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.

Oxygen limitation fails to explain upper chronic thermal limits and the temperature size rule in mayflies.

An inability to adequately meet tissue oxygen demands has been proposed as an important factor setting upper thermal limits in ectothermic invertebrates (especially aquatic species) as well as explaining the observed decline in adult size with increased rearing temperature during the immature stages (a phenomenon known as the temperature size rule, or TSR). We tested this by rearing three aquatic insects (the mayflies Neocloeon triangulifer and two species of the Cloeon dipterum complex) through their entire larval life under a range of temperature and oxygen concentrations. Hyperoxia did not extend upper thermal limits, nor did it prevent the loss of size or fertility experienced near upper chronic thermal limits. At moderate temperatures, the TSR pattern was observed under conditions of hyperoxia, normoxia and hypoxia, suggesting little or no influence of oxygen on this trend. However, for a given rearing temperature, adults were smaller and less fecund under hypoxia as a result of a lowering of growth rates. These mayflies greatly increased the size of their gills in response to lower dissolved oxygen concentrations but not under oxygen-saturated conditions over a temperature range yielding the classic TSR response. Using ommatidium diameter as a proxy for cell size, we found the classic TSR pattern observed under moderate temperature conditions was due primarily to a change in the number of cells rather than cell size. We conclude overall that a failure to meet tissue oxygen demands is not a viable hypothesis for explaining either the chronic thermal limit or TSR pattern in these species.

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.

Temperature affects acute mayfly responses to elevated salinity: implications for toxicity of road de-icing salts.

Salinity in freshwater ecosystems has increased significantly at numerous locations throughout the world, and this increase often reflects the use or production of salts from road de-icing, mining/oil and gas drilling activities, or agricultural production. When related to de-icing salts, highest salinity often occurs in winter when water temperature is often low relative to mean annual temperature at a site. Our study examined acute (96 h) responses to elevated salinity (NaCl) concentrations at five to seven temperature treatments (5-25°C) for four mayfly species (Baetidae: Neocloeon triangulifer, Procloeon fragile; Heptageniidae: Maccaffertium modestum; Leptophlebiidae: Leptophlebia cupida) that are widely distributed across eastern North America. Based on acute LC50s at 20°C, P. fragile was most sensitive (LC50 = 767 mg l-1, 1447 µS cm-1), followed by N. triangulifer (2755 mg l-1, 5104 µS cm-1), M. modestum (2760 mg l-1, 5118 µS cm-1) and L. cupida (4588 mg l-1, 8485 µS cm-1). Acute LC50s decreased as temperature increased for all four species (n = 5-7, R2 = 0.65-0.88, p = 0.052-0.002). Thus, acute salt toxicity is strongly temperature dependent for the mayfly species we tested, which suggests that brief periods of elevated salinity during cold seasons or in colder locations may be ecologically less toxic than predicted by standard 20 or 25°C laboratory bioassays.This article is part of the theme issue 'Salt in freshwaters: causes, ecological consequences and future prospects'.

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.

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.

Colonization and diversification of aquatic insects on three Macaronesian archipelagos using 59 nuclear loci derived from a draft genome.

The study of processes driving diversification requires a fully sampled and well resolved phylogeny, although a lack of phylogenetic markers remains a limitation for many non-model groups. Multilocus approaches to the study of recent diversification provide a powerful means to study the evolutionary process, but their application remains restricted because multiple unlinked loci with suitable variation for phylogenetic or coalescent analysis are not available for most non-model taxa. Here we identify novel, putative single-copy nuclear DNA (nDNA) phylogenetic markers to study the colonization and diversification of an aquatic insect species complex, Cloeon dipterum L. 1761 (Ephemeroptera: Baetidae), in Macaronesia. Whole-genome sequencing data from one member of the species complex were used to identify 59 nDNA loci (32,213 base pairs), followed by Sanger sequencing of 29 individuals sampled from 13 islands of three Macaronesian archipelagos. Multispecies coalescent analyses established six putative species. Three island species formed a monophyletic clade, with one species occurring on the Azores, Europe and North America. Ancestral state reconstruction indicated at least two colonization events from the mainland (to the Canaries, respectively Azores) and one within the archipelago (between Madeira and the Canaries). Random subsets of the 59 loci showed a positive linear relationship between number of loci and node support. In contrast, node support in the multispecies coalescent tree was negatively correlated with mean number of phylogenetically informative sites per locus, suggesting a complex relationship between tree resolution and marker variability. Our approach highlights the value of combining genomics, coalescent-based phylogeography, species delimitation, and phylogenetic reconstruction to resolve recent diversification events in an archipelago species complex.

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.

Elevated major ion concentrations inhibit larval mayfly growth and development.

Anthropogenic disturbances, including those from developing energy resources, can alter stream chemistry significantly by elevating total dissolved solids. Field studies have indicated that mayflies (Order Ephemeroptera) are particularly sensitive to high total dissolved solids. In the present study, the authors measured 20-d growth and survivorship of larval Neocloeon triangulifer exposed to a gradient of brine salt (mixed NaCl and CaCl2 ) concentrations. Daily growth rates were reduced significantly in all salt concentrations above the control (363 µS cm(-1) ) and larvae in treatments with specific conductance >812 µS cm(-1) were in comparatively earlier developmental stages (instars) at the end of the experiment. Survivorship declined significantly when specific conductance was >1513 µS cm(-1) and the calculated 20-d 50% lethal concentration was 2866 µS cm(-1) . The present study's results provide strong experimental evidence that elevated ion concentrations similar to those observed in developing energy resources, such as oil and gas drilling or coal mining, can adversely affect sensitive aquatic insect species.

