Behavioral Responses of Chironomus aprilinus Larvae as Proxies for Cyanobacterial Metabolite Interactions: Insights from Ternary Combinations.

This study aimed to assess the behavioral responses (immobilization, horizontal and vertical motility, and response to light) of Chironomus aprilinus larvae exposed to individual cyanobacterial metabolites aeruginosin 98B (AER-B), anabaenopeptin-B (ANA-B), and cylindrospermopsin (CYL), and their binary and ternary mixtures. The investigation revealed that single metabolites ANA-B and CYL exhibited the highest potency in immobilizing the larvae. Notably, the binary mixture AER-B+CYL induced a remarkably strong synergistic interaction, while other tested binary and ternary mixtures demonstrated antagonistic effects. Both individual metabolites and their mixtures led to a decrease in larval movement speed, with the AER-B+CYL combination showing a very synergistic effect, and strong antagonistic interactions between the oligopeptides in the ternary mixture. Conversely, while AER-B and the binary mixture ANA-B+CYL stimulated vertical movement, other single metabolites and binary and ternary mixtures decreased this parameter. Antagonistic interactions were observed in all mixtures. ANA-B emerged as the most potent inhibitor, yet all tested metabolites and their mixtures decreased larval response to light, displaying synergistic interactions, except for the AER-B+ANA-B mixture at 250 µg L-1 + 250 µg L-1. These findings underscore the sensitivity of Chironomus larvae behavioral parameters as indicators of environmental stressors and mixtures. Consequently, they are recommended for assessing toxic effects induced by cyanobacterial products and other bioactive chemicals.

Characterization and Expression of the Cytochrome P450 Genes in Daphnia magna Exposed to Cerium Oxide Nanoparticles.

As cerium oxide nanoparticles (nCeO2) continue to infiltrate aquatic environments, the resulting health risks to exposed aquatic organisms are becoming evident. Cytochrome P450 (CYP) enzymes are integral to the detoxification processes in these species. Herein, we conducted a genomic analysis of CYPs in Daphnia magna, encompassing phylogenetic relationships, gene structure, and chromosomal localization. We identified twenty-six CYPs in D. magna, categorizing them into four clans and seven families, distributed across six chromosomes and one unanchored scaffold. The encoded CYP proteins varied in length from 99 to 585 amino acids, with molecular weights ranging from 11.6 kDa to 66.4 kDa. A quantitative real-time PCR analysis demonstrated a significant upregulation of CYP4C1.4, CYP4C1.5, CYP4C1.6, CYP4c3.3, and CYP4c3.6 in D. magna exposed to 150 mg/L nCeO2 for 24 h. The transcript levels of CYP4C1.3, CYP18a1, CYP4C1.1, and CYP4c3.9 were notably downregulated in D. magna exposed to 10 mg/L nCeO2 for 48 h. A further transcriptomic analysis identified differential expression patterns of eight CYP genes, including CYP4C1.3, in response to nCeO2 exposure. The differential regulation observed across most of the 26 CYPs highlights their potential role in xenobiotic detoxification in D. magna, thereby enhancing our understanding of CYP-mediated toxicological responses to metal nanoparticles in aquatic invertebrates.

Beaver-related restoration: An opportunity for sandy lowland streams in a human-dominated landscape.

