receiving recycled nutrients. Researchers have since hypothesized that Microcystis aggregates represent a community of synergistic and interacting species, an interactome, each with unique metabolic capabilities that are critical to the growth, maintenance, and demise of Microcystis blooms. Research has also shown that aggregate-associated bacteria are taxonomically different from free-living bacteria in the surrounding water. Moreover, research has identified little overlap in functional potential between Microcystis and members of its microbiome, further supporting the interactome concept. However, we still lack verification of general interaction and know little about the taxa and metabolic pathways supporting nutrient and metabolite cycling within Microcystis aggregates. RESULTS: During a 7-month study of bacterial communities comparing free-living and aggregate-associated bacteria in Lake Taihu, China, we found that aerobic anoxygenic phototrophic (AAP) bacteria were significantly more abundant within Microcystis aggregates than in free-living samples, suggesting a possible functional role for AAP bacteria in overall aggregate community function. We then analyzed gene composition in 102 high-quality metagenome-assembled genomes (MAGs) of bloom-microbiome bacteria from 10 lakes spanning four continents, compared with 12 complete Microcystis genomes which revealed that microbiome bacteria and Microcystis possessed complementary biochemical pathways that could serve in C, N, S, and P cycling. Mapping published transcripts from Microcystis blooms onto a comprehensive AAP and non-AAP bacteria MAG database (226 MAGs) indicated that observed high levels of expression of genes involved in nutrient cycling pathways were in AAP bacteria. CONCLUSIONS: Our results provide strong corroboration of the hypothesized Microcystis interactome and the first evidence that AAP bacteria may play an important role in nutrient cycling within Microcystis aggregate microbiomes. Video Abstract.

Ground-based remote sensing provides alternative to satellites for monitoring cyanobacteria in small lakes.

Cyanobacteria are the most prevalent bloom-forming harmful algae in freshwater systems around the world. Adequate sampling of affected systems is limited spatially, temporally, and fiscally. Remote sensing using space- or ground-based systems in large water bodies at spatial and temporal scales that are cost-prohibitive to standard water quality monitoring has proven to be useful in detecting and quantifying cyanobacterial harmful algal blooms. This study aimed to identify a regional 'universal' multispectral reflectance model that could be used for rapid, remote detection and quantification of cyanoHABs in small- to medium-sized productive reservoirs, such as those typical of Oklahoma, USA. We aimed to include these small waterbodies in our study as they are typically overlooked in larger, continental wide studies, yet are widely distributed and used for recreation and drinking water supply. We used Landsat satellite reflectance and in-situ pigment data spanning 16 years from 38 reservoirs in Oklahoma to construct empirical linear models for predicting concentrations of chlorophyll-a and phycocyanin, two key algal pigments commonly used for assessing total and cyanobacterial algal abundances, respectively. We also used ground-based hyperspectral reflectance and in-situ pigment data from seven reservoirs across five years in Oklahoma to build multispectral models predicting algal pigments from newly defined reflectance bands. Our Oklahoma-derived Landsat- and ground-based models outperformed established reflectance-pigment models on Oklahoma reservoirs. Importantly, our results demonstrate that ground-based multispectral models were far superior to Landsat-based models and the Cyanobacteria Index (CI) for detecting cyanoHABs in highly productive, small- to mid-sized reservoirs in Oklahoma, providing a valuable tool for water management and public health. While satellite-based remote sensing approaches have proven effective for relatively large systems, our novel results indicate that ground-based remote sensing may offer better cyanoHAB monitoring for small or highly dendritic turbid lakes, such as those throughout the southern Great Plains, and thus prove beneficial to efforts aimed at minimizing public health risks associated with cyanoHABs in supply and recreational waters.

Microcystis pangenome reveals cryptic diversity within and across morphospecies.