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.

Part 2: Sensitivity comparisons of the mayfly Centroptilum triangulifer to Ceriodaphnia dubia and Daphnia magna using standard reference toxicants; NaCl, KCl and CuSO4.

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.

Bioconcentration and biotransformation of selenite versus selenate exposed periphyton and subsequent toxicity to the Mayfly Centroptilum triangulifer.

Little is known about the bioaccumulation dynamics, biotransformation processes, or subsequent toxicity to consumers of dissolved selenite (SeO3) versus selenate (SeO4) uptake into aquatic primary producer communities. To address these data gaps, we examined SeO3 and SeO4 bioconcentration into complex freshwater periphyton communities under static and static-renewal conditions. Further, we explored periphyton biotransformation of Se species using X-ray absorption near edge structure (XANES) spectroscopy analysis and changes in the periphyton associated microbial consortium using denaturing gradient gel electrophoresis (DGGE). Last, we fed differentially treated periphyton to the mayfly Centroptilum triangulifer in full life cycle exposures to assess toxicity. Selenite exposed periphyton readily bioconcentrated Se while, in contrast, initial periphyton uptake of SeO4 was negligible, but over time periphyton [Se] increased steadily in conjunction with the formation of dissolved SeO3. XANES analyses revealed that both SeO3 and SeO4 treated periphyton biotransformed Se similarly with speciation dominated by organo-selenide (∼61%). Mayfly survival, secondary production, and time to emergence were similar in both SeO3 and SeO4 treated periphyton exposures with significant adverse effects at 12.8 µg g(-1) ((d.w.) secondary production) and 36 µg g(-1) ((d.w.) survival and development time). Overall, dissolved selenium speciation, residence time, and organisms at the base of aquatic food webs appear to be the principal determinants of Se bioaccumulation and toxicity.

A DNA barcode library for North American Ephemeroptera: progress and prospects.

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional species of mayflies (Insecta: Ephemeroptera) from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some Caenis. Minimum interspecific distances ranged from 0.3-24.7% (mean: 12.5%), while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species.

Trophic transfer of Cd from natural periphyton to the grazing mayfly Centroptilum triangulifer in a life cycle test.

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 microg 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.

Selenium bioaccumulation and maternal transfer in the mayfly Centroptilum triangulifer in a life-cycle, periphyton-biofilm trophic assay.

Selenium contamination in aquatic ecosystems provides management challenges because bioaccumulation in animals is largely a function of dietary exposure, whereas regulatory entities have traditionally focused on direct water to organism interactions. Selenium is known to be readily absorbed by primary producers and can potentially biomagnify in food webs and elicit adverse effects in higher trophic levels. However, selenium bioaccumulation in the invertebrate prey of many predatory animals is poorly understood. Here, we used 75Se (as selenite) as a radiotracer to characterize Se bioaccumulation into natural periphyton biofilms and subsequent dietary and maternal transfer in the mayfly, Centroptilum triangulifer, in a life-cycle assay. On average periphyton biofilms bioconcentrated selenium 1113 (+/-430)-fold following 7-9 days of exposure to a range of environmentally relevant dissolved concentrations (2.4-13.9 microg L(-1)). Mayflies grown to adulthood on these diets further biomagnified Se with trophic transfer factors averaging 22 (+/-0.4)-fold in postpartum maternal tissues. Adults then transferred 46.5 (+/-8.8)% of their body burdens to eggs with an observed reduction in fecundity for mayflies fed on diets greater than approximately 11 microg g(-1). These results suggest that at environmentally feasible dietary Se concentrations insects are potentially affected by Se exposure, and that the current presumption that insects are simply conduits of Se to higher trophic levels is inaccurate.

Mercury(II) bioaccumulation and antioxidant physiology in four aquatic insects.

We examined Hg(II) bioaccumulation and compartmentalization patterns in conjunction with antioxidant responses in four aquatic insect species: two caddisflies (Chimarra sp. and Hydropsyche betteni) and two mayflies (Maccaffertium modestum and Isonychia sp). Total antioxidant capabilities differed among unexposed larvae, with both caddisfly species exhibiting elevated antioxidant activities relative to the mayflies. We were able to account for these differences by examining the constitutive activities of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), and superoxide dismutase (SOD), in the four species. We also examined levels of reduced and oxidized glutathione and cysteine in the insects. Glutathione peroxidase and SOD were the most responsive to Hg exposure, with GPx catalytic activity increasing between 50 and 310%. Superoxide dismutase activity decreased between 35 and 50%. This SOD suppression was shown to be dose-dependent in both caddisflies, butthe strength of this suppression did not appear to be related to rates of uptake. Surprisingly, little Hg (<10%) was found in the heat-stable cytosolic protein subcellular compartment in each of the four species, suggesting that Hg was not well detoxified. By combining bioaccumulation studies with other physiological measures, we can begin to better understand the consequences of trace metal pollutants in nature.