Abstract Scheibener, Shane Arthur

ABSTRACT

SCHEIBENER, SHANE ARTHUR. Osmoregulatory Physiology in Aquatic Insects: Implications for Major Ion Toxicity in a Saltier World. (Under the direction of Dr. David Buchwalter).

The salinization of freshwater ecosystems has emerged as an important 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. As freshwater systems are increasingly threatened by salinization it is important to understand fundamental aspects of aquatic insect physiology (e.g. osmoregulatory processes) that contribute to insect responses to these stressors. These osmoregulatory processes are also important determinants of trace metal exposure and toxicity.

I compared the uptake dynamics of Na in two species thought to differ in their sensitivity to salinity – the tolerant caddisfly Hydropsyche betteni and a more sensitive mayfly Maccaffertium sp. Across a range of Na concentrations encompassing the vast majority of North American freshwater ecosystems (0.06 – 15.22 mM), both species appeared to tightly regulate their whole body sodium concentrations (at ~ 47 ±1.8 µmol/g wet weight) over 7 days. However, at the highest Na concentration (15.22 mM), Na uptake rates in a related species, Hydropsyche sparna (419.1 nM Na g-1 hr-1 wet weight), appeared close to saturation while Na uptake rates in Maccaffertium sp. were considerably faster (715 nM Na g-1 hr-1 wet weight) and unlikely close to saturation. Thus, ion turnover rates (and their associated energetic costs) may play a role in determining sensitivity differences across species. A comparison of Na uptake rates (at 0.57 mM Na) across 9 species representing 4 major orders (Ephemeroptera, Plecoptera, Trichoptera and Diptera) demonstrated large physiological differences across species after accounting for 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 suppressing it. This finding may explain why aquatic insects appear to be tolerant to these metals in traditional aqueous toxicity tests.

As no information about sulfate uptake dynamics in stream insects was available in the literature, I characterized SO4 uptake kinetics in 5 mayflies and assessed the influence of different ions on SO4 uptake. When SO4 was held constant and Na was increased, a dramatic decrease in SO4 transport was observed. Experiments with other salts demonstrated that Na

(and not Cl or HCO3) is antagonistic to SO4 transport. This is a novel discovery that has not been observed in other aquatic taxa. Based on this observation, I tested the hypothesis that increasing Na would protect against SO4 induced toxicity in a Na-dependent manner.

Increasing Na from 0.7 to 10.9 mM improved 96-hr survivorship associated with 20.8 mM

SO4 from 44% to 73% in a concentration dependent manner. However, when Na reached

21.8 mM, 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, these results highlight the need to better understand fundamental physiological processes in this ecologically important faunal group.

by Shane Arthur Scheibener

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science

Zoology

Raleigh, North Carolina

2017

APPROVED BY:

______David Buchwalter Russell Borski Committee Chair

______Bradley Taylor

BIOGRAPHY

Shane Scheibener was born on February 24, 1991 to Tom and Lorraine Scheibener in

Holland, NJ. Most people have a different impression of NJ, typically in the shadows of New

York City or the “glitz and glamor” of the Jersey shore. However true this may be, his upbringing was a different shade of lifestyle. In rural Holland Township, there was not (and is still not) a single traffic light. The town sits directly next to the Delaware River on the border of Pennsylvania and has many beautiful attractions that are surrounded by nature.

From this early lifestyle, he began to love the outdoors, but also knew that there was a strain being placed upon these landscapes. As he entered as an undergraduate at NCSU, he began to study heavily in the biological and ecological sciences. As his college career advanced, he wanted a more in-depth understanding of environmental issues, especially pertaining to water contamination. He began undergraduate research in Dr. David Buchwalter’s lab at NCSU, initially focused on the TVA Kingston Coal Ash Spill in Tennessee. The research honed his laboratory skills and gave him a better understanding of aquatic insect ecology and water chemistry. He graduated with his B.S. in Environmental Sciences in 2013 and continued to work in Dr. Buchwalter’s lab until he decided to go back to school. He was accepted into the

Zoology program at NCSU in 2015 and has focused his research freshwater salinization and aquatic insect osmoregulatory physiology.

ACKNOWLEDGMENTS

I would like to sincerely thank my advisor Dr. David Buchwalter. Although he is my advisor, he is also my friend and a person I look up to in every aspect of life. He has made me a better scientist, writer, thinker and most importantly, a better person. Thank you Dave, cheers!

I would like to thank my committee members, Dr. Bradley Taylor and Dr. Russell

Borski for their assistance and guidance throughout my graduate education and research. I would also like to thank Justin Conley, Monica Poteat, Adeline Lopez, Allison Camp,

Mackenzie Pidgeon, Vinicius Richardi and Hsuan (Tina) Chou for their valuable assistance, expertise and enjoyment in the lab.

Last, I would like to thank my family. A special thanks to my sister Kali, who is always there to listen and make my day a little brighter. And to my mom and dad (Lorraine and Tom), who have been overwhelmingly supportive throughout my undergraduate and graduate career. I would not be where I am today without their love and guidance.

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TABLE OF CONTENTS

List of Tables…………………………………………………………………………………vi List of Figures………………………………………………………………………………..vii Figures from Chapter 1………………………………………………………………vii Figures from Chapter 2……………………………………………………...………viii General Introduction……………………………………………………….………………….1 Freshwater Salinization…………………………………….…….…………………....1 Aquatic Insects……………………………………………………………..………….3 Osmoregulation………………………………………………………….…………….7 Research Needs………..……………………………………………………………..10 References……………………………………………………………………………12 CHAPTER 1: COMPARITIVE SODIUM TRANSPORT PATTERNS PROVIDE CLUES FOR UNDERSTANDING TDS AND METAL RESPONSES IN AQUATIC INSECTS….18 Abstract…………………………………………………………………………..…..19 Introduction…………………………………………………………………………..21 Methods…………………………………………………………………….…...……23 Sample Collection and Preparation…………………………………….…….23 Radioactivity Measurement……………………………...…………….…….24 [Na] Uptake Kinetics in Hydropsyche betteni using three different Na salts

(NaCl, NaHCO3 and Na2SO4)……………………………………………...... 24 Na turnover dynamics in Hydropsyche sparna and Maccaffertium sp. ……..25 Efflux kinetics in Hydropsyche sparna………………………………………27 Assessing the influence of MOPS on Na uptake…………………………….27 Comparative Sodium Uptake Kinetics………………...... 28 Copper and Silver effects on sodium transport………………………………28 Data Analysis………………………………………………………………...30 Results………………………………………………………………………………..31 [Na] Uptake Kinetics in Hydropsyche betteni using three different Na salts