Microcystis, a common harmful algal bloom (HAB) taxon, threatens water supplies and human health, yet species delimitation is contentious in this taxon, leading to challenges in research and management of this threat. Historical and common morphology-based classifications recognize multiple morphospecies, most with variable and diverse ecologies, while DNA sequence-based classifications indicate a single species with multiple ecotypes. To better delimit Microcystis species, we conducted a pangenome analysis of 122 genomes. Core- and non-core gene phylogenetic analyses placed 113 genomes into 23 monophyletic clusters containing at least two genomes. Overall, genome-related indices revealed that Microcystis contains at least 16 putative genospecies. Fifteen genospecies included at least one Microcystis aeruginosa morphospecies, and 10 genospecies included two or more morphospecies. This classification system will enable consistent taxonomic identification of Microcystis and thereby aid in resolving some of the complexities and controversies that have long characterized eco-evolutionary research and management of this important HAB taxon.

Global data set of long-term summertime vertical temperature profiles in 153 lakes.

Climate change and other anthropogenic stressors have led to long-term changes in the thermal structure, including surface temperatures, deepwater temperatures, and vertical thermal gradients, in many lakes around the world. Though many studies highlight warming of surface water temperatures in lakes worldwide, less is known about long-term trends in full vertical thermal structure and deepwater temperatures, which have been changing less consistently in both direction and magnitude. Here, we present a globally-expansive data set of summertime in-situ vertical temperature profiles from 153 lakes, with one time series beginning as early as 1894. We also compiled lake geographic, morphometric, and water quality variables that can influence vertical thermal structure through a variety of potential mechanisms in these lakes. These long-term time series of vertical temperature profiles and corresponding lake characteristics serve as valuable data to help understand changes and drivers of lake thermal structure in a time of rapid global and ecological change.

Global co-occurrence of methanogenic archaea and methanotrophic bacteria in Microcystis aggregates.

Global warming and eutrophication contribute to the worldwide increase in cyanobacterial blooms, and the level of cyanobacterial biomass is strongly associated with rises in methane emissions from surface lake waters. Hence, methane-metabolizing microorganisms may be important for modulating carbon flow in cyanobacterial blooms. Here, we surveyed methanogenic and methanotrophic communities associated with floating Microcystis aggregates in 10 lakes spanning four continents, through sequencing of 16S rRNA and functional marker genes. Methanogenic archaea (mainly Methanoregula and Methanosaeta) were detectable in 5 of the 10 lakes and constituted the majority (~50%-90%) of the archaeal community in these lakes. Three of the 10 lakes contained relatively more abundant methanotrophs than the other seven lakes, with the methanotrophic genera Methyloparacoccus, Crenothrix, and an uncultured species related to Methylobacter dominating and nearly exclusively found in each of those three lakes. These three are among the five lakes in which methanogens were observed. Operational taxonomic unit (OTU) richness and abundance of methanotrophs were strongly positively correlated with those of methanogens, suggesting that their activities may be coupled. These Microcystis-aggregate-associated methanotrophs may be responsible for a hitherto overlooked sink for methane in surface freshwaters, and their co-occurrence with methanogens sheds light on the methane cycle in cyanobacterial aggregates.

Widespread deoxygenation of temperate lakes.

The concentration of dissolved oxygen in aquatic systems helps to regulate biodiversity1,2, nutrient biogeochemistry3, greenhouse gas emissions4, and the quality of drinking water5. The long-term declines in dissolved oxygen concentrations in coastal and ocean waters have been linked to climate warming and human activity6,7, but little is known about the changes in dissolved oxygen concentrations in lakes. Although the solubility of dissolved oxygen decreases with increasing water temperatures, long-term lake trajectories are difficult to predict. Oxygen losses in warming lakes may be amplified by enhanced decomposition and stronger thermal stratification8,9 or oxygen may increase as a result of enhanced primary production10. Here we analyse a combined total of 45,148 dissolved oxygen and temperature profiles and calculate trends for 393 temperate lakes that span 1941 to 2017. We find that a decline in dissolved oxygen is widespread in surface and deep-water habitats. The decline in surface waters is primarily associated with reduced solubility under warmer water temperatures, although dissolved oxygen in surface waters increased in a subset of highly productive warming lakes, probably owing to increasing production of phytoplankton. By contrast, the decline in deep waters is associated with stronger thermal stratification and loss of water clarity, but not with changes in gas solubility. Our results suggest that climate change and declining water clarity have altered the physical and chemical environment of lakes. Declines in dissolved oxygen in freshwater are 2.75 to 9.3 times greater than observed in the world's oceans6,7 and could threaten essential lake ecosystem services2,3,5,11.

