A Glyphosate-Based Herbicide Cross-Selects for Antibiotic Resistance Genes in Bacterioplankton Communities.

Agrochemicals often contaminate freshwater bodies, affecting microbial communities that underlie aquatic food webs. For example, the herbicide glyphosate has the potential to indirectly select for antibiotic-resistant bacteria. Such cross-selection could occur if the same genes (encoding efflux pumps, for example) confer resistance to both glyphosate and antibiotics. To test for cross-resistance in natural aquatic bacterial communities, we added a glyphosate-based herbicide (GBH) to 1,000-liter mesocosms filled with water from a pristine lake. Over 57 days, we tracked changes in bacterial communities with shotgun metagenomic sequencing and annotated metagenome-assembled genomes (MAGs) for the presence of known antibiotic resistance genes (ARGs), plasmids, and resistance mutations in the enzyme targeted by glyphosate (enolpyruvyl-shikimate-3-phosphate synthase; EPSPS). We found that high doses of GBH significantly increased ARG frequency and selected for multidrug efflux pumps in particular. The relative abundance of MAGs after a high dose of GBH was predictable based on the number of ARGs in their genomes (17% of variation explained) and, to a lesser extent, by resistance mutations in EPSPS. Together, these results indicate that GBHs can cross-select for antibiotic resistance in natural freshwater bacteria. IMPORTANCE Glyphosate-based herbicides (GBHs) such as Roundup formulations may have the unintended consequence of selecting for antibiotic resistance genes (ARGs), as demonstrated in previous experiments. However, the effects of GBHs on ARGs remain unknown in natural aquatic communities, which are often contaminated with pesticides from agricultural runoff. Moreover, the resistance provided by ARGs compared to canonical mutations in the glyphosate target enzyme, EPSPS, remains unclear. Here, we performed a freshwater mesocosm experiment showing that a GBH strongly selects for ARGs, particularly multidrug efflux pumps. These selective effects were evident after just a few days, and the ability of bacteria to survive and thrive after GBH stress was predictable by the number of ARGs in their genomes and, to a lesser extent, by mutations in EPSPS. Intensive GBH application may therefore have the unintended consequence of selecting for ARGs in natural freshwater communities.

Current water quality guidelines across North America and Europe do not protect lakes from salinization.

Human-induced salinization caused by the use of road deicing salts, agricultural practices, mining operations, and climate change is a major threat to the biodiversity and functioning of freshwater ecosystems. Yet, it is unclear if freshwater ecosystems are protected from salinization by current water quality guidelines. Leveraging an experimental network of land-based and in-lake mesocosms across North America and Europe, we tested how salinization-indicated as elevated chloride (Cl-) concentration-will affect lake food webs and if two of the lowest Cl- thresholds found globally are sufficient to protect these food webs. Our results indicated that salinization will cause substantial zooplankton mortality at the lowest Cl- thresholds established in Canada (120 mg Cl-/L) and the United States (230 mg Cl-/L) and throughout Europe where Cl- thresholds are generally higher. For instance, at 73% of our study sites, Cl- concentrations that caused a ≥50% reduction in cladoceran abundance were at or below Cl- thresholds in Canada, in the United States, and throughout Europe. Similar trends occurred for copepod and rotifer zooplankton. The loss of zooplankton triggered a cascading effect causing an increase in phytoplankton biomass at 47% of study sites. Such changes in lake food webs could alter nutrient cycling and water clarity and trigger declines in fish production. Current Cl- thresholds across North America and Europe clearly do not adequately protect lake food webs. Water quality guidelines should be developed where they do not exist, and there is an urgent need to reassess existing guidelines to protect lake ecosystems from human-induced salinization.

Warming winters in lakes: Later ice onset promotes consumer overwintering and shapes springtime planktonic food webs.