(NaCl, NaHCO3 and Na2SO4)……………………………………….……….31

Na turnover dynamics in Hydropsyche sparna and Maccaffertium sp………31 Efflux kinetics in Hydropsyche sparna……………………………………....33 Assessing the influence of MOPS on Na uptake………………………….…33 Comparative sodium uptake kinetics………………………………………...34 Copper and Silver effects on sodium transport………………………...….…34 Discussion……………………………………………………………………………35 Chapter 1 Figures…………………………………………………………….….…...42 References…………………………………………………………………….….…..51 CHAPTER 2: SULFATE TRANSPORT KINETICS AND TOXICITY ARE MODULATED BY SODIUM IN AQUATIC INSECTS………………………………………………...... …57 Abstract……………………………………………………………………………....58 Introduction…………………………………………………………………………..60 Methods………………………………………………………………………...…….62 Sample Collection and Preparation…………………………………….…….62 Radioactivity Measurement…………………………………….………..…..63 Assessing effects of Mg on Ca uptake………………………………...……..64 Sulfate Uptake Kinetics in 5 mayfly species……………………………...…64

Effects of ionic composition on Na and SO4 uptake…………………...…….65

Influence of [Na] on SO4 and Na uptake…………………………………….65

Can Na ameliorate SO4 toxicity in N. triangulifer? ………………………....66 Data Analysis………………………………………………………….……..67 Results………………………………………………………………………………..68 Assessing effects of Mg on Ca uptake……………………………...………..68 Sulfate Uptake Kinetics in 5 mayfly species……………………………...…69

Effects of ionic composition on Na and SO4 uptake………………….…..….69

Influence of [Na] on SO4 and Na uptake…………………………………….70

Can Na ameliorate SO4 toxicity in N. triangulifer? ………………………....71 Discussion……………………………………………………………………………72 Chapter 2 Figures………………………………………………………………...…..81 References…………………………………………………………………..………..86 v

LIST OF TABLES

Tables from Chapter 1

Table 1. Sodium concentration in solution, uptake rate (mean ± S.E.), whole body concentration (mean ± S.E.), and estimates of the daily acquisition of Na for two species (H. sparna and Maccaffertium sp.) of aquatic insects collected from the Eno River, NC…...….45

Tables from Chapter 2

LIST OF FIGURES

Figures from Chapter 1

Figure 1. Comparison of sodium salts (NaCl, NaHCO3, and Na2SO4) on Na transport dynamics in H. betteni. Estimated maximum transport rates (Vmax) are as followed: In -1 -1 NaCl=2.194, In NaHCO3=1.39, and In Na2SO4= 0.784 µmol Na g hr w.w. Estimated affinity constants (Km) are as followed: In NaCl=7.71, In NaHCO3=3.321, and In Na2SO4=2.106. Error bars represent mean ± S.E of 5 individuals…………………………..42

Figure 2. Time course radiotracer study of sodium uptake at different dissolved Na concentrations versus time for the species H. sparna (Trichoptera: Hydropsychidae) (A) and Maccaffertium sp. (Ephemeroptera: Heptageniidae) (B). Each point represents the mean ± S.E. of 10-15 individuals…………………………………………………………………….43

Figure 3. Michaelis-Menten type kinetics for the species H. sparna and Maccaffertium sp. -1 -1 Estimated maximum transport rates (Vmax) are as followed: H. sparna=521.3 nmol Na g hr w.w. Estimated affinity constants (Km) are as followed: H. sparna=3.664 mM Na. Vmax and Km values could not be accurately quantified for Maccaffertium sp. Error bars represent mean ± S.E. of 10-15 individuals for H. sparna and 5 individuals for Maccaffertium sp…………44

Figure 4. The influence of ambient Na concentration and prior exposure history on the loss rates of previously acquired 22Na. Raw efflux data (mean ± S.E.) versus time for the species H. sparna, where initial Na uptake was performed in .06mM Na (A) or 5.09mM Na (B). Efflux was either performed in 0.06mM Na (represented by the circles) or performed in 5.09mM Na (represented by the squares)……………………………………………...…….46

Figure 5. Comparison of Na uptake in the presence or absence of MOPS. Na uptake rates across species (A) and in the species H. betteni under various ambient Na concentrations (B). Error bars represent mean ± S.E of 5 individuals……………………………………..……..47

Figure 6. Sodium uptake rates (mean± S.E.) on a per mass basis (A) or per individual basis (B) plotted against body weight (mean ± S.E.) for 9 species of aquatic insects. Symbols represent the following species:  A. irrorata,  Atherix sp.,  H. sparna,  Diplectrona sp.,  P. immarginata,  M. pudicum,  Paraleptophlebia sp.,  Pteronarcys sp., and  Tipula sp……………………………………………………………………………………..48

Figure 7. The influence of dissolved metal concentrations on sodium uptake rates in three species of aquatic insects………………………………………………………………….…49

Figure 8. The influence of dissolved metal concentrations on sodium uptake rates in either no presence of metal (control), 741.65 nM Ag, or 1483.18 nM Cu for three species of aquatic insects……………………………………………………………………………..………….50

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Figures from Chapter 2

Figure 1. Mean survival (%) for the mayfly N. triangulifer versus conductivity (µS cm-1) in 3 reconstituted waters mimicking waters downstream of coal mining operations (data adapted from Kunz et al. (2013)).………………….………………………………………………....78

-1 -1 Figure 2. Rates of SO4 uptake (µmol g d (dry wt.)) as a function of ambient [SO4] in 5 mayfly genera. The lines are Michaelis-Menten curves (Graphpad 6.0), and the inset -1 -1 summarizes SO4 uptake kinetic parameters Vmax (µmol g d (dry wt.)) and Km (mM). Values are means ± SEM (n=5) for each data point…………………………………………81

-1 -1 Figure 3. Influence of the cation on SO4 uptake rates (µmol SO4 g d (wet wt.)) for a caddisfly H. betteni. Four different sulfate salts (MgSO4, CaSO4, K2SO4 and Na2SO4) were used at 3 different concentrations. SO4 uptake rates were obtained at only 5 mM for Na2SO4 due to availability of larvae. Values are means ± SEM (n=5, * represents cation treatment differences (p≤0.05, One-way ANOVA)) for each bar…………………………………..….82

Figure 4. Influence of [Na] on SO4 uptake in three aquatic insect species (P. biloba, H. betteni and E. vitreus) held in either low (0.69 mM Na) or high (4.96 mM Na) Na solutions. [SO4] in solution was held constant (5 mM) for each species. Values are means ± SEM (n=5, * represents Na treatment differences (p≤0.05, Student’s unpaired t-test)) for each bar……83

Figure 5. SO4 (Panel A) and Na (Panel B) in dual-label uptake kinetic experiments in H. betteni (open circles) and N. triangulifer (closed triangles). In these experiments, [SO4] was held constant (5 mM) while Na was varied. The graphical inset (in Panel A) shows SO4 uptake at elevated Na (10.9 mM) for two different Na salts (NaCl and NaHCO3). All values are means ± SEM (n=5) for each point…………………………………………………...….84

Figure 6. Influence of ambient Na on SO4 toxicity in the mayfly N. triangulifer. All SO4 treatments (shaded bars) were held constant at 20.8 mM as a mixture of MgSO4 and CaSO4. Control treatment (white bar) was a modified artificial soft water. An apparent protective effect of Na is observed up to 10.9 mM Na, but toxicity is exacerbated at 21.8 mM Na. Bars represent mean ± SEM (n=5 replicates; 10 individuals/replicate, * represents SO4 treatment differences (p≤0.05, One-way ANOVA) relative to the 0.7mM Na treatment)……………..85

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GENERAL INTRODUCTION

Freshwater Salinization

Freshwater salinization has emerged as an important ecological issue (Cañedo-

Argüelles et al., 2013; Kaushal et al., 2005). The majority of increased salinity levels in freshwater ecosystems result from human activities such as de-icing of roads (Karraker et al.,

2008), mining and weathering of mine tailings (Pond et al., 2008), hydraulic fracturing

(Entrekin et al., 2011) and irrigational practices (Williams, 2001a, 2001b). Additionally, alterations in climate can increase freshwater salt concentrations due to sea level rise

(saltwater intrusion) and altered precipitation patterns.