The expansion of beavers into human-dominated landscapes can help improve the ecological status of degraded streams. This study aimed to assess the changes in the ecological status of a degraded sandy-bottom stream under the influence of processes generated by the activity of a European beaver. We assumed that the processes in the beaver dam-and-pond complex significantly improved the physical, chemical, and bacteriological properties of water and improved the ecological status of a small lowland stream in a human-dominated landscape. To achieve these objectives, water for quality assessment, benthic invertebrates, and fish samples were collected, and the forms of land use and the natural values of the stream were analysed. In the upstream zone of the sandy-bottom stream flowing in a human-dominated landscape, the inflow of pollutants caused a significant reduction in water quality. The amount of nutrients was high and the amount of dissolved oxygen was low. Similarly, the number of meso- and psychrophilic bacteria was significantly higher than in the reference stream. The building activity of beavers initiated a series of positive environmental changes, and beaver ponds improved the self-purification of water in a polluted stream. In the lower part of the studied stream, after the stream passed through the beaver wetland system, the water quality improved, which was indicated by a significant improvement in physicochemical, microbiological, and biotic indicators, that is based on macrozoobenthos and fish. By constructing dams, beavers flooded large areas and created habitats for plant and animal species associated with wetlands, including amphibians, birds, and mammals. The improvement of the ecological status of the stream, combined with the preservation and restoration of wetland habitats in the catchment area, enables the use of ecosystem services provided by the streams in the human-dominated landscape and their surroundings.

Speed it up: How temperature drives toxicokinetics of organic contaminants in freshwater amphipods.

The acceleration of global climate change draws increasing attention towards interactive effects of temperature and organic contaminants. Many studies reported a higher sensitivity of aquatic invertebrates towards contaminant exposure with increasing or fluctuating temperatures. The hypothesis of this study was that the higher sensitivity of invertebrates is associated with the changes of toxicokinetic processes that determine internal concentrations of contaminants and consequently toxic effects. Therefore, the influence of temperature on toxicokinetic processes and the underlying mechanisms were studied in two key amphipod species (Gammarus pulex and Hyalella azteca). Bioconcentration experiments were carried out at four different temperatures with a mixture of 12 exposure relevant polar organic contaminants. Tissue and medium samples were taken in regular intervals and analysed by online solid-phase extraction liquid chromatography high-resolution tandem mass spectrometry. Subsequently, toxicokinetic rates were modelled and analysed in dependence of the exposure temperature using the Arrhenius equation. An exponential relationship between toxicokinetic rates versus temperature was observed and could be well depicted by applying the Arrhenius equation. Due to a similar Arrhenius temperature of uptake and elimination rates, the bioconcentration factors of the contaminants were generally constant across the temperature range. Furthermore, the Arrhenius temperature of the toxicokinetic rates and respiration was mostly similar. However, in some cases (citalopram, cyprodinil), the bioconcentration factor appeared to be temperature dependent, which could potentially be explained by the influence of temperature on active uptake mechanisms or biotransformation. The observed temperature effects on toxicokinetics may be particularly relevant in non-equilibrated systems, such as exposure peaks in summer as exemplified by the exposure modelling of a field measured pesticide peak where the internal concentrations increased by up to fourfold along the temperature gradient. The results provide novel insights into the mechanisms of chemical uptake, biotransformation and elimination in different climate scenarios and can improve environmental risk assessment.

Respirometry reveals major lineage-based differences in the energetics of osmoregulation in aquatic invertebrates.

All freshwater organisms are challenged to control their internal balance of water and ions in strongly hypotonic environments. We compared the influence of external salinity on the oxygen consumption rates (MO2) of three species of freshwater insects, one snail and two crustaceans. Consistent with available literature, we found a clear decrease in MO2 with increasing salinity in the snail Elimia sp. and crustaceans Hyalella azteca and Gammarus pulex (r5=-0.90, P=0.03). However, we show here for the first time that metabolic rate was unchanged by salinity in the aquatic insects, whereas ion transport rates were positively correlated with higher salinities. In contrast, when we examined the ionic influx rates in the freshwater snail and crustaceans, we found that Ca uptake rates were highest under the most dilute conditions, while Na uptake rates increased with salinity. In G. pulex exposed to a serially diluted ion matrix, Ca uptake rates were positively associated with MO2 (r5=-0.93, P=0.02). This positive association between Ca uptake rate and MO2 was also observed when conductivity was held constant but Ca concentration was manipulated (1.7-17.3 mg Ca l-1) (r5=0.94, P=0.05). This finding potentially implicates the cost of calcium uptake as a driver of increased metabolic rate under dilute conditions in organisms with calcified exoskeletons and suggests major phyletic differences in osmoregulatory physiology. Freshwater insects may be energetically challenged by higher salinities, while lower salinities may be more challenging for other freshwater taxa.