Global climate warming is causing the loss of freshwater ice around the Northern Hemisphere. Although the timing and duration of ice covers are known to regulate ecological processes in seasonally ice-covered ecosystems, the consequences of shortening winters for freshwater biota are poorly understood owing to the scarcity of under-ice research. Here, we present one of the first in-lake experiments to postpone ice-cover onset (by ≤21 d), thereby extending light availability (by ≤40 d) in early winter, and explicitly demonstrate cascading effects on pelagic food web processes and phenologies. Delaying ice-on elicited a sequence of events from winter to spring: 1) relatively greater densities of algal resources and primary consumers in early winter; 2) an enhanced prevalence of winter-active (overwintering) consumers throughout the ice-covered period, associated with augmented storage of high-quality fats likely due to a longer access to algal resources in early winter; and 3) an altered trophic structure after ice-off, with greater initial springtime densities of overwintering consumers driving stronger, earlier top-down regulation, effectively reducing the spring algal bloom. Increasingly later ice onset may thus promote consumer overwintering, which can confer a competitive advantage on taxa capable of surviving winters upon ice-off; a process that may diminish spring food availability for other consumers, potentially disrupting trophic linkages and energy flow pathways over the subsequent open-water season. In considering a future with warmer winters, these results provide empirical evidence that may help anticipate phenological responses to freshwater ice loss and, more broadly, constitute a case of climate-induced cross-seasonal cascade on realized food web processes.

Widespread agrochemicals differentially affect zooplankton biomass and community structure.

Anthropogenic environmental change is causing habitat deterioration at unprecedented rates in freshwater ecosystems. Despite increasing more rapidly than many other agents of global change, synthetic chemical pollution-including agrochemicals such as pesticides-has received relatively little attention in freshwater community and ecosystem ecology. Determining the combined effects of multiple agrochemicals on complex biological systems remains a major challenge, requiring a cross-field integration of ecology and ecotoxicology. Using a large-scale array of experimental ponds, we investigated the response of zooplankton community properties (biomass, composition, and diversity metrics) to the individual and joint presence of three globally widespread agrochemicals: the herbicide glyphosate, the neonicotinoid insecticide imidacloprid, and nutrient fertilizers. We tracked temporal variation in zooplankton biomass and community structure along single and combined pesticide gradients (each spanning eight levels), under low (mesotrophic) and high (eutrophic) nutrient-enriched conditions, and quantified (1) response threshold concentrations, (2) agrochemical interactions, and (3) community resistance and recovery. We found that the biomass of major zooplankton groups differed in their sensitivity to pesticides: ≥0.3 mg/L glyphosate elicited long-lasting declines in rotifer communities, both pesticides impaired copepods (≥3 µg/L imidacloprid and ≥5.5 mg/L glyphosate), whereas some cladocerans were highly tolerant to pesticide contamination. Strong interactive effects of pesticides were only recorded in ponds treated with the combination of the highest doses. Overall, glyphosate was the most influential driver of aggregate community properties of zooplankton, with biomass and community structure responding rapidly but recovering unequally over time. Total community biomass showed little resistance when first exposed to glyphosate, but rapidly recovered and even increased with glyphosate concentration over time; in contrast, taxon richness decreased in more contaminated ponds but failed to recover. Our results indicate that the biomass of tolerant taxa compensated for the loss of sensitive species after the first exposure, conferring greater community resistance upon a subsequent contamination event; a case of pollution-induced community tolerance in freshwater animals. These findings suggest that zooplankton biomass may be more resilient to agrochemical pollution than community structure; yet all community properties measured in this study were affected at glyphosate concentrations below common water quality guidelines in North America.

Resistance, resilience, and functional redundancy of freshwater bacterioplankton communities facing a gradient of agricultural stressors in a mesocosm experiment.

Agricultural pollution with fertilizers and pesticides is a common disturbance to freshwater biodiversity. Bacterioplankton communities are at the base of aquatic food webs, but their responses to these potentially interacting stressors are rarely explored. To test the extent of resistance and resilience in bacterioplankton communities faced with agricultural stressors, we exposed freshwater mesocosms to single and combined gradients of two commonly used pesticides: the herbicide glyphosate (0-15 mg/L) and the neonicotinoid insecticide imidacloprid (0-60 µg/L), in high or low nutrient backgrounds. Over the 43-day experiment, we tracked variation in bacterial density with flow cytometry, carbon substrate use with Biolog EcoPlates, and taxonomic diversity and composition with environmental 16S rRNA gene amplicon sequencing. We show that only glyphosate (at the highest dose, 15 mg/L), but not imidacloprid, nutrients, or their interactions measurably changed community structure, favouring members of the Proteobacteria including the genus Agrobacterium. However, no change in carbon substrate use was detected throughout, suggesting functional redundancy despite taxonomic changes. We further show that communities are resilient at broad, but not fine taxonomic levels: 24 days after glyphosate application the precise amplicon sequence variants do not return, and tend to be replaced by phylogenetically close taxa. We conclude that high doses of glyphosate - but still within commonly acceptable regulatory guidelines - alter freshwater bacterioplankton by favouring a subset of higher taxonomic units (i.e., genus to phylum) that transiently thrive in the presence of glyphosate. Longer-term impacts of glyphosate at finer taxonomic resolution merit further investigation.