Salt concentrations in freshwater are dominated by seven major ions, calcium (Ca2+),

2+ + + - - magnesium (Mg ), sodium (Na ), potassium (K ), chloride (Cl ), bicarbonate (HCO3 ) and

2- sulfate (SO4 ). There are typically three different ways to report ionic strength; total conductivity (µS cm-1), total dissolved solids (TDS) (mg L-1) or as salinity (ppt).

Conductivity is a measurement of the ability for water to pass an electric current and is often used as a proxy for salinity concentration in field measurements because it is a relatively easy measurement and is inexpensive. However, conductivity measurements do not provide any information about the ions present in solution. TDS measurements provide an overall mass of

- + 3- - dissolved ions, but can include other dissolved solids such as Si(OH)4, NO3 , NH4 , PO4 , F and dissolved organic matter (DOM). As a result, TDS can be an overestimate of salinity concentration of the seven major ions. Salinity (ppt) is similar to TDS measurements, but is

2+ 2+ + + - - solely a measurement of the total cations and anions: Ca , Mg , Na , K , Cl , HCO3 and

2- SO4 . These three measurements are often used interchangeably and can give an overall understanding of the ionic strength of a stream.

A survey of wadeable reference streams throughout the U.S. showed that 73% of the ecoregions had stream conductivities < 200 µS cm-1 (Griffith, 2014). Although these are averages, variability occurs in natural stream water conductivity and ionic concentrations.

For example, elevated background conductivities are seen in the Salt River, Arizona (1,200

µS cm-1) and dilute background conductivities are seen in Appalachia streams (86 µS cm-1)

(Pond et al., 2008). Streams impacted by anthropogenic salinization have conductivities ranging from 1,000-10,000 µS cm-1 (Cañedo-Argüelles et al., 2013; Johnson et al., 2015).

For example, streams downstream of mountain top mining operations can have conductivities as high as 3,000 µS cm-1 (Lindberg et al., 2011; Pond et al., 2008). On rare instances in agricultural systems, conductivities can increase dramatically to levels seen in saline lakes (>

100,000 µS cm-1) (Kay et al., 2001). Although conductivity measurements are quick and reliable, they lack specific details on ion composition and concentration.

The composition of ionic concentrations in stream waters can be quite variable, but a recent synopsis of average stream ionic concentrations showed: 0.69 mM Na, 0.03 mM K,

0.30 mM Ca, 0.14 mM Mg, 0.39 mM Cl, 0.72 mM HCO3 and 0.24 mM SO4 (David Mount, personal communication). Depending on local land uses/activities and geology, the ionic matrix in salinized waters can be quite variable. For example, waters impacted by road de- icing may have elevated Na+ and Cl- in some areas (Ramakrishna and Viraraghavan, 2005), or elevated Mg2+ and Cl- in others (Lewis, 1999). These conditions are typically seen in northern latitudes and higher elevations as snow and ice events are seasonal (winter months).

For example, streams impacted by road de-icing in Maryland, New York and New

Hampshire can have 100-fold increase in Cl- compared to reference streams (Kaushal et al.,

2005). Streams impacted by mountaintop coal mining operations are typically enriched in

2+ 2+ - 2- Ca , Mg , HCO3 and SO4 (Bryant et al., 2002; Pond et al., 2008), although variation (i.e. elevated Na and reduced SO4) within ecoregions has been observed (Kunz et al., 2013). As a result of freshwater salinization, widespread biodiversity loss, alterations in ecosystem processes and community composition have been observed (Cañedo-Argüelles et al., 2013;

Kefford et al., 2012; Pond et al., 2008; Short et al., 1991; Szöcs et al., 2014).

Aquatic Insects

Aquatic insects are the predominant faunal group in most freshwater ecosystems, with

~8,600 North American species (Merritt et al., 2008) and ~76,000 species globally (Balian et al., 2008). In a typical freshwater ecosystem, aquatic insects can comprise 70-95% of total invertebrate species (Arscott et al., 2006; Minshall, 1969). Diptera (true flies), Coleoptera

(true bugs) and Trichoptera (caddisflies) are the most abundant orders with 43%, 18% and

15% of total taxa, respectively (Balian et al., 2008). Given the diversity of aquatic insect species, they are used extensively in ecological monitoring programs. The orders

Ephemeroptera (mayflies), Plecoptera (stoneflies) and Trichoptera (EPT taxa) are relied on most heavily for these programs due to their unique sensitivity to ecosystem alteration and importance to ecosystem function (Covich et al., 1999; Huryn and Wallace, 2000; Jonsson and Malmqvist, 2000; Wallace and Webster, 1996a). Aquatic insects are extremely important in the processing of organic matter and nutrient cycling and they are a major food source for most fish and other aquatic vertebrates (e.g. salamanders and birds).

Aquatic insects are quite diverse in terms of where they live in a stream and what type of food resource they eat. Researchers often group aquatic insects into the functional feeding groups: grazers, shredders, filter-feeders, gatherers and predators. The distribution of these functional feeding groups vary spatially in lotic environments, both locally (riffles, runs or pools) and longitudinally (first order streams vs third order streams). This phenomena is part of a bigger systematic understanding of most lotic environments, called the River Continuum

Concept (Vannote et al., 1980). This concept describes how physical variables (e.g. substrate, channel width and depth and stream slope) vary continuously along the gradient of a river, from head waters to the mouth. It also describes how these variables affect energy inputs/storage, nutrient cycling, decomposition and the composition of biological communities.