Methylation and Demethylation of Emerging Contaminants Changed Bioaccumulation and Acute Toxicity in Daphnia magna.

Contaminants of emerging concern (CECs) in the environment undergo various transformations, leading to the formation of transformation products (TPs) with a modified ecological risk potential. Although the environmental significance of TPs is increasingly recognized, there has been relatively little research to understand the influences of such transformations on subsequent ecotoxicological safety. In this study, we used four pairs of CECs and their methylated or demethylated derivatives as examples to characterize changes in bioaccumulation and acute toxicity in Daphnia magna, as a result of methylation or demethylation. The experimental results were further compared to quantitative structure-activity relationship (QSAR) predictions. The methylated counterpart in each pair generally showed greater acute toxicity in D. magna, which was attributed to their increased hydrophobicity. For example, the LC50 values of methylparaben (34.4 ± 4.3 mg L-1) and its demethylated product (225.6 ± 17.3 mg L-1) differed about eightfold in D. magna. The methylated derivative generally exhibited greater bioaccumulation than the demethylated counterpart. For instance, the bioaccumulation of methylated acetaminophen was about 33-fold greater than that of acetaminophen. In silico predictions via QSARs aligned well with the experimental results and suggested an increased persistence of the methylated forms. The study findings underline the consequences of simple changes in chemical structures induced by transformations such as methylation and demethylation and highlight the need to consider TPs to achieve a more holistic understanding of the environmental fate and risks of CECs.

Elucidating the spatial distribution of organic contaminants and their biotransformation products in amphipod tissue by MALDI- and DESI-MS-imaging.

The application of mass spectrometry imaging (MSI) is a promising tool to analyze the spatial distribution of organic contaminants in organisms and thereby improve the understanding of toxicokinetic and toxicodynamic processes. MSI is a common method in medical research but has been rarely applied in environmental science. In the present study, the suitability of MSI to assess the spatial distribution of organic contaminants and their biotransformation products (BTPs) in the aquatic invertebrate key species Gammarus pulex was studied. Gammarids were exposed to a mixture of common organic contaminants (carbamazepine, citalopram, cyprodinil, efavirenz, fluopyram and terbutryn). The distribution of the parent compounds and their BTPs in the organisms was analyzed by two MSI methods (MALDI- and DESI-HRMSI) after cryo-sectioning, and by LC-HRMS/MS after dissection into different organ compartments. The spatial distribution of contaminats in gammarid tissue could be successfully analyzed by the different analytical methods. The intestinal system was identified as the main site of biotransformation, possibly due to the presence of biotransforming enzymes. LC-HRMS/MS was more sensitive and provided higher confidence in BTP identification due to chromatographic separation and MS/MS. DESI was found to be the more sensitive MSI method for the analyzed contaminants, whereas additional biomarkers were found using MALDI. The results demonstrate the suitability of MSI for investigations on the spatial distribution of accumulated organic contaminants. However, both MSI methods required high exposure concentrations. Further improvements of ionization methods would be needed to address environmentally relevant concentrations.

Drivers of copper sensitivity in copepods: A meta-analysis of LC50s.

Copper is both an essential trace element and a potent pesticide. The use of copper as an antifoulant has increased in the last decades in line with the expanding aquaculture and shipping industries. In aquatic environments, it also affects non-target taxa. One of which are copepods, which constitute the central link in the marine food web. Despite their ecological importance, there are no systematic reviews of the lethal concentration range and drivers of copper toxicity in this taxon. Here, we combined literature data from 31 peer-reviewed articles recording the Lethal Concentration 50 (LC50) for copper in copepods and the experiments' respective environmental, developmental, and taxonomic parameters. The LC50 is a traditional endpoint for toxicity testing used in standardized toxicity testing and many ecological studies. In total, we were able to extract 166 LC50 entries. The variability in the metadata allowed for a general analysis of the drivers of copper sensitivity in copepods. Using a generalized additive modeling approach, we find that temperature increases copper toxicity when above approximately 25℃. Counter to our expectations; salinity does not influence copper sensitivity across copepod species. Unsurprisingly, nauplii are more susceptible to copper exposure than adult copepods, and benthos-associated harpacticoids are less sensitive to copper than pelagic calanoids. Our final model can predict sensible specific-specific copper concentrations for future experiments, thus giving an informed analytical approach to range testing in future dose-response experiments. Our model can also potentially improve ecological risk assessment by accounting for environmental differences. The approach can be applied to other toxicants and taxa, which may reveal underlying patterns otherwise obscured by taxonomic and experimental variability.