Deeper waters are changing less consistently than surface waters in a global analysis of 102 lakes.

Globally, lake surface water temperatures have warmed rapidly relative to air temperatures, but changes in deepwater temperatures and vertical thermal structure are still largely unknown. We have compiled the most comprehensive data set to date of long-term (1970-2009) summertime vertical temperature profiles in lakes across the world to examine trends and drivers of whole-lake vertical thermal structure. We found significant increases in surface water temperatures across lakes at an average rate of + 0.37 °C decade-1, comparable to changes reported previously for other lakes, and similarly consistent trends of increasing water column stability (+ 0.08 kg m-3 decade-1). In contrast, however, deepwater temperature trends showed little change on average (+ 0.06 °C decade-1), but had high variability across lakes, with trends in individual lakes ranging from - 0.68 °C decade-1 to + 0.65 °C decade-1. The variability in deepwater temperature trends was not explained by trends in either surface water temperatures or thermal stability within lakes, and only 8.4% was explained by lake thermal region or local lake characteristics in a random forest analysis. These findings suggest that external drivers beyond our tested lake characteristics are important in explaining long-term trends in thermal structure, such as local to regional climate patterns or additional external anthropogenic influences.

The global Microcystis interactome.

Bacteria play key roles in the function and diversity of aquatic systems, but aside from study of specific bloom systems, little is known about the diversity or biogeography of bacteria associated with harmful cyanobacterial blooms (cyanoHABs). CyanoHAB species are known to shape bacterial community composition and to rely on functions provided by the associated bacteria, leading to the hypothesized cyanoHAB interactome, a coevolved community of synergistic and interacting bacteria species, each necessary for the success of the others. Here, we surveyed the microbiome associated with Microcystis aeruginosa during blooms in 12 lakes spanning four continents as an initial test of the hypothesized Microcystis interactome. We predicted that microbiome composition and functional potential would be similar across blooms globally. Our results, as revealed by 16S rRNA sequence similarity, indicate that M. aeruginosa is cosmopolitan in lakes across a 280° longitudinal and 90° latitudinal gradient. The microbiome communities were represented by a wide range of operational taxonomic units and relative abundances. Highly abundant taxa were more related and shared across most sites and did not vary with geographic distance, thus, like Microcystis, revealing no evidence for dispersal limitation. High phylogenetic relatedness, both within and across lakes, indicates that microbiome bacteria with similar functional potential were associated with all blooms. While Microcystis and the microbiome bacteria shared many genes, whole-community metagenomic analysis revealed a suite of biochemical pathways that could be considered complementary. Our results demonstrate a high degree of similarity across global Microcystis blooms, thereby providing initial support for the hypothesized Microcystis interactome.

Contrasting Network Features between Free-Living and Particle-Attached Bacterial Communities in Taihu Lake.