Each aquatic insect functional feeding group has different strategies (both morphological and behavioral) in order to maximize nutrient intake and survival (Wallace and Webster, 1996b). Grazers typically collect food resources from substrates (e.g. submerged rocks) in the form of periphyton, algae and microbiota. The abundance of algal production in a stream can be directly related to grazer abundance and vice versa. Certain families classified as grazers have morphological adaptations that have enabled them to thrive in certain habitats. For example, aquatic insects in the family Heptageniidae have adapted flat bodies, which enables them to cling to rocks in fast flowing waters, thus enabling them to graze on algal resources that other taxa cannot. Shredders typically consume allochthonous litter inputs (e.g. leaves and woody debris) and are vital in converting course particulate organic matter (CPOM) into fine particulate organic matter (FPOM). The

conversion of CPOM to FPOM has benefits to stream nutrient cycling and can be a major food source for groups such as gatherers. Gatherers primarily feed on FPOM and are often the most abundant group of macroinvertebrates in a stream ecosystem (Benke et al., 1984;

Huryn and Wallace, 1987). Filter-feeders primarily remove particles from moving waters through adaptations such as silk net production. Certain families of filter-feeders (e.g.

Hydropsychidae) can also be considered predators, whereby they collect prey that have drifted into their silk nets (Benke and Wallace, 1980). It should be noted that functional feeder classifications are not mutually exclusive (i.e. a grazer can also be a predator).

Predators can have strong top-down effects on prey populations, however this is not the case in all ecosystems (Cooper et al., 1990; Sih and Wooster, 1994). Predators can also alter growth and reproduction strategies for various types of prey populations. For example,

Baetidae mayflies in the presence of stonefly predators will mature at smaller sizes, which in return has effects on fecundity (i.e. lower amounts of eggs) (Peckarsky et al., 1993).

We know that aquatic insects have evolved from terrestrial origins and have done so in separate and distinct invasions (Bradley et al., 2009; Kristensen, 1981). The vast majority of aquatic insects have successfully adapted to hypotonic freshwater environments and are typically not observed in marine and/or saline environments (Bradley, 2008; Bradley et al.,

2009; Maddrell, 1998; Pruthi, 1932). The exclusion of aquatic insects from these environments has been attributed to ecological and physiological processes. Ecological reasons include being outcompeted by crustaceans and predated on by fish, the latter being more likely because crustaceans have successfully adapted strategies that cope against fish predation (Maddrell, 1998). Causal physiological relationships have been understudied at this

point. In naturally freshwater environments that now have higher salinity concentrations

(primarily anthropogenic in nature), loss of aquatic insect taxa (especially Ephemeroptera,

Plecoptera and Trichoptera (EPT)) has been widely reported (U.S. EPA, 2011).

Field-based observations of aquatic insect taxa loss are typically reported as a function of conductivity level. For a given conductivity, the specific ionic matrix (ionic concentrations) can be dramatically different and is primarily due to the cause of increased salinity concentration (see above). Lab-based toxicity tests reveal that relatively equal conductivities with different ionic matrices are not similarly toxic for a given species (Kunz et al., 2013). The study compared two standard crustacean bioassay organisms, the amphipod

(Hyalella azteca) and the cladoceran (Ceriodaphnia dubia), and two lesser used taxa for toxicity testing, the mussel (Lampsilis siliquoidea) and the mayfly (Neocloeon triangulifer).

This study showed that waters containing elevated Ca, Mg and SO4 were toxic to the mayfly

Neocloeon triangulifer and waters containing elevated Na and marginally reduced SO4 were less toxic to the same species at comparable conductivities. The study also showed that aquatic taxa vary in their susceptibility to salinity toxicity for a given ionic matrix, the mayfly and mussel being more sensitive. Other studies show that the salt used (NaCl, MgSO4 or NaHCO3) can alter mayfly drift, abundance and community metabolism to different degrees (Clements and Kotalik, 2016). Clearly, effects from freshwater salinization can have effects on ecosystem function, but a more rigorous physiological understanding of osmoregulatory processes in aquatic insects is needed to elucidate salinity tolerance in this ecologically important faunal group.

Osmoregulation

Aquatic insects, like all other insects, have an internal hemolymph which consists of plasma. The plasma contains blood cells (hemocytes), water and many other organic and inorganic constituents which are necessary for basic physiological processes. Organic constituents include amino acids, proteins, carbohydrates (primarily trehalose) and lipids.

The major inorganic ions in the hemolymph include Ca, Mg, Na, K, Cl, HCO3 and SO4 along with lesser amounts of PO4 and metals (i.e. Cu, Fe, Zn and Mn).

Freshwater insects have a tendency to lose major ions to the external environment, and as such, they must compensate for this loss in order to maintain hemolymph concentrations. There are two distinct strategies for doing this. The first process involves the facilitative transport of major ions through specialized mitochondrial-rich structures into the hemolymph. These structures are often scattered on the epidermis or gill of an insect in the form of chloride cells, or they can be in discrete complexes such as the chloride epithelia or on the anal papillae (Chapman, 1998; Komnick, 1977; Komnick and Wichard, 1975).

Chloride cells are responsible for the transport of chloride, but they are also responsible for the transport of other major ions (e.g. Ca and Na). The structure of a chloride cell is complex, with a deeply folded plasma membrane in order to maximize the surface area for transport.

Chloride cells are found loosely scattered on mayfly and stonefly larvae, including on the gill surface. Trichoptera (caddisflies) have adapted distinct regions (chloride epithelia) on the dorsal side of their abdomen where the chloride cells are located. Mosquito larvae have adapted anal papillae which are also rich with chloride cells. Interestingly, if mosquito larvae are raised in solutions containing elevated salt (as NaCl), the anal papillae of the mosquito

larvae decreases in size (Wigglesworth, 1938). This is most likely a morphological and/or physiological response due to a reduced requirement of major ion uptake through the chloride cells located on the anal papillae.

The second strategy for ionic hemolymph regulation involves the Malpighian tubules and the hindgut. The Malpighian tubules are responsible for removing toxic materials from the insect via the production of urine. This process involves two steps: (1) the production of primary urine and (2) the selective modification of this urine for reabsorption into the hemolymph (Bradley, 1987; Chapman, 1998). The production of primary urine involves the movement of water, major ions and solutes (e.g. sucrose and insulin) out of the hemolymph and into the Malpighian tubules. This process is highly unselective and can result in the loss of too many ions from the hemolymph. This is exacerbated by the tendency for aquatic insects to lose water to their environment as a result of a high cuticle permeability (Chapman,

1998). In order to counterbalance this loss, freshwater insects (and all insects for that matter) reabsorb major ions in the hindgut as the primary urine travels from the Malpighian tubules.

Salt tolerant and salt-water insects (e.g. Aedes detritus and Coelopa frigida) are often considered osmoconformers. These taxa are able to alter their hemolymph concentration at different salinities (osmoconform) by producing concentrated urine (Bradley and Philips,

1977; Bradley and Phillips, 1977, 1975) or via the expression of compatible organic solutes

(Patrick and Bradley, 2000a, 2000b). Most freshwater insects are generally considered strict osmoregulators (Komnick, 1977; Stobbart and Shaw, 1974). This is because the ionic composition in the hemolymph tends to stay constant, even at elevated ionic concentrations.

