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Relative to a growing body of knowledge about the negative consequences of freshwater salinization, little is known about how aquatic insects respond to progressively ion-poor conditions. Here, we examined life-history and physiological acclimation in

Ecologists have observed declines in the biodiversity of sensitive freshwater organisms in response to increasing concentrations of major ions (salinization). Yet, how changing salinities physiologically challenge aquatic organisms, such as mayflies, remains remarkably understudied. Moreover, it is not well understood the degree to which species respond and acclimate to salinity changes. Our lab is developing the Baetid mayfly, N. triangulifer, as a model organism for physiological research. We have previously described acclimatory changes in both ion flux rates and altered mRNA transcript levels in response to chronic exposures to elevated major ion concentrations at the whole animal level. In the present study, we use shotgun proteomics to identify the specific proteins associated with apical ion transport and how their abundance changes in response to chronic salinity exposures in gills. Gills were isolated from the penultimate nymphal stage of N. triangulifer reared under control culture conditions, elevated NaCl (157 mg L

Increased temperatures and exposure to agricultural insecticides are increasingly threatening freshwater ecosystems worldwide. However, their combined effects are still poorly understood. We invest...

Freshwater salinization is a rapidly emerging ecological issue and is correlated with significant declines in aquatic biodiversity. It remains unclear how changing salinity regimes affect the physiology of sensitive aquatic insects. We used the parthenogenetic mayfly, Neocloeon triangulifer, to ask how ionic exposure history alters physiological processes and responses to subsequent major ion exposures. Using radiotracers (22Na, 35SO4, and 45Ca), we observed that mayflies chronically reared in elevated sodium or sulfate (157 mg L−1 Na or 667 mg L−1 SO4) had 2-fold (p

To better understand the effects of transient thermal stress in an aquatic insect, we first identified static temperatures associated with fitness deficits, and then reared larvae from egg hatch to adulthood under diurnally variable regimens including daily forays into deleterious temperatures. We sampled mature larvae at the coolest and warmest portions of their respective regimens for RNA-seq analysis. Few transcripts (28) were differentially expressed when larvae oscillated between favorable temperatures, while 614 transcripts were differentially expressed when experiencing daily transient thermal stress. Transcripts associated with N-glycan processing were downregulated while those associated with lipid catabolism and chitin turnover were significantly upregulated in heat stressed larvae. An across-regimen comparison of differentially expressed transcripts among organisms sampled at comparable temperatures demonstrated that the effects of daily thermal stress persisted even when larvae were sampled at a more optimal temperature (806 differentially expressed transcripts). The chronically stressed population had reduced expression of transcripts related to ATP synthesis, mitochondrial electron chain functions, gluconeogenesis and glycolytic processes while transcripts associated with cell adhesion, synaptic vesicle transport, regulation of membrane potential and lipid biosynthesis increased. Comparisons of constant vs. variable temperatures revealed that the negative consequences of time spent at stressful temperatures were not offset by more time spent at optimal temperatures.

OBJECTIVE Human obesity is a complex metabolic disorder disproportionately affecting people of lower socioeconomic strata, and ethnic minorities, especially African Americans and Hispanics. Although genetic predisposition and a positive energy balance are implicated in obesity, these factors alone do not account for the excess prevalence of obesity in lower socioeconomic populations. Therefore, environmental factors, including exposure to pesticides, heavy metals, and other contaminants, are agents widely suspected to have obesogenic activity, and they also are spatially correlated with lower socioeconomic status. Our study investigates the causal relationship between exposure to the heavy metal, cadmium (Cd), and obesity in a cohort of children and in a zebrafish model of adipogenesis. DESIGN An extensive collection of first trimester maternal blood samples obtained as part of the Newborn Epigenetics Study (NEST) was analyzed for the presence of Cd, and these results were cross analyzed with the weight-gain trajectory of the children through age five years. Next, the role of Cd as a potential obesogen was analyzed in an in vivo zebrafish model. RESULTS Our analysis indicates that the presence of Cd in maternal blood during pregnancy is associated with increased risk of juvenile obesity in the offspring, independent of other variables, including lead (Pb) and smoking status. Our results are recapitulated in a zebrafish model, in which exposure to Cd at levels approximating those observed in the NEST study is associated with increased adiposity. CONCLUSION Our findings identify Cd as a potential human obesogen. Moreover, these observations are recapitulated in a zebrafish model, suggesting that the underlying mechanisms may be evolutionarily conserved, and that zebrafish may be a valuable model for uncovering pathways leading to Cd-mediated obesity in human populations.