Free-living (FL) and particle-attached (PA) bacterial communities play critical roles in nutrient cycles, metabolite production, and as a food source in aquatic systems, and while their community composition, diversity, and functions have been well studied, we know little about their community interactions, co-occurrence patterns, and niche occupancy. In the present study, 13 sites in Taihu Lake were selected to study the differences of co-occurrence patterns and niches occupied between the FL and PA bacterial communities using correlation-based network analysis. The results show that both FL and PA bacterial community networks were non-random and significant differences of the network indexes (average path length, clustering coefficient, modularity) were found between the two groups. Furthermore, the PA bacterial community network consisted of more correlations between fewer OTUs, as well as higher average degree, making it more complex. The results of observed (O) to random (R) ratios of intra- or inter-phyla connections indicate more relationships such as cross-feeding, syntrophic, mutualistic, or competitive relationships in the PA bacterial community network. We also found that four OTUs (OTU00074, OTU00755, OTU00079, and OTU00454), which all had important influences on the nutrients cyclings, played different roles in the two networks as connectors or module hubs. Analysis of the relationships between the module eigengenes and environmental variables demonstrated that bacterial groups of the two networks favored quite different environmental conditions. These findings further confirmed the different ecological functions and niches occupied by the FL and PA bacterial communities in the aquatic ecosystem.

Ecological Patterns Among Bacteria and Microbial Eukaryotes Derived from Network Analyses in a Low-Salinity Lake.

Microbial communities are comprised of complex assemblages of highly interactive taxa. We employed network analyses to identify and describe microbial interactions and co-occurrence patterns between microbial eukaryotes and bacteria at two locations within a low salinity (0.5-3.5 ppt) lake over an annual cycle. We previously documented that the microbial diversity and community composition within Lake Texoma, southwest USA, were significantly affected by both seasonal forces and a site-specific bloom of the harmful alga, Prymnesium parvum. We used network analyses to answer ecological questions involving both the bacterial and microbial eukaryotic datasets and to infer ecological relationships within the microbial communities. Patterns of connectivity at both locations reflected the seasonality of the lake including a large rain disturbance in May, while a comparison of the communities between locations revealed a localized response to the algal bloom. A network built from shared nodes (microbial operational taxonomic units and environmental variables) and correlations identified conserved associations at both locations within the lake. Using network analyses, we were able to detect disturbance events, characterize the ecological extent of a harmful algal bloom, and infer ecological relationships not apparent from diversity statistics alone.

Dynamics of an experimental microbial invasion.

The ecological dynamics underlying species invasions have been a major focus of research in macroorganisms for the last five decades. However, we still know little about the processes behind invasion by unicellular organisms. To expand our knowledge of microbial invasions, we studied the roles of propagule pressure, nutrient supply, and biotic resistance in the invasion success of a freshwater invasive alga, Prymnesium parvum, using microcosms containing natural freshwater microbial assemblages. Microcosms were subjected to a factorial design with two levels of nutrient-induced diversity and three levels of propagule pressure, and incubated for 7 d, during which P. parvum densities and microbial community composition were tracked. Successful invasion occurred in microcosms receiving high propagule pressure whereas nutrients or community diversity played no role in invasion success. Invaded communities experienced distinctive changes in composition compared with communities where the invasion was unsuccessful. Successfully invaded microbial communities had an increased abundance of fungi and ciliates, and decreased abundances of diatoms and cercozoans. Many of these changes mirrored the microbial community changes detected during a natural P. parvum bloom in the source system. This role of propagule pressure is particularly relevant for P. parvum in the reservoir-dominated southern United States because this species can form large, sustained blooms that can generate intense propagule pressures for downstream sites. Human impact and global climate change are currently causing widespread environmental changes in most southern US freshwater systems that may facilitate P. parvum establishment and, when coupled with strong propagule pressure, could put many more systems at risk for invasion.

Gene expression in the mixotrophic prymnesiophyte, Prymnesium parvum, responds to prey availability.