These insects appear to be able to tightly control their osmolality by active transport of major

ions, production of copious amounts of dilute urine (hypotonic to the hemolymph) and the ability to drink the external medium (Bradley, 1987; Ramsay, 1950). We know that internal hemolymph osmolality in freshwater insects is hyperosmotic and can range from ~200-400 mmol kg-1 (>10 g L-1) in natural waters (Sutcliffe, 1963). For osmoregulators, hemolymph ion concentrations do not increase at severely elevated salinities (Chadwick et al., 2002;

Dowse et al., 2017; Kapoor, 1979). However, once the concentration in solution becomes iso-osmotic to the hemolymph, regulation appears to break down. This does not directly translate to mortality because toxicity is quite common at salinities much lower than that of the insect hemolymph (Dowse et al., 2017; Dunlop et al., 2008). Additionally, field based observations of taxa loss occur at far lower salinities (0.1-0.7 g L-1) (Pond, 2010; Pond et al.,

2008; U.S. EPA, 2011).

The extirpation of freshwater insects from salinized environments has been attributed to ecological and physiological processes. However, a rigorous understanding of basic osmoregulatory physiology (ion transport) in freshwater insects has not been examined and the majority of what we do know comes from fish (Bury et al., 1999; Laurén and McDonald,

1985), crustaceans (Bianchini and Wood, 2008) and saline-tolerant or saline-dwelling insects

(Maddrell and Phillips, 1978, 1975; Patrick et al., 2001). However, recent work with freshwater insects has shed light on Ca transport in aquatic insects. Body weight and clade influenced Ca uptake after examining 12 different species within two families

(Ephemerellidae and Hydropsychidae) (Poteat and Buchwalter, 2014). Additionally, divalent metals (Cd and Zn), which have been shown to reduce Ca uptake in other aquatic organisms through shared transport systems (Hogstrand et al., 1996; Tan and Wang, 2008), did not

reduce Ca uptake at environmentally relevant metal concentrations for Hydropsychidae or

Ephemerellidae (Poteat and Buchwalter, 2014). In some instances, Ca uptake actually increased in the presence of Cd or Zn. This research highlights the fact that aquatic insect physiology is different from other aquatic organisms (i.e. fish and crustaceans) and may be in part the reason why freshwater insects are vulnerable to ecosystem alterations. Other metals

(Ag and Cu) have been shown to inhibit Na transport in fish (Morgan et al., 1997) and crustaceans (Bianchini et al., 2002; Bianchini and Wood, 2003), yet no work to date has examined the possible effects of these metals on Na transport in freshwater insects.

Research Needs

Freshwater salinization has emerged as an important ecological issue and results in widespread biodiversity loss. Some aquatic insects appear especially vulnerable, yet little research has focused on their osmoregulatory physiology. The primary objective of this research was to compare transport kinetics in aquatic insect taxa (primarily for two major ions: Na and SO4) to identify any relationships between ion transport and salinity tolerance/toxicity.

Here, we compared Na uptake across a multitude of aquatic insect species using 22Na as a radiotracer. We first compared Na uptake dynamics among different Na salts under a wide range of ambient Na concentrations. We further made comparisons of Na turnover rates in 2 common aquatic insects—the caddisfly H. sparna, and the mayfly Maccaffertium sp. We then asked if species found in similar habitats varied significantly in their Na uptake rates by

comparing Na uptake among 9 aquatic insect species representing 4 orders. Additionally, we asked if Na transport was a target of Cu or Ag exposure under a range of concentrations ranging from environmentally relevant to extreme.

Additionally, we used a radiotracer approach to determine if ionic interactions might help explain observations described above by Kunz et al. (2013). Briefly, synthetic waters with low Ca:Mg ratios and high SO4:Na ratios were toxic to mayflies, whereas waters with higher Ca:Mg ratios and lower SO4:Na ratios were not toxic at comparable conductivities.

We first asked if Mg was antagonistic to Ca uptake in four aquatic insect taxa. We then characterized Michalis-Menten type SO4 uptake kinetics for 5 mayfly species to understand the extent to which SO4 transport varies across taxa. We further assessed ionic interactions in relation to SO4 uptake and tested the hypothesis that SO4 toxicity could be modulated by other major ions.

References

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CHAPTER 1

COMPARATIVE SODIUM TRANSPORT PATTERNS PROVIDE CLUES FOR UNDERSTANDING TDS AND METAL RESPONSES IN AQUATIC INSECTS

S. A. Scheibener1, V.S. Richardi2, and D.B. Buchwalter1

1 Department of Biological Sciences. Environmental and Molecular Toxicology. North

Carolina State University.

2 Department of Zoology. Entomology Program. Universidade Federal do Parana.

Published in Aquatic Toxicology (2016) 171:20-29

Abstract

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 suppressing 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.

1.1 Introduction

Macroinvertebrate life in freshwater ecosystems is typically dominated by insects

(approximately 8,600 species in North America)(Merritt, 2008). Because of their ecological diversity and importance, aquatic insects play important roles in freshwater food webs and ecosystem function (Wallace et al. 1996; Huryn et al. 2000; Covich et al. 1999; Johnson et al. 2000). Aquatic insects evolved from terrestrial ancestors (Bradley et al. 2009;

Kristensen, 1981) and have successfully adapted to dilute, hypotonic (freshwater) environments (~ 20 to 650 mg salts/L). The paucity of insect species in more saline habitats has been attributed to both ecological and physiological processes (Bradley et al. 2009;

Bradley, 2013; Maddrell, 1998; Pruthi, 1932), though physiological processes remain poorly studied.

Understanding aquatic insect osmoregulation is important because a growing number of human activities are increasing the salinity of freshwater habitats (Canedo-Arguelles et al.

2013; Kaushal et al. 2005; Kefford et al. 2012; Pond et al. 2008; Williams, 2001a;

Williams, 2001b). Increasing salinization is occurring in conjunction with several activities, including the de-icing of roads (Karraker et al. 2008), mining and weathering of mine tailings (Pond et al. 2008), hydraulic fracturing (Entrekin et al. 2011), and return flows from irrigated landscapes (Williams, 2001b; Williams, 2001a). Climate related causes of freshwater salinization include sea level rise and reduced precipitation patterns that lower stream and lake volumes and concentrate salts. These shifts in salinity are associated with biodiversity loss and alterations in ecosystem function (Canedo-Arguelles et al. 2013;

Kefford et al. 2012; Pond et al. 2008).

Almost all aquatic insects express specialized, mitochondrial rich osmoregulatory structures in the form of ionocytes (termed chloride cells), chloride epithelia, or anal papillae

(Komnick, 1977) on the body surface. The primary role of these structures is to take up physiologically important ions directly from the surrounding water (typically against concentration gradients) (Poteat et al. 2014a). These structures are also important sites of trace metal uptake from water (Buchwalter et al. 2005), with large variation among taxa in rates of ion and metal uptake (Buchwalter et al. 2008; Poteat et al. 2014c). Our working understanding is that once ions are taken up into the hemocoel, ion concentrations are tightly regulated via the insect renal system (Malpighian tubules) and hindgut (Bradley, 1987).