Synergistic effect of chloroquine and copper to the euryhaline rotifer Proales similis.

Chloroquine (CQ) has been widely used for many years against malaria and various viral diseases. Its important use and high potential to being persistent make it of particular concern for ecotoxicological studies. Here, we evaluated the toxicity of CQ alone and in combination with copper (Cu) to the euryhaline rotifer Proales similis. All experiments were carried out using chronic toxicity reproductive five-day tests and an application factor (AF) of 0.05, 0.1, 0.3, and 0.5 by multiplying the 24-h LC50 values of CQ (4250 µg/L) and Cu (68 µg/L), which were administered in solution. The rate of population increase (r, d-1) ranged from 0.50 to 52 (controls); 0.20 to 0.40 (CQ); 0.09 to 0.43 (Cu); and -0.03 to 0.30 (CQ-Cu) and showed significant decrease as the concentration of both chemicals in the medium increased. Almost all tested mixtures induced synergistic effects, mainly as the AF increased. We found that the presence of Cu intensifies the vulnerability of organisms to CQ and vice versa. These results stress the potential hazard that these combined chemicals may have on the aquatic systems. This research suggests that P. similis is sensitive to CQ as other standardized zooplankton species and may serve as a potential test species in the risk assessment of emerging pollutants in marine environments.

Bicarbonate alone does not totally explain the toxicity from major ions of coal bed derived waters to freshwater invertebrates.

Concentrations of major ions in coal mine discharge waters and unconventional hydrocarbon produced waters derived from coal bed methane (CBM) production, are potentially harmful to freshwater ecosystems. Bicarbonate is a major constituent of produced waters from CBM and coal mining. However, little is known about the relative toxicity of differing ionic proportions, especially bicarbonate, found in these CBM waters. As all freshwater invertebrates tested are more acutely sensitive to sodium bicarbonate (NaHCO3) than sodium chloride (NaCl) or synthetic sea water, we tested the hypotheses that toxicity of CBM waters are driven by bicarbonate concentration, and waters containing a higher proportion of bicarbonate are more toxic to freshwater invertebrates than those with less bicarbonate. We compared the acute (96 h) lethal toxicity to six freshwater invertebrate species of NaHCO3 and two synthetic CBM waters, with ionic proportions representative of water from CBM wells across New South Wales (NSW) and Queensland (Qld), in Australia. The ranking of LC50 values expressed as total salinity was consistent with the hypotheses. However, when toxicity was expressed as bicarbonate concentration, the hypothesis that the toxicity of coal bed waters would be explained by bicarbonate concentration was not well supported, and other ionic components were either ameliorating or exacerbating the NaHCO3 toxicity. Our findings showed NaHCO3 was more toxic than NaCl and that the NaHCO3 proportion of synthetic CBM waters drives toxicity, however other ions are altering the toxicity of bicarbonate.

Urbanisation process generates more independently-acting stressors and ecosystem functioning impairment in tropical Andean streams.