The mixotrophic prymnesiophyte, Prymnesium parvum, is a widely distributed alga with significant ecological importance. It produces toxins and can form ecosystem disruptive blooms that result in fish kills and changes in planktonic food web structure. However, the relationship between P. parvum and its prey on the molecular level is poorly understood. In this study, we used RNA-Seq technology to study changes in gene transcription of P. parvum in three treatments with different microbial populations available as potential prey: axenic P. parvum (no prey), bacterized P. paruvm, and axenic P. parvum with ciliates added as prey. Thousands of genes were differentially expressed among the three treatments. Most notably, transcriptome data indicated that P. parvum obtained organic carbon, including fatty acids, from both bacteria and ciliate prey for energy and cellular building blocks. The data also suggested that different prey provided P. parvum with macro- and micro-nutrients, namely organic nitrogen in the form of amino acids from ciliates, and iron from bacteria. However, both transcriptomic data and growth experiments indicated that P. parvum did not grow faster in the presence of prey despite the gains in nutrients, although algal abundances attained in culture were slightly greater in the presence of prey. The relationship between phototrophy, heterotrophy and growth of P. parvum is discussed.

The niche of an invasive marine microbe in a subtropical freshwater impoundment.

Growing attention in aquatic ecology is focusing on biogeographic patterns in microorganisms and whether these potential patterns can be explained within the framework of general ecology. The long-standing microbiologist's credo 'Everything is everywhere, but, the environment selects' suggests that dispersal is not limiting for microbes, but that the environment is the primary determining factor in microbial community composition. Advances in molecular techniques have provided new evidence that biogeographic patterns exist in microbes and that dispersal limitation may actually have an important role, yet more recent study using extremely deep sequencing predicts that indeed everything is everywhere. Using a long-term field study of the 'invasive' marine haptophyte Prymnesium parvum, we characterize the environmental niche of P. parvum in a subtropical impoundment in the southern United States. Our analysis contributes to a growing body of evidence that indicates a primary role for environmental conditions, but not dispersal, in the lake-wide abundances and seasonal bloom patterns in this globally important microbe.

Seasonality and disturbance: annual pattern and response of the bacterial and microbial eukaryotic assemblages in a freshwater ecosystem.

High-throughput pyrosequencing of SSU rDNA genes was used to obtain monthly snapshots of eukaryotic and bacterial diversity and community structure at two locations in Lake Texoma, a low salinity lake in the south central United States, over 1 year. The lake experienced two disturbance events (i) a localized bloom of Prymnesium parvum restricted to one of the locations that lasted from January to April, and (ii) a large (17 cm), global rain event in the beginning of May, overlaid onto seasonal environmental change. Eukaryotic species richness as well as both eukaryotic and bacterial community similarity exhibited seasonal patterns, including distinct responses to the rain event. The P. parvum bloom created a natural experiment in which to directly explore the effects of an Ecosystem Disruptive Algal Bloom (EDAB) on the microbial community separated from seasonal changes. Microbial species richness was unaffected by the bloom, however, the eukaryotic community structure (evenness) and the patterns of both eukaryotic and bacterial community similarity at bloom and non-bloom sites were statistically distinct during the 4 months of the bloom. These results indicate that physical and biological disturbances as well as seasonal environmental forces contribute to the structure of both the eukaryotic and bacterial communities.

Toxin-assisted micropredation: experimental evidence shows that contact micropredation rather than exotoxicity is the role of Prymnesium toxins.

Blooms of Prymnesium parvum can severely harm fish and zooplankton, presumably through the release of allelopathic exotoxins that offer advantages for Prymnesium in its interactions with competitors and prey. We show that Prymnesium attaches to zooplankton and fish, causing mortality, whereas exposure of these organisms to Prymnesium across a permeable membrane does not cause mortality. We also show that Prymnesium exotoxins are released independently of contact toxicity only in response to experimental procedures or natural causes of stress. Our results are consistent with the idea that toxins have evolved for release during cell-to-cell contact in support of heterotrophy. The evolution of toxin-assisted micropredation would be consistent with mechanisms of natural selection favouring individual fitness as opposed to broadcast allelopathy from which the benefits are more dispersed. Research into the toxicity of Prymnesium and other harmful algal species may profit from focus on processes following physical contact with potential prey.

Variation in resource consumption across a gradient of increasing intra- and interspecific richness.

