Phylogeny and Size Differentially Influence Dissolved Cd and Zn

Article pubs.acs.org/est

Phylogeny and Size Differentially Influence Dissolved Cd and Zn Bioaccumulation Parameters among Closely Related Aquatic Insects Monica D. Poteat and David B. Buchwalter* Environmental and Molecular Toxicology Program, Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina 27695, United States S Supporting Information *

ABSTRACT: Evolutionarily distinct lineages can vary markedly in their accumulation of, and sensitivity to, contaminants. However, less is known about variability among closely related species. Here, we compared dissolved Cd and Zn bioaccumulation in 19 species spanning two species-rich aquatic insect families: Ephemerellidae (order Ephemeroptera (mayflies)), generalized to be metal sensitive, and Hydropsychidae (order Trichoptera (caddisflies)), generalized to be metal tolerant. Across all species, Zn and Cd uptake rate constants (k u s), efflux rate constants (k e s) and bioconcentration factors (BCFs) strongly covaried, suggesting that these metals share transport pathways in these distinct lineages. Kus and BCFs were substantially larger in Ephemerellidae than in Hydropsychidae, whereas kes did not dramatically differ between the two families. Body size played an important role in driving ku differences among species, but had no influence on kes. While familial differences in metal bioconcentration were striking, each family exhibited tremendous variability in all bioaccumulation parameters. At finer levels of taxonomic resolution (within families), phylogeny did not account for differences in metal bioaccumulation. These findings suggest that intrafamily variability can be profound and have important practical implications in that we need to better understand how well “surrogate species” represent their fellow congeners and family members.

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context.7−10 Previous work analyzed the uptake and efflux of Cd across 21 aquatic insect species representing orders Ephemeroptera, Plecoptera, and Trichoptera (EPT) and found metal fluxes to vary drastically across the three orders.7 Importantly, variability across species was largely driven by phylogeny, with insects of the same taxonomic lineage generally having more similar metal flux parameters than insects from different taxonomic lineages.7 Thus, while highly variable, metal bioaccumulation parameters in these aquatic insects were heavily influenced by evolutionary history and generally followed phylogeny. Here we made an attempt to understand the variability of aquatic insect responses to trace metal pollution by examining dissolved Cd and Zn fluxes in closely related aquatic insect species. We examined Cd and Zn bioaccumulation specifically because they co-occur in ores in the earth’s crust and therefore commonly co-occur in metal contaminated environments. Both are borderline transition metals,11 though Zn is an essential element and typically more abundant than Cd, a nonessential element. Further, these ions have similar biochemical and physical properties.11−13

INTRODUCTION Aquatic insects are the most dominant invertebrate faunal group in freshwater ecosystems, often accounting for between 75 and 90% of the invertebrate species pool.1,2 They are also an inherently diverse faunal group, with global biodiversity estimates of 100 000 species3 stemming from multiple freshwater invasions by terrestrial ancestors throughout evolutionary history.4 The sheer dominance of aquatic insects in freshwater systems coupled with their differential responses to pollutants has led to their widespread use in biomonitoring and bioassessment programs worldwide. While biomonitoring and bioassessment programs take advantage of the variable pollution responsiveness of these species, our understanding of the physiological drivers of these sensitivity differences remains poorly understood. Further, our understanding of physiological variability among species (and species groups) remains surprisingly limited. Similarly, regulatory toxicology programs are reliant on the widespread use of surrogate species to represent larger groups of species without a clear understanding of how much variability might occur at broader levels of biological organization. For example, the requirement to have only a single aquatic insect represented in toxicity data sets used to establish water quality criteria in the United States5 functionally means that a single species often represents the entire class (∼6500 North American species6). We can begin to understand variability across species by examining physiological/toxicological traits in a comparative © XXXX American Chemical Society