The tropical Andes are experiencing rapid population growth and urbanisation has become a major driver impairing stream ecosystems. However, knowledge about multiple-stressors effects on urbanised Andean streams is lacking. In southern Ecuador, we assessed how multiple stressors determine the structural (aquatic invertebrate metrics) and functional (organic matter breakdown and delta N of primary consumers) attributes of streams in a densely populated watershed without wastewater treatment and with contrasting land uses. We found that urbanised streams exhibited individual-stressor effects and that stressor interactions were rare. While structural and function attributes responded negatively to urbanisation, ecosystem functioning metrics were influenced most. Stream ecosystem functions were influenced by water-chemistry stressors, whereas aquatic invertebrate metrics were influenced by physical-habitat stressors. We suggest that managers of urbanised streams in the Andes immediately focus on the most important stressors by reducing inputs of inorganic N and P, re-establishing stream flow and substrate heterogeneity, and restoring riparian vegetation instead of attempting to elucidate intricate interactions among stressors. Our result also demonstrate that stream biomonitoring programs would benefit from a combination of structural and functional indicators to assess anthropogenic effects in a multiple-stressors scenario.

Patterns in benthic macroinvertebrate assemblages in an active region of unconventional shale-gas development in the western Appalachian Plateau of West Virginia, USA.

We sampled headwater streams to characterize impacts of unconventional shale gas development (SGD) on aquatic ecosystems. The study area was relatively un-impacted by confounding activities. Intensity of SGD over the study decreased then increased again but not to levels seen the first year. Shale gas development was associated with increased, but non-impaired, water pH and specific conductance during the latter part of the study. Metrics summarizing macroinvertebrate assemblages were better on average in un-impacted reaches. A genus-level multimetric index of biotic integrity was statistically lower downstream of impacts compared to upstream, but only in the year when SGD activity was most intense. Multivariate analyses indicated that assemblages diverged in similarity downstream compared to upstream of impacts in the first and last years of the study when SGD activity was elevated. Assemblage divergence was related to variation in water quality. Indicator species analysis linked a few key taxa to un-impacted conditions in the first year of the study; tolerant taxa were indicators for impacted conditions later in the study. Our study links SGD to weak negative changes in water quality and benthic macroinvertebrates, which may have negative consequences to food quality for wildlife that rely on aquatic prey within forested systems.

Substantial long-term loss of alpha and gamma diversity of lake invertebrates in a landscape exposed to a drying climate.

Many regions across the globe are shifting to more arid climates. For shallow lakes, decreasing rainfall volume and timing, changing regional wind patterns and increased evaporation rates alter water regimes so that dry periods occur more frequently and for longer. Drier conditions may affect fauna directly and indirectly through altered physicochemical conditions in lakes. Although many studies have predicted negative effects of such changes on aquatic biodiversity, empirical studies demonstrating these effects are rare. Global warming has caused severe climatic drying in southwestern Australia since the 1970s, so we aimed to determine whether lakes in this region showed impacts on lake hydroperiod, water quality, and α, ß and γ diversity of lake invertebrates from 1998 to 2011. Seventeen lakes across a range of salinities were sampled biennially in spring in the Wheatbelt and Great Southern regions of Western Australia. Multivariate analyses were used to identify changes in α, ß and γ diversity and examine patterns in physicochemical data. Salinity and average rainfall partially explained patterns in invertebrate richness and assemblage composition. Climatic drying was associated with significant declines in lake depth, increased frequency of dry periods, and reduced α and γ diversity (γ declined from ~300 to ~100 taxa from 1998 to 2011 in the 17 wetlands). In contrast, ß diversity remained consistently high, because each lake retained a distinct fauna. Mean α diversity per-lake declined both in lakes that dried and lakes that did not dry out, but lakes which retained a greater proportion of their maximum depth retained more α diversity. Accumulated losses in α diversity caused the decline in γ diversity likely through shrinking habitat area, fewer stepping stones for dispersal and loss of specific habitat types. Biodiversity loss is thus likely from lakes in drying regions globally. Management actions will need to sustain water depth in lakes to prevent biodiversity loss.

Drivers of long-term invertebrate community stability in changing Swedish lakes.

