Variation in Freshwater Insect Osmoregulatory Traits: A Comparative Approach.

AbstractFreshwater salinity regimes vary naturally and are changing in response to anthropogenic activities. Few insect species tolerate saline waters, and biodiversity losses are associated with increasing salinity in freshwater. We used radiotracers (22Na, 35SO4, and 45Ca) to examine ion uptake rates across concentration gradients in mayflies (Ephemeroptera), caddis flies (Trichoptera), and mosquitoes (Diptera) and made observations for some traits in seven other taxa representing mayflies, stone flies (Plecoptera), true flies (Diptera), and true bugs (Hemiptera). We further assessed the permeability of the cuticle to 3H2O influx and 22Na efflux when faced with deionized water in these same taxa. We hypothesized a relationship between uptake rates and reported saline tolerances, but our data did not support this hypothesis, likely because acclimatory responses were not part of this experimental approach. However, we found several common physiological traits across the taxa studied, including (i) ionic uptake rates that were always positively correlated with dissolved concentrations, (ii) generally low Ca uptake rates relative to other freshwater taxa, (iii) greater Na loss than Na uptake in dilute conditions, (iv) ion uptake that was more variable in ion-rich conditions than in dilute conditions, and (v) 3H2O influx that occurs quickly (but this rapidly exchangeable pool of body water accounts for a surprisingly small percentage of the water content of species tested). There remains much to learn about the physiology of these important organisms in the face of changing salinity regimes worldwide.

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.

Salinity-induced ionoregulatory changes in the gill proteome of the mayfly, Neocloeon triangulifer.

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-1 Na), elevated CaCl2 (121 mg L-1 Ca), elevated Ca/MgSO4 (735 mg L-1 SO4). These conditions mirrored those from previously published physiological work. We also acutely exposed nymphs to dilute (50% dilution of culture water with deionized water) to explore proteomic changes in the gills in response to dilute conditions. We report 710 unique peptide sequences among treatment groups, including important apical ion transporters such as Ca-ATPase, Na/K ATPase, and V-ATPase. Treatment with elevated NaCl and Ca/MgSO4 appeared to cause more significant differential protein expression (452 and 345, respectively) compared to CaCl2 and dilute groups (134 and 17, respectively). Finally, we demonstrated the breadth of physiological functions in gills by exploring non-transport related pathways found in our dataset, including ATP synthesis, calcium signaling, and oxidative stress response. We discuss our results in the context of freshwater salinization and the challenges of working with non-model species without fully sequenced and annotated genomes.

The acclimatory response of the mayfly Neocloeon triangulifer to dilute conditions is linked to the plasticity of sodium transport.

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 Neocloeon triangulifer by rearing nymphs from 1-day post-egg hatch to adulthood across a gradient of decreasing Na concentrations (15, 8, 4, 2 and 1 mg l-1 Na). We found no significant changes in survival, growth, development time and whole-body Na content across these treatments. Radiotracer data revealed that nymphs acclimated to their dilute exposures by increasing their rates of Na uptake and were able to maintain a relatively narrow range of uptake rates (±s.e.m.) of 38.5 ± 4.2 µg Na g-1 h-1 across all treatments. By contrast, the Na uptake rates observed in naive nymphs were much more concentration dependent. This acclimatory response is partially explained by differences in ionocyte counts on the gills of nymphs reared under different salinities. Acclimated nymphs were surprisingly less retentive of their sodium composition when subjected to deionized water challenge. By contrasting our findings with a previous N. triangulifer salinity acclimation study, we show a physiological affinity for dilute conditions in this emerging mayfly model.

Assessing the Pcrit in relation to temperature and the expression of hypoxia associated genes in the mayfly, Neocloeon triangulifer.