Received: March 4, 2014 Revised: April 8, 2014 Accepted: April 14, 2014

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catawba, Ephemerella crenula, Ephemerella hispida, Ephemerella invaria, Ephemerella rossi, Ephemerella subvaria, Eurylophella verisimilis, Teleganopsis def iciens)in methods described previously.18 Briefly, three dual-labeled bulk solutions of the following environmentally relevant Zn and Cd concentrations were prepared for each species: 3 μg L−1 Zn and 0.3 μg L−1 Cd, 9 μg L−1 Zn and 0.9 μg L−1 Cd, and 27 μg L−1 Zn and 2.7 μg L−1 Cd. Solutions all contained between 104−207 Bq L−1 Zn and 30−59 Bq L−1 Cd, with the remaining dissolved metal being stable Zn (as ZnCl2) and stable Cd (as CdCl2). Water samples (1 mL) were counted to verify radiotracer concentrations. The pH of all solutions was adjusted to 7.2 ± 0.02 using 0.1 N NaOH. A total of 5−10 replicates were utilized per concentration level within each species, with each replicate consisting of a single larva within a high-density polyethylene (HDPE) cup containing 80 mL aerated solution, Teflon mesh as substrate and Parafilm to reduce evaporative loss. Therefore, 15−30 individual insect larvae were used to determine kus for each species. Larvae were exposed to solutions for a total of 9 h. After 3 and 6 h of exposure, larvae were removed from the exposure, rinsed with VSW, assayed in vivo for radioactivity and returned to the exposure solution. After 9 h of exposure, insects were removed from the exposure, rinsed with VSW, assayed for radioactivity, blotted dry and weighed to obtain the wet weight of individuals. Uptake rates over time were determined at each concentration measured for Zn and Cd. The slope of the line of uptakes rates versus the concentration at which the uptake rate was derived was taken as the uptake rate constant (ku) for each species. Determination of Efflux Rate Constants. Efflux rate constants (ke) were determined for all insects used in the above uptake experiments (except for T. def iciens, due to a lack of sufficient quantities collected) using methods described previously.7,19−21 Briefly, insect larvae from each species were exposed to a dual labeled solution containing 3 μg L−1 Zn and 0.3 μg L−1 Cd for a total of 4−5 days to ensure adequate uptake of radiolabel. The solutions contained 104−207 Bq L−1 Zn and 30−59 Bq L−1 Cd, and the remaining dissolved metal was stable Zn (as ZnCl2) and Cd (as CdCl2). The pH of all solutions was adjusted with 0.1 N NaOH to 7.20 ± 0.02. Insects were exposed individually in 80 mL aerated solution in HDPE beakers containing Teflon mesh as substrate and Parafilm to reduce evaporative loss. After 4−5 days, insects were removed from the exposure solution, rinsed with VSW, and assayed to ensure the adequate uptake of radiolabel. Each larva (5−10 replicates per species) was then placed in an individual 1 L HDPE container with 500 mL aerated VSW, Teflon mesh as substrate and Parafilm to reduce evaporative loss. Larvae were assayed in vivo daily for 10 days, and solutions were checked for radioactivity to ensure that there was no appreciable metal available for reuptake. After larvae were assayed on day 10, wet weights were obtained. Efflux rate constants were determined as the slope of the natural log of the proportion of metal retained in the body tissue and the time of depuration22 after excluding days 0 and 1 to minimize the confounding influence of desorbed metals as follows:

We measured Cd and Zn uptake and efflux rate constants (ku and ke, respectively) in 19 and 18 species, respectively, with species representing two common aquatic insect families previously suggested to be especially variable in Cd fluxes.7 The mayfly family Ephemerellidae (order Ephemeroptera) is generalized to be metal sensitive, whereas the caddisfly family Hydropsychidae (order Trichoptera) is generalized to be metal tolerant.14 We explored whether the trafficking patterns of Zn are similar to those of Cd across species tested, and assessed the influence of body weight on Cd and Zn flux parameters. Lastly, by examining the fluxes of these two metals among several closely related species, we asked if phylogeny could continue to explain interspecies variability within families.

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MATERIALS AND METHODS

Insect Collection and Handling. Larvae of 19 aquatic insect species spanning families Ephemerellidae and Hydropsychidae were collected from Great Smoky Mountains National Park during the time period of July 2010 to August 2013. All larvae were collected using a D-frame kicknet from cool, cobble-bottomed streams and transported back to the laboratory at North Carolina State University as described previously.15 Upon arrival, larvae were held under laboratory conditions for a minimum of 48 h before experimentation. Larvae were unfed for all experiments. Experiments were performed in a walk-in cold room with a controlled climate (12.7 °C, 12 h:12 h light:dark photoperiod). All experiments utilized American Society for Testing Materials (ASTM) very soft water (VSW) (mg L−1: 12 NaHCO3, 7.5 CaSO4·2H2O, 7.5 MgSO4, 0.5 KCl) because all insects were collected from streams with low ambient calcium (∼1 mg L−1). Only larvae that appeared healthy were used for experimentation. All ephemerellid larval identifications were determined using morphological characters, and some identifications were confirmed using DNA barcoding when data were available.16 Hydropsychid larval identifications were performed using morphological characters.17 Radioactivity Measurement. The γ-emitting isotopes 65 Zn and 109Cd were used to measure metal fluxes across insect species. Isotopes 65Zn (as 65ZnCl2 in HCl) and 109Cd (as 109 CdCl2 in HCl) were diluted in 0.1 N HNO3 for working stock solutions. Methods for the simultaneous counting of 109 Cd and 65Zn were established and verified against single and dual standards. Protocols for counting dual labeled exposures were utilized because previous work in ephemerellids (Poteat, unpublished) and Hydropsyche sparna15 showed no difference in the metal uptake or efflux of insects exposed to single or dual metal exposures at environmentally relevant concentrations. Water and larvae samples were counted in 20 mL scintillation vials using a PerkinElmer Wallac Wizard 1480 Automatic Gamma Counter. The nondestructive nature of in vivo γcounting allowed us to count individual larvae multiple times throughout experimental time periods in 15 mL VSW within scintillation vials. All samples were counted for 3 min to ensure low counting errors ( 0.05) (SI Figure S1). Log-transformed Zn BCFs were negatively correlated with log-transformed body weight (r = −0.65, p = 0.0035) (Figure 4a). Multiple linear regression analysis found family to be an