Research on ecosystem stability has had a strong focus on local systems. However, environmental change often occurs slowly at broad spatial scales, which requires regional-level assessments of long-term stability. In this study, we assess the stability of macroinvertebrate communities across 105 lakes in the Swedish "lakescape." Using a hierarchical mixed-model approach, we first evaluate the environmental pressures affecting invertebrate communities in two ecoregions (north, south) using a 23 year time series (1995-2017) and then examine how a set of environmental and physical variables affect the stability of these communities. Results show that lake latitude, size, total phosphorus and alkalinity affect community composition in northern and southern lakes. We find that lake stability is affected by species richness and lake size in both ecoregions and alkalinity and total phosphorus in northern lakes. There is large heterogeneity in the patterns of community stability of individual lakes, but relationships between that stability and environmental drivers begin to emerge when the lakescape, composed of many discrete lakes, is the focal unit of study. The results of this study highlight that broad-scale comparisons in combination with long time series are essential to understand the effects of environmental change on the stability of lake communities in space and time.

Methylmercury-total mercury ratios in predator and primary consumer insects from Adirondack streams (New York, USA).

Mercury (Hg) is a global pollutant that affects biota in remote settings due to atmospheric deposition of inorganic Hg, and its conversion to methylmercury (MeHg), the bioaccumulating and toxic form. Characterizing biotic MeHg is important for evaluating aquatic ecosystem responses to changes in Hg inputs. Aquatic insects possess many qualities desired for MeHg biomonitoring, but are not widely used, largely because of limited information regarding percentages of total mercury (THg) composed of MeHg (i.e., MeHg%) in various taxa. Here, we examine taxonomic, spatial, and seasonal variation in MeHg% of stream-dwelling predator and primary-consumer insects from nine streams in the Adirondack region (NY, USA). Predator MeHg% was high (median 94%) and did not differ significantly among five taxa. MeHg% in selected dragonflies (the most abundant predators, Odonata: Aeshnidae and Libellulidae) exhibited little seasonal and spatial variation, and THg concentration was strongly correlated with aqueous (filtered) MeHg (FMeHg; rs = 0.76). In contrast, MeHg% in primary consumers-shredders (northern caddisflies [Trichoptera: Limnephilidae]) and scrapers (flathead mayflies [Ephemeroptera: Heptageniidae]), were lower (medians 52% and 35%, respectively), and differed significantly between taxa, among sites, and seasonally. Correlations of THg with FMeHg were weak (shredders, rs = 0.45, p = 0.09) or not significant (scrapers, p = 0.89). The higher MeHg% of predators corresponded with their higher trophic positions (indicated by nitrogen stable isotopes). Results suggest obligate predators hold the most promise for the use of THg as a surrogate for MeHg biomonitoring with aquatic insects within the Adirondack region.

To composite or replicate: how sampling method and protocol differences alter collected stream invertebrates and associated bioassessment metrics.

Aquatic invertebrates are excellent indicators of ecosystem quality; however, choosing a sampling method can be difficult. Each method and associated protocol has advantages and disadvantages, and finding the approach that minimizes biases yet fulfills management objectives is crucial. To test the effects of both sampling methods and sample handling-i.e., to composite samples or leave them as replicates-we collected aquatic invertebrates from the Niobrara River at Agate Fossil Beds National Monument, Nebraska, using three methods and two sample handling protocols. We compared aquatic invertebrate assemblages collected with a Hester-Dendy multi-plate sampler, Hess sampler, and a D-frame dipnet. We calculated six common bioassessment metrics from composite (combined) and replicate (separate) samples. Hess samples contained the highest taxonomic richness (capturing 77% of all taxa observed) and dipnet samples the least (47%). Hester-Dendy samples had the greatest proportion of Ephemeroptera, and Ephemeroptera, Plecoptera, and Trichoptera (EPT). Dipnet samples had the lowest evenness values. In terms of sample handling, composite samples had inflated richness, diversity, and evenness compared with replicate samples, but bioassessment metrics calculated from proportions or averages (i.e., Hilsenhoff's Biotic Index and the proportion of EPT taxa) did not differ between them. The proportion of invertebrate groups from composite samples were not statistically different among sampling methods, but several groups differed between replicate samples collected by different methods. Ultimately, we recommend collecting replicate samples with a Hess sampler when the goal of the study is to detect ecosystem change, among locations or differences in variables of interest.