Previous studies have demonstrated that metals antagonistic to Ca transport (e.g. Cd) in fish and crustaceans do not appear to elicit this response in aquatic insects (Gillis et al. 2008b;

Gillis et al. 2008a; Poteat et al. 2014a). We are not aware of comparable studies in insects with metals (e.g. Ag and Cu) that are known to act as sodium antagonists in other aquatic organisms.

Sodium is a major ion in most freshwater ecosystems and also an important osmolyte in almost all cells (Evans, 2008a; Evans, 2008b; Potts et al. 1964). However, sodium transport remains poorly understood in aquatic insects, and comparisons of sodium transport rates among different freshwater insect species currently do not exist. Understanding fundamental difference in osmoregulatory process across different insect groups has the potential to shed light on patterns of biodiversity loss associated with salinized waters. Sodium transport is also of interest because it has been identified as a mechanism by which metals such as silver and copper can cause acute toxicity in freshwater organisms. For example, exposure to

waterborne copper has been shown to reduce Na influx rates in fish (Grosell M et al. 2002;

Laurén et al. 1985; Pelgrom et al. 1995; Sola et al. 1995) and crustaceans (Brooks et al.

2003), as well as reduce blood Na concentrations in fish (Laurén et al. 1985; Pelgrom et al.

1995). Similarly, exposure to waterborne silver has been shown to target Na transport in a variety of aquatic species such as rainbow trout (Oncorhynchus mykiss) (Bianchini et al.

2002; Morgan et al. 1997), crayfish (Cambarus diogenes diogenes) (Bianchini et al. 2002) and daphnids (Daphnia magna) (Bianchini et al. 2002).

In this study, we first compared Na uptake dynamics among different Na salts under a wide range of ambient Na concentrations. We further made comparisons of Na turnover rates in 2 common aquatic insects – the caddisfly H. sparna, and the mayfly Maccaffertium sp. We then asked if species found in similar habitats varied significantly in their Na uptake rates by comparing Na uptake among 9 aquatic insect species representing 4 orders. Finally, we asked if Na transport was a target of Cu or Ag exposure under a range of concentrations ranging from environmentally relevant to extreme.

MATERIALS AND METHODS

2.1 Sample Collection and Preparation

Insect larvae were collected from the Eno River, NC (36°5’.39”N, 79°08” W); Basin

Creek, NC (36°22’27”N, 81°8’32” W); or Cataloochee Creek, NC (35°38’48”N, 83°4’31”

W) (details below). All insects were obtained using a D-frame kick-net and transported to the laboratory using coolers filled with aerated stream water, cold packs, and mesh substrate. The larvae were acclimated in a cold room set to the temperature of the stream at time of

sampling (14-16°C, 12 hour: 12 hour light: dark photoperiod) for a minimum of 48 hours.

Larvae were not fed prior to or during any experiments. Base waters for all of the experiments were comprised of American Society for Testing and Materials (ASTM) artificial soft water (ASW) (mM: 0.57 NaHCO3, 0.17 CaSO4*2H2O, 0.25 MgSO4, and 0.03

KCl; pH=7.8±0.02).

2.2 Radioactivity Measurement

The γ-emitting isotope 22Na was obtained as 22NaCl (Perkin Elmer, Billerica, Ma, USA) and diluted in deionized water to make a working stock solution. For all experiments, solutions ranged from 156.25 Bq mL-1 to 260 Bq mL-1. Measurement of 22Na in working solutions (1 mL subsamples) and in insect larvae was performed on a PerkinElmer Wallac

Wizard 1480 Automatic Gamma Counter. All larvae were rinsed in ASW containing stable

Na to displace loosely adsorbed 22Na from the exoskeleton. Larval samples were counted in

15 mL of clean water in 20 mL glass scintillation vials. All samples were counted for 3 minutes and all samples had counting errors less than 5%.

2.3 [Na] Uptake Kinetics in Hydropsyche betteni using three different Na salts (NaCl,

NaHCO3 and Na2SO4)

Larvae were field collected from the Eno River and acclimated for 48hrs in ASW, but with varying Na concentrations as NaCl, NaHCO3, or Na2SO4. All experiments were performed at 0.06, 0.19, 0.57, 1.70, 5.09 and 15.22 mM Na. pH was maintained at 7.4±0.02 for the duration of the experiments using 0.1 M KOH. 22Na radiotracer (260 Bq mL-1of

exposure solution) was used for all treatments. Exposure solutions were made in bulk and distributed into 5 replicate 25 mL treatments in 50 mL aerated HDPE beakers with Teflon® mesh as a substrate. An individual larva was placed into each replicate beaker and Parafilm™ was used to reduce evaporative loss. Larvae were assayed for radioactivity in vivo at 4,8,12, and 24 hours and returned to their respective exposure chambers after each measurement.

Linear uptake rates were determined for each concentration of Na and for each salt used.

2.4 Na turnover dynamics in Hydropsyche sparna and Maccaffertium sp.

Larvae of both species were field collected from the Eno River and acclimated for 48hrs in ASW, but with varying Na concentrations as NaHCO3. NaHCO3 was chosen as the sodium

- salt because assessments of inland streams in the U.S. have demonstrated that HCO3 is typically the predominant anion in Central Appalachia (Pond et al. 2008) and the majority of wadeable streams in the U.S.(Griffith, 2014). An initial experiment with H. sparna examined uptake kinetics at 0.06, 0.19, 0.57, 1.70, and 5.09 mM Na. That experiment exhibited apparent saturation type kinetics, but higher Na concentrations were needed to achieve a robust estimate of maximum transport (Vmax) and affinity (Km). A second set of experiments with H. sparna were run at 1.70, 5.09 and 15.22 mM Na. Subsequent experiments with

Maccaffertium sp. were conducted at 0.06, 0.19, 0.57, 1.70, 5.09 and 15.22 mM Na. The buffer MOPS (3-(N-Morpolino) Propane-Sulfonic Acid) (5mM) (Fischer Scientific, Fair

Lawn, NJ, USA) maintained pH at 7.40±0.02 for all treatments. MOPS stock solution was buffered using 1M KOH (total additional KOH in test treatments=3.56 mM). 22Na radiotracer

(260 Bq mL-1of exposure solution) was used for all treatments. Individual larvae were placed

in aerated High Density Poly-Ethylene (HDPE) beakers with 25 mL of the spiked treatment waters. In H. sparna, ten larvae were used for each treatment except for 0.06 and 5.09 mM in, which used 15 larvae to allow for reciprocal transfer efflux experiments (see below). For

Maccaffertium sp., five individuals were used for each Na concentration as fewer larvae were collected from the field. A Teflon square substrate was added for the larvae and a Parafilm™ cover was placed to reduce evaporative loss. Larvae were assayed for radioactivity in vivo at

24, 44, 76.5 and 100 hours and returned to their respective exposure chambers. After 120 hours, the final uptake time point, larvae were rinsed, assayed, and transferred to clean water for efflux studies.