Figure 5. Zn BCFs for 18 species plotted onto their phylogeny. Pagel’s arbitrary branch lengths47 are depicted. The phylogeny was constructed using Dendroscope.48

Zn kes ranging 0.031−0.18 d−1 and Cd kes ranging 0.019−0.18 d−1 (Table 1). Zn and Cd BCFs were variable across all genera for which we were able to measure multiple species. Across the hydropsychid genus Hydropsyche (n = 3), Cd BCFs ranged 256−4297 and Zn BCFs ranged 265−1139. The genus Drunella had BCFs spanning almost the entire ephemerellid range, with Cd BCFs spanning 1486−88 423 and Zn BCFs spanning 1966− 56 089. Across the genus Ephemerella, BCFs were also quite variable, with Cd BCFs ranging 1053−41 764 and Zn BCFs ranging 1524−19 140 (Table 1).

Figure 4. Log-transformed Zn (a) and Cd (b) BCFs of 18 species versus the average log-transformed body weight of individuals. Letters represent the names of species identified in Table 1.

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important predictor of Zn BCF values (p = 0.03), but body weight was not a significant predictor of Zn BCF values (p = 0.21). Log-transformed Cd BCFs were negatively correlated with log-transformed body weight (r = −0.65, p = 0.0037) (Figure 4b). Multiple linear regression revealed neither body weight (p = 0.12) nor family (p = 0.16) were significant predictors of Cd BCFs. Phylogenetic Analyses of Metal Bioaccumulation Parameters. While familial differences in Zn and Cd kus and BCFs were apparent, the variance seen within families was not explained by evolutionary history. Across Zn and Cd kus, kes, and BCFs, no K-statistic was found to be statistically significant. This signified that all traits analyzed failed to exhibit phylogenetic signal after accounting for the effects of body weight (p > 0.05) within the species tested in this study (for example, Figure 5). This lack of phylogenetic influence over metal bioaccumulation parameters can be partially explained by the very large variation seen in metal bioaccumulation within genera, specifically in the family Ephemerellidae. Species within the ephemerellid genus Drunella (n = 4) had Zn kus ranging 0.21− 1.58 L g−1 d−1 and Cd kus ranging 0.28−1.96 L g−1 d−1, values which cover almost the entire range of kus for ephemerellid species. Species within the ephemerellid genus Ephemerella (n = 6) also had highly variable kus, with Zn kus ranging 0.26−0.91 L g−1 d−1 and Cd kus ranging 0.27−0.98 L g−1 d−1 (Table 1). Zn and Cd kes were also especially variable in ephemerellid genera. Across the genus Drunella, Zn kes ranged 0.05−0.11 d−1 and Cd kes ranged 0.01−0.19 d−1. Interestingly, the Drunella Cd kes spanned the entire range of values for ephemerellids. The genus Ephemerella also had a wide range of ke values, with

DISCUSSION One objective of this study was to compare Cd and Zn bioaccumulation parameters in two species-rich aquatic insect families known to be especially variable in metal fluxes.7 We focused on the dissolved route of exposure to expediently make comparisons across several taxa, though we acknowledge that dietary routes of exposure often drive bioaccumulation differences among species.20,27 Evidence for shared dissolved uptake pathways of Cd and Zn is found in a range of freshwater fauna including aquatic insects,15,18,28 mussels,29 and crustaceans,30 though other studies have shown a lack of evidence for shared uptake pathways across aquatic taxa.31,32 The strong covariation observed in Zn and Cd fluxes is suggestive of shared transport systems in aquatic insects, however at environmentally relevant concentrations, we have no evidence that they compete. Strong covariances in Zn and Cd efflux have been observed across aquatic organisms spanning mollusks to fish to arthropods.21 In fact, the covariation between Cd and Zn kes in aquatic insects was so strong that Cd kes from 11 ephemerellid and hydropsychid species successfully predicted Zn kes from known Cd kes in five of six EPT taxa using a simple linear regression.21 This is the first study to our knowledge to examine Cd and Zn BCFs across aquatic insect species. Here we used a time independent (steady state) estimate of BCF based on rate constants of uptake and elimination to avoid common pitfalls associated with the use of this term. This BCF estimate is useful to compare the relative tendencies of these species to accumulate Cd and Zn exclusively from solution. While we E