Small-scale phenotypic differentiation along complex stream gradients in a non-native amphipod.

BACKGROUND: Selective landscapes in rivers are made up by an array of selective forces that vary from source to downstream regions or between seasons, and local/temporal variation in fitness maxima can result in gradual spatio-temporal variation of phenotypic traits. This study aimed at establishing freshwater amphipods as future model organisms to study adaptive phenotypic diversification (evolutionary divergence and/or adaptive plasticity) along stream gradients. METHODS: We collected Gammarus roeselii from 16 sampling sites in the Rhine catchment during two consecutive seasons (summer and winter). Altogether, we dissected n = 1648 individuals and quantified key parameters related to morphological and life-history diversification, including naturally selected (e.g., gill surface areas) as well as primarily sexually selected traits (e.g., male antennae). Acknowledging the complexity of selective regimes in streams and the interrelated nature of selection factors, we assessed several abiotic (e.g., temperature, flow velocity) and biotic ecological parameters (e.g., conspecific densities, sex ratios) and condensed them into four principal components (PCs). RESULTS: Generalized least squares models revealed pronounced phenotypic differentiation in most of the traits investigated herein, and components of the stream gradient (PCs) explained parts of the observed differences. Depending on the trait under investigation, phenotypic differentiation could be ascribed to variation in abiotic conditions, anthropogenic disturbance (influx of thermally polluted water), or population parameters. For example, female fecundity showed altitudinal variation and decreased with increasing conspecific densities, while sexual dimorphism in the length of male antennae-used for mate finding and assessment-increased with increasing population densities and towards female-biased sex ratios. CONCLUSIONS: We provide a comprehensive protocol for comparative analyses of intraspecific variation in life history traits in amphipods. Whether the observed phenotypic differentiation over small geographical distances reflects evolutionary divergence or plasticity (or both) remains to be investigated in future studies. Independent of the mechanisms involved, variation in several traits is likely to have consequences for ecosystem functions. For example, leaf-shredding in G. roeselii strongly depends on body size, which varied in dependence of several ecological parameters.

Acute and chronic toxicity of magnesium to the early life stages of two tropical freshwater mussel species.

Magnesium (Mg) is a common contaminant in mine water discharges. Although Mg is an essential element in biological processes, increased concentrations from anthropogenic sources can stress aquatic ecosystems. Additionally, studies evaluating the effects of Mg on north Australian freshwater species have indicated that in very soft waters there is a high risk to some species. Freshwater mussels are an ecologically and culturally important taxon in many freshwater environments, but knowledge of their sensitivity to Mg is limited. In the present study, the acute and chronic sensitivity of two freshwater mussel species, Velesunio angasi and an undescribed Velesunio species, to Mg was assessed (using MgSO4) on their early life stages, larval glochidia and post-parasitic juveniles. Acute 24-h exposures with glochidia generated a mean median lethal (LC50) toxicity estimate of 284mg/L for the five tests with V. angasi, and a mean LC50 of 300mg/L for the three tests with Velesunio sp. Mean chronic 14-d toxicity estimates resulting in 50% (EC50) and 10% (EC10) growth rate reductions for juveniles were 241 and 88mg/L respectively for the three tests with V. angasi juveniles, and 232 and 87mg/L respectively for the three tests with Velesunio sp. juveniles. The results represent the first acute and chronic Mg toxicity data for tropical freshwater mussels, and indicated that V. angasi and Velesunio sp. exhibited similar sensitivity and were moderately sensitive to Mg when compared to other tropical species. These results are a valuable contribution to the small existing dataset for Mg toxicity to tropical freshwater species, which can be used to inform water management in areas where Mg is a contaminant of concern, and ensure the protection of these taxa.