The salinization of freshwater ecosystems is emerging as a major ecological issue. Several anthropogenic causes of salinization (e.g. surface coal mining, hydro-fracking, road de-icing, irrigation of arid lands, etc.) are associated with biodiversity losses in freshwater ecosystems. Because insects tend to dominate freshwater ecology, it is important that we develop a better understanding of how and why different species respond to salinity matrices dominated by different major ions. This study builds upon previous work demonstrating that major ion toxicity to the mayfly Neocloeon triangulifer was apparently due to the ionic composition of water rather than specific conductance. Synthetic waters with low Ca:Mg ratios and high SO4:Na ratios produced toxicity, whereas waters with higher Ca:Mg ratios and lower SO4:Na ratios were not toxic to mayflies at comparable conductivities. Here we used a radiotracer approach to show that Mg did not competitively exclude Ca uptake at environmentally realistic ratios in 4 aquatic insect species. We characterized SO4 uptake kinetics in 5 mayflies and assessed the influence of different ions on SO4 uptake. Dual label experiments show an inverse relationship between SO4 and Na transport rates as SO4 was held constant and Na was increased, suggesting that Na (and not Cl or HCO3) is antagonistic to SO4 transport. Based on this observation, we tested the hypothesis that increasing Na would protect against SO4 induced toxicity in a Na-dependent manner. Increasing Na from 0.7 to 10.9mM improved 96-h survivorship associated with 20.8mM SO4 from 44% to 73% in a concentration dependent manner. However, when Na reached 21.8mM, survivorship decreased to 16%, suggesting that other interactive effects of major ions caused toxicity under those conditions. Thus, the combination of elevated sulfate and low sodium commonly observed in streams affected by mountaintop coal mining has the potential to cause toxicity in sensitive aquatic insects. Overall, it is important that we develop a better understanding of major ion toxicity to effectively mitigate and protect freshwater biodiversity from salinization.

Understanding species' thermal limits and their physiological determinants is critical in light of climate change and other human activities that warm freshwater ecosystems. Here, we ask whether oxygen limitation determines the chronic upper thermal limits in larvae of the mayfly Neocloeon triangulifer, an emerging model for ecological and physiological studies. Our experiments are based on a robust understanding of the upper acute (∼40°C) and chronic thermal limits of this species (>28°C, ≤30°C) derived from full life cycle rearing experiments across temperatures. We tested two related predictions derived from the hypothesis that oxygen limitation sets the chronic upper thermal limits: (1) aerobic scope declines in mayfly larvae as they approach and exceed temperatures that are chronically lethal to larvae; and (2) genes indicative of hypoxia challenge are also responsive in larvae exposed to ecologically relevant thermal limits. Neither prediction held true. We estimated aerobic scope by subtracting measurements of standard oxygen consumption rates from measurements of maximum oxygen consumption rates, the latter of which was obtained by treating with the metabolic uncoupling agent carbonyl cyanide-4-(trifluoromethoxy) pheylhydrazone (FCCP). Aerobic scope was similar in larvae held below and above chronic thermal limits. Genes indicative of oxygen limitation (LDH, EGL-9) were only upregulated under hypoxia or during exposure to temperatures beyond the chronic (and more ecologically relevant) thermal limits of this species (LDH). Our results suggest that the chronic thermal limits of this species are likely not driven by oxygen limitation, but rather are determined by other factors, e.g. bioenergetics costs. We caution against the use of short-term thermal ramping approaches to estimate critical thermal limits (CTmax) in aquatic insects because those temperatures are typically higher than those that occur in nature.

Arsenic is an important environmental pollutant whose speciation and mobility in freshwater food webs is complex. Few studies have characterized uptake and efflux rates of arsenic in aquatic benthic invertebrates. Further, we lack a fundamental understanding of how pH influences uptake kinetics in these organisms or how this key environmental variable could alter dietary exposure for primary consumers. Here we used a radiotracer approach to characterize arsenate accumulation dynamics in benthic invertebrates, the influence of pH on uptake in a subset of these organisms, and the influence of pH on uptake of arsenate by periphyton - an important food source at the base of aquatic food webs. Uptake rate constants (K u ) from aqueous exposure were modest, ranging from ∼0.001 L g −1 d −1 in three species of mayfly to 0.06 L g −1 d −1 in Psephenus herricki . Efflux rate constants ranged from ∼0.03 d −1 in Corbicula fluminea to ∼0.3 d −1 in the mayfly Isonychia sp , and were generally high. Arsenate uptake decreased with increasing pH, which may be a function of increased adsorption at lower pHs. A similar but much stronger correlation was observed for periphyton where K u decreased from ∼3.0 L g −1 d −1 at 6.5 pH to ∼0.7 L g −1 d −1 at 8.5 pH, suggesting that site specific pH could significantly alter arsenic exposure, particularly for primary consumers. Together, these findings shed light on the complexity of arsenic bioavailability and help explain observed differences reported in the literature.

The fields of behavioral ecology, conservation science, and environmental toxicology individually aim to protect and manage the conservation of wildlife in response to anthropogenic stressors, including widespread anthropogenic pollution. Although great emphasis in the field of toxicology has been placed on understanding how single pollutants affect survival, a comprehensive, interdisciplinary approach that includes behavioral ecology is essential to address how anthropogenic compounds are a risk for the survival of species and populations in an increasingly polluted world. We provide an integrative framework for behavioral ecotoxicology using Tinbergen’s four postulates (causation and mechanism, development and ontogeny, function and fitness, and evolutionary history and phylogenetic patterns). The aims of this review are: 1) to promote an integrative view and re-define the field of integrative behavioral ecotoxicology; 2) to demonstrate how studying ecotoxicology can promote behavior research; and 3) to identify areas of behavioral ecotoxicology that require further attention to promote the integration and growth of the field.