Hypoxia is a growing concern in aquatic ecosystems. Historically, scientists have used the Pcrit (the dissolved oxygen level below which an animal can no longer oxyregulate) to infer hypoxia tolerance across species. Here, we tested the hypothesis that the Pcrit is positively correlated with temperature in the mayfly, Neocloeon triangulifer. Cross-temperature comparisons showed a modest (r = 0.47), but significant (p < 0.0001) association between temperature and Pcrit despite relatively large interindividual variability (Coefficient of Variance (CV) = 39.9% at 18 °C). We used the expression of hypoxia-responsive genes EGL-9 (an oxygen sensing gene and modulator of HIF-1a activity) and LDH (a hypoxia indicator) to test whether oxygen partial pressure near the Pcrit stimulates expression of hypoxia-responsive genes. Neither gene was upregulated at oxygen levels above the estimated Pcrit, however, at or below the Pcrit estimates, expression of both genes was stimulated (~20- and ~3-fold change for EGL-9 and LDH, respectively). Finally, we evaluated the influence of hypoxic exposure time and pretreatment conditions on the mRNA expression levels of hypoxia-responsive genes. When larvae were exposed to a gradual reduction of DO, hypoxic gene expression was more robust than during instantaneous exposure to hypoxia. Our data provide modest support for traditional interpretation of the Pcrit as a physiologically meaningful shift from aerobic to anaerobic metabolism in N. triangulifer. However, we also discuss limitations of the Pcrit as a proxy measure of hypoxia tolerance at the species level.

Physiological plasticity and acclimatory responses to salinity stress are ion-specific in the mayfly, Neocloeon triangulifer.

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 < 0.0001) and 8-fold (p < 0.0001) lower ion uptake rates than mayflies reared in dilute control water (16 mg L-1 Na and 23 mg L-1 SO4) and subsequently transferred to elevated salinities, respectively. These acclimatory ion transport changes provided protection in 96-h toxicity bioassays for sodium, but not sulfate. Interestingly, calcium uptake was uniformly much lower and minimally influenced by exposure history, but was poorly tolerated in the toxicity bioassays. With qRT-PCR, we observed that the expression of many ion transporter genes in mayflies was influenced by elevated salinity in an ion-specific manner (general upregulation in response to sulfate, downregulation in response to calcium). Elevated sodium exposure had minimal influence on the same genes. Finally, we provide novel light microscopic evidence of histomorphological changes within the epithelium of the Malpighian tubules (insect primary excretory system) that undergoes cellular degeneration and necrosis secondary to calcium toxicity. We conclude that physiological plasticity to salinity stress is ion-specific and provide evidence for ion-specific toxicity mechanisms in N. triangulifer.

Periphyton enhances arsenic release and methylation at the soil-water interface of paddy soils.

Periphyton is ubiquitous in rice paddy fields, however its role in paddy soil arsenic (As) biogeochemistry remains unexplored. In this study, microcosm incubations and extensive field sampling were used to better understand the roles of periphyton on As mobility and transformation at the soil-water interface. Microcosm incubations revealed that periphyton on the paddy soil surface enhanced As release to water and increased methylated As contents at the soil-water interface. Experimental additions of dissolved phosphate did not significantly affect these processes. The presence of periphyton increased the dissolved organic carbon (DOC) content of the surface soil which may have played a role in the increased As mobility. However, the increase in methylated As species at the soil-water interface is indicative of detoxification processes of As by periphyton. The results from the field study revealed a high abundance and diversity of As biotransformation and detoxification genes in periphyton. Genera of Kineosporia, Limisphaera, Ornatilinea, Ktedonosporobacter and Anaerolinea played key roles in shaping arsM harboring microbe communities in field periphyton. These results highlight the importance of periphyton in the behavior of As in paddy soils and can potentially facilitate improved management of As contamination in paddy soils.

Transcriptomic and life history responses of the mayfly Neocloeon triangulifer to chronic diel thermal challenge.

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.

Energetics as a lens to understanding aquatic insect's responses to changing temperature, dissolved oxygen and salinity regimes.

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.

It's all about the fluxes: Temperature influences ion transport and toxicity in aquatic insects.

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.

Cadmium exposure increases the risk of juvenile obesity: a human and zebrafish comparative study.

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 5 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.

Periphyton and abiotic factors influencing arsenic speciation in aquatic environments.