Salinity tolerance of a rare and endangered unionid mussel, Popenaias popeii (Texas Hornshell) and its implications for conservation and water management.

Unionid mussels are considered sensitive to salinity and there is growing concern in arid and semi-arid regions that declining flows coupled with anthropogenic impacts are amplifying natural salinity levels. In this study, we tested the effects of varying salinity concentrations (3.0, 4.0, 5.0, 6.0, 7.0 and 10.0 ppt NaCl) on survival of adult Popenaias popeii, (Texas Hornshell). This species occurs in the Rio Grande basin of Texas and northern Mexico, an arid to semi-arid stream plagued by salinization, and was recently listed as Endangered under the U.S. Endangered Species Act. We performed 2, 4, and 10-day toxicity tests on individuals from two disjunct populations: Laredo, TX, and the Lower Canyons of the Rio Grande near Big Bend National Park. We found no significant differences in LC50 estimates between populations at 96-hrs or 10-days but significant differences in TUD50s at 5 ppt between populations, which indicates that tolerance does not vary but sensitivity may between these populations. Overlaying LC50 estimates at 10-days for both populations on plots of salinity (ppt) measured over time, we show parts of the Rio Grande periodically approach or exceed 4.0 ppt, indicating these reaches are becoming unsuitable for P. popeii and populations within them at risk.

Hemolymph Microbiomes of Three Aquatic Invertebrates as Revealed by a New Cell Extraction Method.

Symbiotic microorganisms have been found in the hemolymph (blood) of many aquatic invertebrates, such as crabs, shrimp, and oysters. Hemolymph is a critical site in the host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies using selective media (e.g., thiosulfate-citrate-bile salts-sucrose [TCBS], 2216E, and LB) for enumerating and isolating microbial cells. However, doubts remain about the "true" representation of the microbial abundance and diversity of symbiotic microorganisms in hemolymph, particularly for uncultivable microorganisms, which are believed to be more abundant than the cultured microorganisms. To explore this, we developed a culture-independent cell extraction method for separating microbial cells from the hemolymph of three aquatic invertebrates (Scylla paramamosain [mud crab], Litopenaeus vannamei [whiteleg shrimp], and Crassostrea angulata [Portuguese oysters]) involving filtration through a 5-µm-pore-size mesh filter membrane (the filtration method). A combination of the filtration method with fluorescence microscopy and high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. More than 2.6 × 104 cells/ml of microbial cells dominated by Escherichia-Shigella and Halomonas, Photobacterium and Escherichia-Shigella, and Pseudoalteromonas and Arcobacter were detected in the hemolymph of Scylla paramamosain, Litopenaeus vannamei, and Crassostrea angulata, respectively. A parallel study for investigating the hemolymph microbiomes by comparing the filtration method and a culture-dependent method (the plate count method) showed significantly higher microbial abundances (between 26- and 369-fold difference; P < 0.05) and less biased community structures of the filtration method than those of the plate count method. Furthermore, hemolymph of the three invertebrates harbored many potential pathogens, including Photobacterium, Arcobacter, and Vibrio species. Finally, the filtration method provides a solution that improves the understanding of the metabolic functions of uncultivable hemolymph microorganisms (e.g., metagenomics) devoid of host hemocyte contamination.IMPORTANCE Microorganisms are found in the hemolymph of invertebrates, a critical site in the host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies. However, doubts remain about the "true" representation of the hemolymph microbiome. This study developed a culture-independent cell extraction method that could separate microbial cells from the hemolymph of three aquatic invertebrates (S. paramamosain, L. vannamei, and C. angulata) based on filtration through a 5-µm-pore-size mesh filter membrane (the filtration method). A combination of the filtration method with fluorescence microscopy and a high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. Our results demonstrate that the hemolymph of aquatic invertebrates harbors a much higher microbial abundance and more distinct microbial community composition than previously estimated. Furthermore, this work provides a less biased solution for studying the metabolic functions of uncultivable hemolymph microbiota devoid of host hemocyte contamination.