Assemblages of aquatic insects are structured by multiple biotic and abiotic conditions, including temperature, salinity and oxygen. Here we highlight recent developments in our understanding of how high temperatures, elevated salinities and low oxygen levels affect physiological processes, responses at the organismal level, and impacts on species interaction and community assembly. As aquatic insects may be exposed to multiple stressors, we review their sensitivity to interactive effects of multiple stressors. While each of these stressors may operate via different physiological mechanisms, they all influence the overall energy budget as well as the allocation of energy to competing functions such as homeostatic maintenance, growth, development and reproduction. As such, there is potential for interaction whereby one stressor may exacerbate the effect of another stressor. Integrating research on these stressors can provide a powerful approach for delineating the sensitivity of aquatic insects to multiple stressors and developing sound management practices.

The development of water quality criteria (WQC) for the protection of aquatic life is a fundamental component of the Clean Water Act-the primary US legislation responsible for protecting aquatic ecosystems from pollution. Water quality criteria define acceptable levels of contamination in the environment and thus play an important role in society. Rules for how science is used to develop WQC were created in 1985. Most rely on only data and knowledge obtained through a single methodology, the single-species laboratory toxicity test. Since 1985, understanding of the fate and effects of environmental contaminants has advanced markedly from multiple perspectives and disciplines. However, many of these advances are routinely discarded in WQC development because they do not adhere to data limits imposed by the 1985 guidelines. The present Focus article outlines how multiple lines of inquiry have played important roles in improving understanding of the ecological implications of environmental contaminants. The authors focus on gains in understanding that would not have been possible through traditional toxicity bioassays alone and argue that more robust scientific understanding can be used to modernize WQC development. In particular, the present article highlights ways to increase the relevance of toxicity testing (at different spatiotemporal scales) and incorporate all relevant lines of evidence into WQC modernization. Environ Toxicol Chem 2017;36:285-291. © 2017 SETAC.

Neonicotinoid insecticide usage has increased globally in recent decades. Neonicotinoids, such as imidacloprid, are potent insect neurotoxicants that may pose a threat to non-target aquatic organisms, such as aquatic insects. In nature, insects typically live in thermally fluctuating conditions, which may significantly alter both contaminant exposures and affects. Here we investigate the relationship between temperature and time-to-effect for imidacloprid toxicity with the aquatic insect Isonychia bicolor, a lotic mayfly. Additionally, we examined the mechanisms driving temperature-enhanced toxicity including metabolic rate, imidacloprid uptake rate, and tissue bioconcentration. Experiments included acute toxicity tests utilizing sublethal endpoints and mortality, as well as respirometry and radiotracer assays with [(14)C] imidacloprid. Further, we conducted additional uptake experiments with a suite of aquatic invertebrates (including I. bicolor, Neocloeon triangulifer, Macaffertium modestum, Pteronarcys proteus, Acroneuria carolinensis, and Pleuroceridae sp) to confirm and contextualize our findings from initial experiments. The 96h EC50 (immobility) for I. bicolor at 15°C was 5.81μg/L which was approximately 3.2 fold lower than concentrations associated with 50% mortality. Assays examining the impact of temperature were conducted at 15, 18, 21, and 24°C and demonstrated that time-to-effect for sublethal impairment and immobility was significantly decreased with increasing temperature. Uptake experiments with [(14)C] imidacloprid revealed that initial uptake rates were significantly increased with increasing temperature for I. bicolor, as were oxygen consumption rates. Further, in the separate experiment with multiple species across temperatures 15, 20, and 25°C, we found that all the aquatic insects tested had significantly increased imidacloprid uptake with increasing temperatures, with N. triangulifer accumulating the most imidacloprid on a mass-specific basis. Our acute toxicity results highlight the importance of evaluating sublethal endpoints, as profound impairments of motor function were evident far before mortality. Further, we demonstrate that temperature is a powerful modulator of sublethal toxicity within a range of environmentally relevant temperatures, impacting both uptake rates and metabolic rates of I. bicolor. Finally, we show that temperature alters imidacloprid uptake across a range of species, highlighting the physiological variation present within aquatic invertebrate communities and the challenge associated with relying solely on surrogate species. Taken together, this research points to the need to consider the role of temperature in toxicity assessments.