To assess Na turnover rates, it was essential to estimate whole body Na concentrations of larvae. A parallel experiment measured whole body Na concentration larvae held at each of the Na concentrations described above. Exposures lasted for 7 days to parallel the 2 day acclimation and 5 day tracer study. Here, larvae (N=3 per beaker) were held in aerated 100 mL beakers, with 5 replicate beakers per Na concentration. A Teflon square substrate was added for the larva and a Parafilm™ cover was placed to reduce evaporative loss. Larvae were then removed from solution and blotted dry to obtain fresh weights then dried in a drying oven (65°C) for 48 hours and weighed on a Sartorius CPA2P microbalance to the nearest 0.001 mg. The three larvae from each exposure beaker were composited and microwave digested (CEM MARSXpress) in 2 mL of OmniTrace Ultra High Purity Nitric

Acid (EMD Chemicals, Darmstadt, Germany). After digestion, Na concentrations were determined via ICP-MS at the Environmental and Agricultural Testing Services Laboratory

(Department of Soil Sciences, North Carolina State University, Raleigh, NC, USA). Quality

control blanks were below detection limits. Standard reference material (NIST 2976 mussle tissue (freeze-dried)) and instrument check standards were within 2.5% of the expected Na concentrations.

2.4.1 Efflux kinetics in Hydropsyche sparna

To measure 22Na efflux, larvae from each Na treatment were placed in fresh HDPE beakers with 50 mL of their respective Na treatment in the absence of radiotracer. Larvae were assayed daily for radioactivity and the water was refreshed to prevent the re-uptake of effluxed 22Na. Larvae that acquired a 22Na signal in our lowest Na treatment (0.06 mM), were split to efflux at either their exposure concentration or at 5.09 mM Na. Similarly, larvae that acquired a 22Na signal at 5.09 mM Na were split to efflux at either their exposure concentration or under low (0.6 mM) Na.

2.5 Assessing the influence of MOPS on Na uptake

We compared Na uptake rates (at 0.57 mM Na, pH 7.4±0.02 ) in the absence and presence of MOPS (5mM in ASW base water) in several field collected species including A. abnormis (Plecoptera), P. immarginata (Plecoptera), P. biloba (Plecoptera), A. carolinensis

(Plecoptera), and H. sparna (Trichoptera). Experiments were performed at 4, 8, and 29 hours.

Subsequent experiments assessed whether the effects of MOPS on Na uptake were dependent on the Na concentration. In these experiments, we used field collected H. betteni to determine the effects of MOPS (conditions as above) at different ambient [Na] (0.187, 0.570,

1.70, 5.09, and 15.22 mM Na). 22Na radiotracer (260 Bq mL-1of exposure solution) was used for all treatments. Larvae were assayed for radioactivity in vivo at 4, 8, 12 and 25 hours and

returned to their respective exposure chambers. Linear uptake rates were determined for each species, either in the presence or absence of MOPS.

2.6 Comparative Sodium Uptake Kinetics

Na uptake rates were compared among field-collected (Cataloochee Creek, NC)

Paraleptophlebia sp., Maccaffertium pudicum, Pteronarcys sp., Paragnetina immarginata,

Arctopsyche irrorata, Hydropsyche sparna, Diplectrona sp., Tipula sp., and Atherix sp. (N=5 for each species). All insects were acclimated for at least 48 hours in ASW. Bulk solutions were created with ASW (pH=7.8±0.02; adjusted with 0.1 M KOH and 0.1 M HNO3) and the

22Na radiotracer (156.25 Bq mL-1) for a total Na concentration of 0.57 mM. Larvae (1 individual per replicate) were placed in aerated HDPE beakers with 50 mL of the exposure solution. A Teflon square substrate was added for the larva and a Parafilm™ cover was placed to reduce evaporative loss. Larvae were assayed for 22Na in vivo after 8, 24, 32, 48, and 72 hours of exposure. At every time point, each individual was removed from solution and rinsed in an ASW bath to remove 22Na that may have been loosely adsorbed to the surface of the larvae. After the final measurement, each individual was blotted dry and a fresh weight was obtained. To obtain our best estimates of unidirectional uptake rates, only the initial linear portion of the uptake curves were used.

2.7 Copper and Silver Effects on Sodium transport

Initial experiments examined the effects of Ag (0.0185, 0.185, and 1.85 µM) and Cu

(0.0314, 0.314, 3.14 µM) on Na uptake in three aquatic insect species - representing

Plecoptera (Isoperla (near namata group)), Trichoptera (H. sparna), and Ephemeroptera (M.

pudicum). Because Isoperla (near namata group) individuals did not survive at the highest

Ag and Cu concentrations, subsequent experiments were conducted at Ag concentrations of

0.030, 0.148, and 0.741 µM and Cu concentrations of 0.148, 0.741, and 1.483 µM. Bulk solutions were created for each treatment with the 22Na radiotracer (156.25 Bq mL-1). Copper was added as CuSO4*5H2O (Sigma-Aldrich, St. Louis, MO, USA) and silver was added as

AgNO3 (EMD Chemicals, Gibbstown, NJ, USA). The buffer MOPS (5 mM) controlled pH at pH of 6.8±0.02. All dissolved metal concentrations were verified within 2.5% accuracy using

ICP-MS analyses. Pre-exposure water samples were taken for metal concentration verification.

We collected sufficient numbers of larvae to perform Na uptake at all Cu and Ag concentrations for three of the six species tested - H. sparna (Eno River), M. pudicum

(Cataloochee Creek), and A. abnormis (Cataloochee Creek). We only collected sufficient numbers of A. irrorata (Cataloochee Creek), Atherix sp. (Cataloochee Creek) and

Pteronarcys sp. (Cataloochee Creek) to measure Na uptake in conjunction with the highest levels of Cu and Ag. All insects were acclimated for at least 48 hours in ASW prior to any exposure.

A replicate consisted of a single larva in individual aerated 50 mL HDPE beakers with 25 mL of the treatment solution. A Teflon square substrate was added for the larva and a

Parafilm™ cover was placed to reduce evaporative loss. In vivo assays were performed at 23,

43, 68 and 99.5 hours. At every time point, each individual was removed from solution and rinsed in an ASW bath to remove 22Na that may have been loosely adsorbed to the surface of the larvae. Each individual was then placed in a scintillation vial with 15 mL ASW for

counting. Larvae were then placed back into exposures until the final time point, where they were blotted dry and fresh weights were obtained. For each species, Na uptake rates were determined as the slopes of Na acquisition over time.

2.8 Data Analysis

Data analysis was performed using GraphPad Prism (v5.04). Unidirectional Na uptake rates were estimated from the initial linear portions of the uptake curves because inclusion of later time points would bias rate estimates due to the physiological loss of radiotracer. For

[Na] kinetics experiments in H. sparna and Maccaffertium sp., nonlinear regression curves were fit using a one-site specific binding equation to estimate Vmax and Km. Student’s t-tests were performed to determine the effects of MOPS on Na uptake rates. For the comparative uptake kinetics at 0.57 mM Na, rates were calculated on a per individual and weight specific basis. All mass specific rates of Na uptake or tissue Na concentrations are reported on a wet weight basis unless explicitly indicated otherwise. Student’s t-tests were performed to determine effects of Cu and Ag on Na uptake relative to controls along with any differences related to weight. Results were significant when P≤0.05. Error bars represent ± SE throughout.