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found Zn and Cd BCFs to covary, we found evidence to suggest that Cd generally bioaccumulates more readily than Zn. Previous work in aquatic insects has shown Cd to have a higher affinity for shared cotransporters of Zn.15 This could be a reason we generally observed higher Cd kus than Zn kus (and higher Cd BCFs than Zn BCFs). A second objective of this study was to compare metal bioaccumulation parameters in two families described as differentially responsive to metal pollution. Ephemerellid mayfly species are commonly observed to be more sensitive to metal contamination in the field, as they account for some of the first species to disappear at contaminated sites.14 Conversely, hydropsychid caddisfly species are able to persist in metal contaminated environments,14 withstanding higher concentrations of dissolved metals.33 Here we see that ephemerellids had elevated uptake of dissolved metals as compared to hydropsychids, leading to higher BCFs. Ephemerellid species are especially known for their comparatively high uptake of metals in relation to other aquatic insects in the laboratory.27,28 We suspect that the ephemerellid tendency to strongly bioconcentrate metals contributes to their observed metal sensitivity in the field.34 However, other important considerations in metal sensitivities such as diet27,35 and the ability to detoxify metals7 were not taken into account in this study. Body weight appeared to influence Zn and Cd kus, and to a lesser extent BCFs, in Ephemerellidae and Hydropsychidae. Allometric scaling is common in physiological traits across aquatic species, including the dissolved metal uptake of freshwater organisms.7,36,37 In this study, ephemerellids were generally smaller larvae and tended to bioaccumulate more metal from dissolved exposures than the larger caddisflies. A third objective of this study was to examine the physiological variation occurring among closely related species. While familial differences in some metal bioaccumulation parameters were apparent within our data set, phylogenetic patterns previously observed across EPT orders7 appeared to break down at the genus/species level of taxonomic identification. Evolutionary patterns in comparative data sets are well documented across a range of physiological traits38 including metal and ion transport traits.7,21 However, most if not all of these data sets include species from a wide range of families, orders, and even phyla. In toxicology, it is often assumed that closely related species, particularly ones within the same genus or family, are physiologically similar. Aquatic insect families and genera known to be tolerant of metal pollution have recently come into use as a way to monitor bioavailable metals as well as predict community effects in polluted freshwater systems.39−42 In particular, total metal body burdens of species of the hydropsychid genus Hydropsyche are often used in this regard due to the taxon’s persistence and ability to reflect metal bioavailabilities in contaminated environments.40,41 However, the inherently variable nature of some species groups (including Hydropsyche) can potentially influence the results of studies which utilize coarser family- or genus- level identifications of test species and should be considered. In general, measuring Cd and Zn bioaccumulation parameters in both ephemerellid mayflies and hydropsychid caddisflies gave us a unique opportunity to observe patterns in the physiological variability of two species-rich families with radically different evolutionary histories. The mayfly family Ephemerellidae has an estimated 75 species in North America43

and is descended from one of the oldest aquatic insect lineages which has been aquatic for up to an estimated 400 million years.44 The caddisfly family Hydropsychidae has an estimated 184 species in North America45 and is descended from a lineage which has been aquatic for up to 234 million years.46 These two families are descendants of different terrestrial ancestors4 which used unique physiological methods to overcome the challenges to living in freshwater, thus enabling each lineage to develop differing physiologies which potentially lead to varying metal bioaccumulation parameters. To our knowledge, this study is the first attempt to comparatively examine the physiologies of closely related aquatic insect species of the same families and genera. Whether variation in metal bioaccumulation across species is caused by underlying evolutionary patterns or differences in body size, it is important to acknowledge the extreme physiological variances seen across even closely related species. Biomonitoring and bioassessment programs often use measures of biodiversity as important end points upon which regulatory decisions are made, and such biodiversity measures are highly dependent on taxonomic resolution (e.g., species-, genus-, family level identifications). It is important to recognize that not all species within a given family (or even genus) will physiologically resemble each other, and even species within the same genera can vary drastically in their metal flux physiologies.

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ASSOCIATED CONTENT

S Supporting Information *

The relationships between log-transformed Zn and Cd kes of 18 species versus the average log-transformed body weight of individuals are shown in Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (919) 513-1129; fax: (919) 515-7169; e-mail: david_ [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Luke Jacobus (Indiana University−Purdue University Columbus) and Eric Fleek (North Carolina Department of Environmental and Natural Resources) for their aid in insect species identification. Allison Camp (NCSU), Justin Conley (NCSU), Gerald LeBlanc (NCSU), and anonymous reviewers provided valuable editorial comments. This work was supported by NSF (IOS 0919614), the ICA Chris Lee Award for Metals Research and the Society of Environmental Toxicology and Chemistry (SETAC). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the ICA or SETAC.

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