Many freshwater ecosystems are becoming saltier and/or warmer, but our understanding of how these factors interact and affect the physiology and life history outcomes of most aquatic species remain unknown. We hypothesize that temperature modulates ion transport rates. Since ion transport is energetically expensive, increases in salinity and/or temperature may influence ion flux rates and ultimately, organismal performance. Radiotracer (22Na+, 35SO4-2, and 45Ca2+) experiments with lab-reared mayflies (N. triangulifer) and other field-collected insects showed that increasing temperature generally increased ion transport rates. For example, increasing temperature from 15 °C to 25 °C, increased 22Na+ uptake rates by two-fold (p < 0.0001) and 35SO4-2 uptake rates by four-fold (p < 0.0001) in the caddisfly, Hydropsyche sparna. Smaller changes in 22Na+ and 35SO4-2 uptake rates were observed in the mayflies, Isonychia sayi and Maccaffertium sp., suggesting species-specific differences in the thermal sensitivity of ion transport. Finally, we demonstrated that the toxicity of SO4 was influenced by temperature profoundly in a 96-h bioassay. Under the saltiest conditions (1500 mg L-1 SO4), mayfly survival was 78 % at 15 °C, but only 44 % at 25 °C (p < 0.0036). Conceivably, the energetic cost of osmoregulation in warmer, saltier environments may cause significant major ion toxicity in certain freshwater insects.

Criteria for establishing water quality standards that are protective for 95% of the native species are generally based upon laboratory toxicity tests. These tests utilize common model organisms that have established test methods. However, for invertebrates these species represent mostly the zooplankton community and are not inclusive of all taxa. In order to examine a potential under-representation in emerging aquatic invertebrates the US Environmental Protection Agency has cultured a parthenogenetic mayfly, Centroptilum triangulifer (Ephemeroptera: Baetidae). This study established a 48h acute and a 14-day short-term chronic testing procedure for C. triangulifer and compared its sensitivity to two model invertebrates, Ceriodaphnia dubia and Daphnia magna. Toxicity tests were conducted to determine mortality and growth effects using standard reference toxicants: NaCl, KCl and CuSO4. In 48-h acute tests, the average LC50 for the mayfly was 659mgL(-1) NaCl, 1957mgL(-1) KCl, and 11μgL(-1) CuSO4. IC25 values, using dry weight as the endpoint, were 228mgL(-1) NaCl, 356mgL(-1) KCl and 5μgL(-1) CuSO4. C. triangulifer was the most sensitive species in NaCl acute and chronic growth tests. At KCl concentrations tested, C. triangulifer was less sensitive for acute tests but was equally or more sensitive than C. dubia and D. magna for growth measurements. This study determined C. triangulifer has great potential and benefits for use in ecotoxicological studies.

1) Many environmental stressors manifest their effects via physiological processes (traits) that can differ significantly among species and species groups. We compiled available data for three traits related to the bioconcentration of the toxic metal cadmium (Cd) from 42 aquatic insect species representing orders Ephemeroptera (mayfly), Plecoptera (stonefly), and Trichoptera (caddisfly). These traits included the propensity to take up Cd from water (uptake rate constant, ku), the ability to excrete Cd (efflux rate constant, ke), and the net result of these two processes (bioconcentration factor, BCF). 2) Ranges in these Cd bioaccumulation traits varied in magnitude across lineages (some lineages had a greater tendency to bioaccumulate Cd than others). Overlap in the ranges of trait values among different lineages was common and highlights situations where species from different lineages can share a similar trait state, but represent the high end of possible physiological values for one lineage and the low end for another. 3) Variance around the mean trait state differed widely across clades, suggesting that some groups (e.g., Ephemerellidae) are inherently more variable than others (e.g., Perlidae). Thus, trait variability/lability is at least partially a function of lineage. 4) Akaike information criterion (AIC) comparisons of statistical models were moremore » often driven by clade than by other potential biological or ecological explanation tested. Clade-driven models generally improved with increasing taxonomic resolution. 5) Altogether, these findings suggest that lineage provides context for the analysis of species traits, and that failure to consider lineage in community-based analysis of traits may obscure important patterns of species responses to environmental change.« less

Aquatic insects play critical roles in freshwater ecosystems and temperature is a fundamental driver of species performance and distributions. However, the physiological mechanisms that determine the thermal performance of species remain unclear. Here we used a metabolomics approach to gain insights into physiological changes associated with a short-term, sublethal thermal challenge in the mayfly Neocloeon triangulifer (Ephemeroptera: Baetidae). Larvae were subjected to a thermal ramp (from 22 to 30 °C at a rate of 1°C/h) and metabolomics analysis (both Nuclear Magnetic Resonance (NMR) Spectroscopy and Gas Chromatography coupled Time-of-Flight Mass Spectrometry (GC-TOF-MS)) indicated that processes related to energetics (sugar metabolism) and membrane stabilization primarily differentiated heat treated larvae from controls. Limited evidence of anaerobic metabolism was observed in the heat treated larvae at 30°C, a temperature that is chronically lethal to larvae.

Anthropogenic salinization of rivers is an emerging issue of global concern, with significant adverse effects on biodiversity and ecosystem functioning. Impacts of freshwater salinization on biota are strongly mediated by evolutionary history, as this is a major factor determining species physiological salinity tolerance. Freshwater insects dominate most flowing waters, and the common lotic insect orders Ephemeroptera (mayflies), Plecoptera (stoneflies) and Trichoptera (caddisflies) are particularly salt-sensitive. Tolerances of existing taxa, rapid adaption, colonization by novel taxa (from naturally saline environments) and interactions between species will be key drivers of assemblages in saline lotic systems. Here we outline a conceptual framework predicting how communities may change in salinizing rivers. We envision that a relatively small number of taxa will be saline-tolerant and able to colonize salinized rivers (e.g. most naturally saline habitats are lentic; thus potential colonizers would need to adapt to lotic environments), leading to depauperate communities in these environments.