Community rescue in experimental phytoplankton communities facing severe herbicide pollution.

Community rescue occurs when ecological or evolutionary processes restore positive growth in a highly stressful environment that was lethal to the community in its ancestral form, thus averting biomass collapse in a deteriorating environment. Laboratory evidence suggests that community rescue is most likely in high-biomass communities that have previously experienced moderate doses of sublethal stress. We assessed this result under more natural conditions, in a mesocosm experiment with phytoplankton communities exposed to the ubiquitous herbicide glyphosate. We tested whether community biomass and prior herbicide exposure would facilitate community rescue after severe contamination. We found that prior exposure to glyphosate was a very strong predictor of the rescue outcome, while high community biomass was not. Furthermore, although glyphosate had negative effects on diversity, it did not influence community composition significantly, suggesting a modest role for genus sorting in this rescue process. Our results expand the scope of community rescue theory to complex ecosystems and confirm that prior stress exposure is a key predictor of rescue.

A meta-analysis of zooplankton functional traits influencing ecosystem function.

The use of functional traits to characterize community composition has been proposed as a more effective way to link community structure to ecosystem functioning. Organismal morphology, body stoichiometry, and physiology can be readily linked to large-scale ecosystem processes through functional traits that inform on interspecific and species-environment interactions; yet such effect traits are still poorly included in trait-based approaches. Given their key trophic position in aquatic ecosystems, individual zooplankton affect energy fluxes and elemental processing. We compiled a large database of zooplankton traits contributing to carbon, nitrogen, and phosphorus cycling and examined the effect of classification and habitat (marine vs. freshwater) on trait relationships. Respiration and nutrient excretion rates followed mass-dependent scaling in both habitats, with exponents ranging from 0.70 to 0.90. Our analyses revealed surprising differences in allometry and respiration between habitats, with freshwater species having lower length-specific mass and three times higher mass-specific respiration rates. These differences in traits point to implications for ecological strategies as well as overall carbon storage and fluxes based on habitat type. Our synthesis quantifies multiple trait relationships and links organisms to ecosystem processes they influence, enabling a more complete integration of aquatic community ecology and biogeochemistry through the promising use of effect traits.

A compilation of quantitative functional traits for marine and freshwater crustacean zooplankton.

This data compilation synthesizes 8609 individual observations and ranges of 13 traits from 201 freshwater and 191 marine crustacean taxa belonging to either Copepoda or Cladocera, two important zooplankton groups across all major aquatic habitats. Most data were gathered from the literature, with the balance being provided by zooplankton ecologists. With the aim of more fully assessing zooplankton effects on elemental processes such as nitrogen (N), phosphorus (P) and carbon (C) stocks and fluxes in aquatic ecosystems, this data set provides information on the following traits: body size (length and mass), trophic group, elemental and biochemical corporal composition (N, P, C, lipid and protein content), respiration rates, N- and P-excretion rates, as well as stoichiometric ratios. Although relationships for zooplankton metabolism as a function of body mass or requirements have been explored in the past three decades, data have not been systematically compiled nor examined from an integrative and large-scale perspective across crustacean taxa and habitat types. While this contribution likely represents the most comprehensive assembly of traits for both marine and freshwater species, this data set is not exhaustive either. As a result, this compilation also identifies knowledge gaps: a fact that should encourage researchers to disclose information they may have to help complete such databases. This trait matrix is made available for the first time in this data paper; prior to its release, the data set has been analyzed in a meta-analysis published as a companion paper. This data set should prove extremely valuable for aquatic ecologists for trait-based characterization of plankton community structure as well as biogeochemical modeling. These data are also well-suited for deriving shortcut relationships that predict more difficult to measure trait values, most of which can be directly related to ecosystem properties (i.e., effect traits), from simpler traits (e.g., body size), and for exploring patterns of trait variation within and amongst taxonomic units or ecosystem types. Overall, this data set is likely to provide new insights into the functional structure of zooplankton communities and increase our mechanistic understanding of the influence of these pivotal organisms on aquatic ecosystems.