Based on the premise that ecosystems with more species will function at more efficient rates, declining biodiversity is expected to alter important ecosystem functions, goods, and services across the globe. However, applicability of this general hypothesis to genetic or clonal richness in assemblages composed of few species is understudied. This illustrates the need to expand the focus of biodiversity-ecosystem-function experiments across all levels of biological diversity (including genetic). To explore this generality, we manipulated intraspecific (clonal) and interspecific (species) richness of a primary consumer, Daphnia, and measured assemblage feeding rate and total resource consumption. Our results showed that greater clonal richness had no effect on Daphnia feeding, and greater species richness decreased feeding-related effects of Daphnia. This suggests that multiclonal Daphnia assemblages may be no more efficient at consuming resources than monocultures, and that monocultures of Daphnia may consume resources more efficiently than more species-rich assemblages. The inhibitory effect of increasing richness observed in this study resulted from chemical and mechanical interference among some of the Daphnia taxa. This suggests that consumer-mediated ecosystem functions could be reduced when assemblages include taxa equipped with adaptations for interference competition.

Factors influencing mercury accumulation in three species of forage fish from Caddo Lake, Texas, USA.

Most studies that have examined mercury (Hg) contamination of fish have focused on game species feeding near the top of the food web, while studies that examine forage fish that feed near the base of the food web are rare. We conducted a survey of Hg contamination in three species of forage fish, brook silverside (Labidesthes sicculus), threadfin shad (Dorosoma petenense) and gizzard shad (Dorosoma cepedianum), from Caddo Lake, Texas, USA and found species-specific differences in Hg concentrations. We examined total length, age, trophic position (determined using delta15N), and growth rate of forage fish as factors that could have influenced within- and between-species differences in Hg concentration. Total length and age were the best predictors of within-species differences in Hg concentration. Between-species differences in Hg concentrations were most strongly influenced by trophic position.

Reassessing the ichthyotoxin profile of cultured Prymnesium parvum (golden algae) and comparing it to samples collected from recent freshwater bloom and fish kill events in North America.

Within the last two decades, Prymnesium parvum (golden algae) has rapidly spread into inland waterways across the southern portion of North America and this organism has now appeared in more northerly distributed watersheds. In its wake, golden algae blooms have left an alarming trail of ecological devastation, namely massive fish kills, which are threatening the economic and recreational value of freshwater systems throughout the United States. To further understand the nature of this emerging crisis, our group investigated the chemical nature of the toxin(s) produced by P. parvum. We approached the problem using a two-pronged strategy that included analyzing both laboratory-grown golden algae and field-collected samples of P. parvum. Our results demonstrate that there is a striking difference in the toxin profiles for these two systems. An assemblage of potently ichthyotoxic fatty acids consisting primarily of stearidonic acid was identified in P. parvum cultures. While the concentration of the fatty acids alone was sufficient to account for the rapid-onset ichthyotoxic properties of cultured P. parvum, we also detected a second type of highly labile ichthyotoxic substance(s) in laboratory-grown golden algae that remains uncharacterized. In contrast, the amounts of stearidonic acid and its related congeners present in samples from recent bloom and fish kill sites fell well below the limits necessary to induce acute toxicity in fish. However, a highly labile ichthyotoxic substance, which is similar to the one found in laboratory-grown P. parvum cultures, was also detected. We propose that the uncharacterized labile metabolite produced by P. parvum is responsible for golden algae's devastating fish killing effects. Moreover, we have determined that the biologically-relevant ichthyotoxins produced by P. parvum are not the prymnesins as is widely believed. Our results suggest that further intensive efforts will be required to chemically define P. parvum's ichthyotoxins under natural bloom conditions.

WITHDRAWN: A revised amino group pK(a) for prymnesins does not provide decisive evidence for a pH-dependent mechanism of Prymnesium parvum's toxicity.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi:10.1016/j.toxicon.2010.02.008. The duplicate article has therefore been withdrawn.