Understanding species' thermal limits and their physiological determinants is critical in light of climate change and other human activities that warm freshwater ecosystems. Here, we ask whether oxygen limitation determines the chronic upper thermal limits in larvae of the mayfly

Elevated major ion concentrations in streams are commonly observed as a consequence of resource extraction, de-icing and other anthropogenic activities. Ecologists report biodiversity losses associated with increasing salinity, with mayflies typically being highly responsive to increases of different major ions. In this study, we evaluated the performance of the mayfly Neocloeon triangulifer reared for its entire larval phase in a gradient of sulfate concentrations. Two natural waters were amended with SO 4 as a blend of CaSO 4 and MgSO 4 and exposures ranged from 5 to 1500 mg l –1 SO 4. Survival (per cent successful emergence to the subimago stage) was significantly reduced at the highest SO 4 concentration in both waters, while development was significantly delayed at 667 mg l −1 SO 4 . Final sub-adult body weights were consistent across treatments, except at the highest treatment concentration. Despite evidence for sulfate uptake rates increasing with exposure concentrations and not being saturated at even extremely high SO 4 concentrations, total body sulfur changed little in subimagos. Together, these results suggest that elevated SO 4 imposes an energetic demand associated with maintaining homeostasis that is manifested primarily as reduced growth rates and associated developmental delays. We identified two genes related to sulfate transport in N. triangulifer . This article is part of the theme issue ‘Salt in freshwaters: causes, ecological consequences and future prospects’.

Freshwater mussels (superfamily Unionoidea) are in serious global decline and in urgent need of protection and conservation. The declines have been attributed to a wide array of human activities resulting in pollution and water-quality degradation, and habitat destruction and alteration. Linkages among poor water quality, pollutant sources, and mussel decline in rivers and streams have been associated with results of laboratory-based tests of specific pollutants. However, uncertainties remain about the relationship of laboratory data to actual contaminant exposure routes for various mussel species, life stages, and in the habitats occupied during these exposures. We evaluated the pathways of exposure to environmental pollutants for all 4 life stages (free glochidia, encysted glochidia, juveniles, adults) of unionoidean mussels and found that each life stage has both common and unique characteristics that contribute to observed differences in exposure and sensitivity. Free glochidia typically are exposed only briefly (e.g., seconds to days) through surface water, whereas adults sustain exposure over years to decades through surface water, pore water, sediment, and diet. Juveniles live largely burrowed in the sediment for the first 0 to 4 y of life. Thus, sediment, pore water, and diet are the predominant exposure routes for this life stage, but surface water also might contribute to exposure during certain periods and environmental conditions. The obligate parasitic stage (encysted glochidia stage) on a host fish might be exposed from surface water while partially encysted or from toxicants in host-fish tissue while fully encysted. Laboratory methods for testing for acute and chronic exposures in water have advanced, and toxicant-specific information has increased in recent years. However, additional research is needed to understand interactions of life history, habitat, and long-term exposure to contaminants through water, pore water, sediment, and diet so that the risks of environmental exposures can be properly assessed and managed.

It has become increasingly apparent that diet can be a major source of trace metal bioaccumulation in aquatic organisms. In this study, we examined cadmium uptake, efflux, and subcellular compartmentalization dynamics in the freshwater oligochaete Lumbriculus variegatus. L. variegatus is an important component of freshwater food webs in Europe and North America and is potentially useful as a standard food source for laboratory-based trophic transfer studies. Cadmium accumulation and depuration were each followed for 10 days. Rate constants of uptake (ku) and efflux (ke) were estimated and subcellular Cd compartmentalization was followed over the course of uptake and efflux. The partitioning of Cd into operationally-defined subcellular compartments was relatively consistent throughout the 20-day experiment, with the majority of Cd accumulating in the cytosol. No major changes in Cd compartmentalization were observed over uptake or depuration, but there appeared to be some exchange between heat-stable and heat-labile cytosolic protein fractions. Cadmium accumulation from solution was strongly affected by ambient calcium concentrations, suggesting competition between Cd and Ca for uptake sites. Finally, we demonstrate the ability to manipulate the whole body calcium content of L. variegatus as a potential tool for examining calcium influences on dietary Cd dynamics. The potential for this species to be an important conduit of Cd to higher trophic levels is discussed, along with its potential as a standardized food source in metal trophic transfer studies. © 2007 Elsevier B.V. All rights reserved.