Benthic periphytic biofilms are important food sources at the base of aquatic ecosystems. These biofilms also sit at the interface of oxic waters and hypoxic sediments, and can be influenced by or influence trace element speciation. In the present study, we compared arsenic (As) enrichment in periphyton exposed to arsenate (As[V]) or arsenite (As[III]) (20 µg/L, static renewal, 7 d), and we found similar accumulation patterns of total As (101 ± 27 and 88 ± 22 mg kg-1 dry wt, respectively). Periphyton As was 6281- and 6684-fold higher than their aqueous exposures and occurred primarily as As(V). When these biofilms were fed to larval mayflies, similar total As tissue concentrations (13.9 and 14.6 mg kg-1 dry wt, respectively) were observed, revealing significant biodilution (∼ 10% of their dietary concentrations). Finally, we investigated the influence of aeration and periphyton presence on As speciation in solutions and solid phases treated with As(III). Predominantly As(III) solutions were slowly oxidized over a 7-d time period, in the absence of periphyton, and aeration did not strongly affect oxidation rates. However, in the presence of periphyton, solution and solid-phase analyses (by microscale x-ray absorption spectroscopy) showed rapid As(III) oxidation to As(V) and an increasing proportion of organo-As forming over time. Thus periphyton plays several roles in As environmental behavior: 1) decreasing total dissolved As concentrations via abiotic and biotic accumulation, 2) rapidly oxidizing As(III) to As(V), 3) effluxing organo-As forms into solution, and 4) limiting trophic transfer to aquatic grazers. Environ Toxicol Chem 2018;37:903-913. © 2017 SETAC.

Metabolomics reveal physiological changes in mayfly larvae (Neocloeon triangulifer) at ecological upper thermal limits.

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.

Physiological responses to short-term thermal stress in mayfly (Neocloeon triangulifer) larvae in relation to upper thermal limits.

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.

Periphyton uptake and trophic transfer of coal fly-ash-derived trace elements.

To determine whether the bioavailability of trace elements derived from coal ash leachates varies with the geochemical conditions associated with their formation, we quantified periphyton bioaccumulation and subsequent trophic transfer to the mayfly Neocloeon triangulifer. Oxic ash incubations favored periphyton uptake of arsenic, selenium, strontium, and manganese, whereas anoxic incubations favored periphyton uptake of uranium. Mayfly enrichment was strongest for selenium, whereas biodilution was observed for strontium, uranium, and arsenic. Environ Toxicol Chem 2017;36:2991-2996. © 2017 SETAC.

Arsenic (V) bioconcentration kinetics in freshwater macroinvertebrates and periphyton is influenced by pH.

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 (Ku) from aqueous exposure were modest, ranging from ∼0.001 L g-1d-1 in three species of mayfly to 0.06 L g-1d-1in 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 Ku decreased from ∼3.0 L g-1d-1 at 6.5 pH to ∼0.7 L g-1d-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.

Modernizing Water Quality Criteria in the United States: A Need to Expand the Definition of Acceptable Data.

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.

Integrative behavioral ecotoxicology: bringing together fields to establish new insight to behavioral ecology, toxicology, and conservation.

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.

Bioaccumulation Dynamics of Arsenate at the Base of Aquatic Food Webs.

Periphyton is an important food source at the base of freshwater ecosystems that tends to bioconcentrate trace elements making them trophically available. The potential for arsenic-a trace element of particular concern due to its widespread occurrence, toxicity, and carcinogenicity-to bioconcentrate in periphyton and thus be available to benthic grazers is less well characterized. To better understand arsenate bioaccumulation dynamics in lotic food webs, we used a radiotracer approach to characterize accumulation in periphyton and subsequent trophic transfer to benthic grazers. Periphyton bioconcentrated As between 3,200-9,700-fold (dry weight) over 8 days without reaching steady state, suggesting that periphyton is a major sink for arsenate. However, As-enriched periphyton as a food source for the mayfly Neocloeon triangulifer resulted in negligible As accumulation in a full lifecycle exposure. Additional studies estimate dietary assimilation efficiency in several primary consumers ranging from 22% in the mayfly N. triangulifer to 75% in the mayfly Isonychia sp. X-ray fluorescence mapping revealed that As was predominantly associated with iron oxides in periphyton. We speculate that As adsorption to Fe in periphyton may play a role in reducing dietary bioavailability. Together, these results suggest that trophic movement of As in lotic food webs is relatively low, though species differences in bioaccumulation patterns are important.

Part 1: Laboratory culture of Centroptilum triangulifer (Ephemeroptera: Baetidae) using a defined diet of three diatoms.

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.