Aquatic insects often dominate lotic ecosystems, yet these organisms are under-represented in trace metal toxicity databases. Furthermore, toxicity data for aquatic insects do not appear to reflect their actual sensitivities to metals in nature, because the concentrations required to elicit toxicity in the laboratory are considerably higher than those found to impact insect communities in the field. New approaches are therefore needed to better understand how and why insects are differentially susceptible to metal exposures. Biodynamic modeling is a powerful tool for understanding interspecific differences in trace metal bioaccumulation. Because bioaccumulation alone does not necessarily correlate with toxicity, we combined biokinetic parameters associated with dissolved cadmium exposures with studies of the subcellular compartmentalization of accumulated Cd. This combination of physiological traits allowed us to make predictions of susceptibility differences to dissolved Cd in three aquatic insect taxa: Ephemerella excrucians, Rhithrogena morrisoni, and Rhyacophila sp. We compared these predictions with long-term field monitoring data and toxicity tests with closely related taxa: Ephemerella infrequens, Rhithrogena hageni, and Rhyacophila brunea. Kinetic parameters allowed us to estimate steady-state concentrations, the time required to reach steady state, and the concentrations of Cd projected to be in potentially toxic compartments for different species. Species-specific physiological traits identified using biodynamic models provided a means for better understanding why toxicity assays with insects have failed to provide meaningful estimates for metal concentrations that would be expected to be protective in nature.

The importance of insects in freshwater ecosystems has led to their extensive use in ecological monitoring programs. As freshwater systems are increasingly challenged by salinization and metal contamination, it is important to understand fundamental aspects of aquatic insect physiology (e.g., osmoregulatory processes) that contribute to insect responses to these stressors. Here we compared the uptake dynamics of Na as NaCl, NaHCO3 and Na2SO4 in the caddisfly Hydropsyche betteni across a range of Na concentrations (0.06-15.22 mM) encompassing the vast majority of North American freshwater ecosystems. Sulfate as the major anion resulted in decreased Na uptake rates relative to the chloride and bicarbonate salts. A comparison of Na (as NaHCO3) turnover rates in the caddisfly Hydropsyche sparna and the mayfly Maccaffertium sp. revealed different patterns in the 2 species. Both species appeared to tightly regulate their whole body sodium concentrations (at ∼47±1.8 μmol/g wet wt) across a range of Na concentrations (0.06-15.22 mM) over 7 days. However, at the highest Na concentration (15.22 mM), Na uptake rates in H. sparna (419.1 μM Na g(-1) hr(-1) wet wt) appeared close to saturation while Na uptake rates in Maccaffertium sp. were considerably faster (715 g μM Na g(-1) hr(-1) wet wt) and appeared to not be close to saturation. Na efflux studies in H. sparna revealed that loss rates are commensurate with uptake rates and are responsive to changes in water Na concentrations. A comparison of Na uptake rates (at 0.57 mM Na) across 9 species representing 4 major orders (Ephemeroptera, Plecoptera, Trichoptera and Diptera) demonstrated profound physiological differences across species after accounting for the influence of body weight. Faster Na uptake rates were associated with species described as being sensitive to salinization in field studies. The metals silver (Ag) and copper (Cu), known to be antagonistic to Na uptake in other aquatic taxa did not generally exhibit this effect in aquatic insects. Ag only reduced Na uptake at extremely high concentrations, while Cu generally stimulated Na uptake in aquatic insects, rather than suppress it. These results help explain the lack of insect responses to dissolved metal exposures in traditional toxicity testing and highlight the need to better understand fundamental physiological processes in this ecologically important faunal group.

Development of methods for assessing exposure and effects of waterborne toxicants on stream invertebrate species is important to elucidate environmentally relevant information. Current protocols for freshwater invertebrate toxicity testing almost exclusively utilize cladocerans, amphipods or chironomids rather than the more typical aquatic insect taxa found in lotic systems. Centroptilum triangulifer is a parthenogenetic mayfly occurring in depositional habitats of streams and rivers of the Eastern U.S. and Canada. C. triangulifer is an ideal stream insect for toxicity testing under field and laboratory conditions because of its short life cycle, parthenogenetic mode of reproduction, and it represents a group considered sensitive to environmental stressors. In this study, a colony of C. triangulifer was reared using a defined diet of three diatoms, Mayamaea atomus var. permitis, Nitzschia cf. pusilla, and Achnanthidium minutissimum. Percent survival (⩾80%), fecundity measurements (⩾1000 eggs) and pre-egg laying weights were used as indicators of overall colony health and fitness in our laboratory water (Lab-line) and in Moderately Hard Reconstituted Water (MHRW). Lab-line reared C. triangulifer had average survival rate of 92.69% for eleven generations and 82.99% over thirteen generations. MHRW reared C. triangulifer had an average survival rate of 80.65% for four generations and three generations of fecundities greater than 1000 eggs per individual. Pre-egg laying weight and fecundity were highly correlated and a best-fit model equation was derived to estimate egg counts for future generations. Establishment of this culturing protocol provides a more ecologically relevant species for toxicity testing and aids in further stressor identification for stream bioassessments.

In situ translocation experiments are advantageous relative to traditional laboratory experiments, particularly for understanding the bioavailability of trace metals like mercury (Hg). Individuals of the polychaete Perinereis gualpensis were translocated from a reference site (Raqui estuary, Chile) to an estuarine site with significant sediment Hg concentrations (Lenga estuary: 1.78–9.89 mg/kg). Individuals were exposed in polluted and non-polluted sediments for 21 days and sampled every 7 days with cages deployed at three different depths. Tissue Hg concentrations were measured in conjunction with oxidative stress responses. Translocated polychaetes rapidly accumulated Hg. Glutathione S-transferase (GST) activities measured from posterior body regions were 2-fold higher than control activities after 21 days of exposure. Other antioxidant measures were idiosyncratic. Distinct burrowing behavior differences were observed; control polychaetes exhibited more homogenous vertical distributions, whereas in Lenga, worms tended to remain in upper layers. These studies demonstrate that under natural conditions, Hg is highly bioavailable to polychaetes affecting both biochemical and behavioral responses after relatively short-term exposure. Fil: Diaz Jaramillo, Mauricio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Concepción; Chile Fil: da Rocha, Alessandra Martins. Universidade Federal do Rio Grande do Sul; Brasil Fil: Chiang, Gustavo. Universidad de Concepción; Chile. University Of New Brunswick; Canadá Fil: Buchwalter, David. University of North Carolina; Estados Unidos Fil: Monserrat, José María. Universidade Federal do Rio Grande do Sul; Brasil Fil: Barra, Ricardo. Universidad de Concepción; Chile

SummaryCalcium sequestration in the hypo-osmotic freshwater environment is imperative in maintaining calcium homeostasis in freshwater aquatic organisms. This uptake process is reported to have the unintended consequence of potentially toxic heavy metal (Cd, Zn) uptake in a variety of aquatic species. However, calcium uptake remains poorly understood in aquatic insects, the dominant invertebrate faunal group on most freshwater ecosystems. Here we examined Ca uptake and interactions with heavy metals (Cd, Zn) at low ambient Ca levels (12.5 μmol l-1) in 12 aquatic insect species within Ephemerellidae (mayfly) and Hydropsychidae (caddis fly), two families differentially responsive to trace metal pollution. We found Ca uptake varied 70-fold across the 12 species studied. Body weight and clade (family) were found to significantly influence both Ca uptake and adsorption (p < 0.05). Zn and Cd uptake rate constants (kus) exhibited a strong correlation (r = 0.96, p < 0.0001), suggesting a shared transport system. Ca uptake failed to significantly correlate with either Zn or Cd kus. Further, neither Zn nor Cd exhibited inhibitory effects toward Ca uptake. In fact we saw evidence of modest stimulation of Ca uptake rates in some metal treatments. This work suggests that insects generally differ from other freshwater taxa in that aqueous Ca uptake does not appear to be compromised by Cd or Zn exposure. It is important to understand the trace metal and major ion physiology of aquatic insects due to their ecological importance and widespread use as ecological indicators.

A simple and robust isotope dilution mass spectrometry-based assay was developed for the determination of free cysteine and glutathione (GSH) in aquatic insects. Several experimental parameters were evaluated and optimized to provide specific and sensitive detection of both compounds by in situ derivatization with N-ethylmaleimide followed by acid alkylation quenching and reverse-phased liquid chromatography coupled with selected reaction monitoring. For both targets, the assay was evaluated over a concentration range of 0.313 to 320 μM and was demonstrated to have a quantitative dynamic range spanning nearly three orders of magnitude, with lower limits of quantification being 0.330 μM for GSH and 0.370 μM for cysteine. Additionally, measurements were observed to be highly reproducible over the course of several days. When applied to the analysis of four different species of insects, large biological variation between and within species was observed. Different feeding regimens were also tested within two species of insects but statistical comparisons revealed no significant difference in the levels of either compound.

Cadmium (Cd) is a toxic metal and an important environmental pollutant that can strongly affect mitochondrial function and bioenergetics in animals. We investigated the mechanisms of Cd action on mitochondrial function of a marine mollusk (the eastern oyster Crassostrea virginica ) by performing a top-down control analysis of the three major mitochondrial subsystems (substrate oxidation, proton leak, and phosphorylation). Our results showed that the substrate oxidation and proton leak subsystems are the main targets for Cd toxicity in oyster mitochondria. Exposure to 12.5 μM Cd strongly inhibited the substrate oxidation subsystem and stimulated the proton conductance across the inner mitochondrial membrane. Proton conductance was also elevated and substrate oxidation inhibited by Cd in the presence of a mitochondrially targeted antioxidant, MitoVitE, indicating that Cd effects on these subsystems were to a large extent ROS independent. Cd did not affect the kinetics of the phosphorylation system, indicating that it has negligible effects on F1, FOATP synthase and/or the adenine nucleotide transporter in oyster mitochondria. Cd exposure altered the patterns of control over mitochondrial respiration, increasing the degree of control conferred by the substrate oxidation subsystem, especially in resting (state 4) mitochondria. Taken together, these data suggest that Cd-induced decrease of mitochondrial efficiency and ATP production are predominantly driven by the high sensitivity of substrate oxidation and proton leak subsystems to this metal.

In streams, periphyton biofilms are important sinks for trace metals such as cadmium and are primary food sources of many invertebrate consumers. To study Cd trophic transfer, we produced differentially contaminated diets by exposing natural periphyton to environmentally relevant dissolved Cd ranging from 0 to 10 μg L−1 for 6–7 days using a radiotracer approach. On average, periphyton grown during three different seasons bioconcentrated Cd similarly – approximately 1315 (±442) -fold above dissolved concentrations. However, mayfly larvae (Centroptilum triangulifer) raised on these differentially contaminated diets (first instar through adulthood) had significantly higher trophic transfer factors from periphyton grown in Aug and Nov 2008 (4.30 ± 1.55) than from periphyton grown in Jan 2009 (0.85 ± 0.21). This Cd bioaccumulation difference is only partially explained by apparent food quality and subsequent growth differences. Taken together, these results suggest that primary producers at the base of food webs drive metal bioaccumulation by invertebrate grazers.

We used a phylogenetically based comparative approach to evaluate the potential for physiological studies to reveal patterns of diversity in traits related to susceptibility to an environmental stressor, the trace metal cadmium (Cd). Physiological traits related to Cd bioaccumulation, compartmentalization, and ultimately susceptibility were measured in 21 aquatic insect species representing the orders Ephemeroptera, Plecoptera, and Trichoptera. We mapped these experimentally derived physiological traits onto a phylogeny and quantified the tendency for related species to be similar (phylogenetic signal). All traits related to Cd bioaccumulation and susceptibility exhibited statistically significant phylogenetic signal, although the signal strength varied among traits. Conventional and phylogenetically based regression models were compared, revealing great variability within orders but consistent, strong differences among insect families. Uptake and elimination rate constants were positively correlated among species, but only when effects of body size and phylogeny were incorporated in the analysis. Together, uptake and elimination rates predicted dramatic Cd bioaccumulation differences among species that agreed with field-based measurements. We discovered a potential tradeoff between the ability to eliminate Cd and the ability to detoxify it across species, particularly mayflies. The best-fit regression models were driven by phylogenetic parameters (especially differences among families) rather than functional traits, suggesting that it may eventually be possible to predict a taxon's physiological performance based on its phylogenetic position, provided adequate physiological information is available for close relatives. There appears to be great potential for evolutionary physiological approaches to augment our understanding of insect responses to environmental stressors in nature.

Early life stages of aquatic organisms tend to be more sensitive to various chemical contaminants than later life stages. This research attempted to identify the key biological factors that determined sensitivity differences among life stages of the aquatic insect Chironomous riparius. Specifically, second to fourth instar larvae were exposed in vivo to both low and high waterborne concentrations of chlorpyrifos to examine differences in accumulation rates, chlorpyrifos biotransformation, and overall sensitivity among instars. In vitro acetylcholinesterase (AChE) assays were performed with chlorpyrifos and the metabolite, chlorpyrifos-oxon, to investigate potential target site sensitivity differences among instars. Earlier instars accumulated chlorpyrifos more rapidly than later instars. There were no major differences among instars in the biotransformation rates of chlorpyrifos to the more polar metabolites, chlorpyrifos-oxon, and chlorpyridinol (TCP). Homogenate AChE activities from second to fourth instar larvae were refractory to chlorpyrifos, even at high concentrations. In contrast, homogenate AChE activities were responsive in a dose-dependent manner to chlorpyrifos-oxon. In general, it appeared that chlorpyrifos sensitivity differences among second to fourth instar C. riparius were largely determined by differences in uptake rates. In terms of AChE depression, fourth instar homogenates were more sensitive to chlorpyrifos and chlorpyrifos-oxon than earlier instars. However, basal AChE activity in fourth instar larvae was significantly higher than basal AChE activity in second to third instar larvae, which could potentially offset the apparent increased sensitivity to the oxon.

The influence of metal exposure history on rates of aqueous Cd accumulation, elimination, and subcellular distribution was examined in the aquatic insect Hydropsyche californica. Specimens were obtained from a reference site and a metal-contaminated site and returned to the laboratory where they were continuously exposed to aqueous Cd (518 ng/L, nominal) for 6 d, followed by 9 d of depuration. Rates of Cd accumulation and elimination were similar in insects from the two sites. Efflux rate constants, k((e), ranged from 0.20 to 0.24/d (t 1/2 approximately 3 d). Immediately following exposure, the cytosol accounted for 40% of the body burden in insects from both sites; however, 89 +/- 2% of the cytosolic Cd was associated with metallothionein-like proteins (MTLP) in insects from the contaminated site, compared to 60 +/- 0% in insects from the reference site. The concentration of Cd bound to non-MTLPs (representing potentially Cd-sensitive proteins) was significantly greater in the insects from the reference site (134 +/- 7 ng/g) than in those from the contaminated site (42 +/- 2 ng/g). At the end of the depuration period, 90% of the accumulated Cd body burden had been eliminated, and Cd concentrations in MTLPs and non-MTLPs were similar between the sites. Results suggested that differences in exposure history had no influence on the bioaccumulation of Cd, but did affect the concentrations of Cd bound to MTLP during Cd exposure in these insects.