3. RESULTS

3.1 [Na] Uptake Kinetics in Hydropsyche betteni using three different Na salts (NaCl,

NaHCO3 and Na2SO4)

Na uptake kinetics in H. betteni were affected by the anionic composition of the

- - exposure water. Relative to the Cl and HCO3 salts, Na uptake rates were consistently lower

2- 2- when the anion was SO4 (Fig. 1). For example, Na uptake was reduced 5-53% with SO4 as the anion relative to Cl- as the major anion. These differences were significant (p≤0.05) in 4 out of 5 of the Na concentrations tested. Two of 5 Na treatments (0.57 and 15.22mM)

- - - differed between Cl and HCO3 , with faster Na uptake rates observed in the Cl salt treatments. No other combinations of treatment groups and Na salt were significantly different.

3.2 Na turnover dynamics in Hydropsyche sparna and Maccaffertium sp.

Radiotracer studies on H. sparna and Maccaffertium sp. demonstrated that Na uptake is time- (Fig. 2) and concentration-dependent (Fig. 2, 3). In H. sparna, estimates of unidirectional Na uptake rates ranged from 12 to 419 nmol Na g-1 hr-1 across the range of Na

2 concentrations tested. Uptake exhibited saturation type kinetics (r =.91) with Vmax and Km estimates of 512.3 nmol Na g-1 hr-1 and 3.664 mM Na, respectively (Fig. 3). In

Maccaffertium sp., estimates of unidirectional Na uptake rates ranged from 5.7 to 715.5 nmol

Na g-1 hr-1. While these data were well fit by a one-site binding model (r2=0.82), these larvae did not appear to be close to exhibiting maximum transport rates at the highest concentration tested (15.22 mM), and it was not possible to reliably estimate Michaelis-Menten values (Fig.

3). Thus, these two species vary widely in their Na acquisition patterns, with H. sparna being more effective than Maccaffertium sp. at acquiring Na at very low Na concentrations, but efficiently halting excess transport under higher Na concentrations. Maccaffertium sp. in contrast appears to take up Na excessively at higher Na concentrations.

Measures of whole body Na were comparable between H. sparna and Maccaffertium sp.

(Table 1). The global average for whole body Na concentration (µM/g wet wt) of H. sparna and Maccaffertium sp. larvae held for 7 days under different Na concentrations (0.06 – 5.09 mM) were 43.7 ± 7.6 and 51.6 ± 3.5, respectively. A regression of Log dissolved Na (0.06 –

5.09 mM) on whole body Na concentrations in H. sparna, did not have a significant slope

(p=0.16), but the whole body Na concentration of 5.09 mM exposed larvae was slightly higher than the global mean of the preceding concentrations (p<0.01). Unfortunately, we did not obtain measures of whole body Na from larvae held at 15.22 mM. In contrast, regressions of Log dissolved Na (0.06 – 5.09 mM or 0.06 - 15.22 mM) on whole body Na concentrations in Maccaffertium sp. had significantly positive (albeit small) and identical slopes (p=0.037 and 0.029 respectively). The % body water was 82% water in H. sparna and 84% in

Maccaffertium sp.

When we link Na uptake rates to measures of whole body Na, it is apparent that Na turnover rates increase with increases in dissolved Na concentrations. Estimates of the daily acquisition of Na relative to whole body Na range from 0.7 to 20.9% in H. sparna and 0.2 to

66.9% in Maccaffertium sp. (Table 1). The differential (concentration gradient) between total body Na and dissolved Na in our treatments ranged from 32.5 to 53.7 mM in H. sparna, and 14.9 to 63.7 mM in Maccaffertium sp. (assuming a body density of 1 g/ml).

3.2.1 Efflux in H. sparna

Since whole body sodium concentrations were relatively consistent among larvae held in

0.06-1.7 mM Na, we hypothesized that efflux rates would have to be commensurate with uptake rates to maintain steady-state type conditions. We observed a general increasing trend in the total amount of radiotracer lost over 7 days with increasing dissolved Na concentrations. However, since we could not simultaneously measure 22Na efflux rates while larvae acquired new unlabeled Na, and monitor whole body Na concentrations in the same larvae, quantitative measures of Na efflux rates were challenging. A transplant experiment revealed that larvae lost tracer faster when transplanted to Na rich water (Fig. 4A). Similarly, larvae transplanted to Na poor water lost tracer more slowly (Fig. 4B). Together, these data suggest that overall Na turnover rates are dependent on ambient Na conditions.

3.3 Assessing the influence of MOPS on Na uptake

MOPS (5mM) consistently reduced Na flux rates across species under stable Na concentrations (0.57 mM, pH 7.4±.02) (Fig. 5A). The percent reductions in Na uptake for each species are as follows: A. abnormis (37.4%), P. immarginata (48.7%), P. biloba

(33.1%), A. carolinensis (46.9%), and H. sparna (26.7%). All differences were statistically significant (P<0.05). Effects of MOPS on Na uptake in H. betteni were dependent on Na concentration (Fig. 5B). H. betteni exhibited increased Na uptake in the presence of MOPS under lower ambient [Na]. At 0.187 and 0.57 mM Na, Na uptake increased by 61.8% and

58.8% in the presence of MOPS, respectively. MOPS had differential effects at higher

ambient Na concentrations. At 1.70 mM Na, MOPS decreased Na uptake by 32.8%.

Additionally, there was a 26.6% decrease in Na uptake at 15.22 mM Na. There was no significant effect of MOPS on Na uptake at 5.09 mM Na.

3.4 Comparative Sodium Uptake Kinetics

When larvae of several species were held at 0.57 mM Na (pH=7.8), weight specific Na uptake rates ranged from 39.9 nmol Na g-1 hr-1 in Atherix sp. to 709 nmol Na g-1 hr-1 in

Paraleptophlebia sp. (Fig. 6A). On a per individual basis, there is a significantly positive association between Na influx rates and body weight across taxa (r=0.78, p=0.013) (Fig. 6B), suggesting that size exerts important influence on Na influx rates across species. However, intra-specific variation in Na flux rates was quite low in all species, even those that varied widely in their body weights. For example, in the stonefly P. immarginata, flux rates varied only 6.2% among individuals spanning 37.5 to 187.7 mg. Similarly, in Tipula sp., Na influx rates varied only 8% among individuals spanning 35.5 to 173.9 mg. Thus, species specific differences in Na influx rates are only partially driven by body weight as physiological differences among taxa are also important.

3.5 Copper and Silver Effects on Sodium transport

The effects of Ag on Na uptake varied with species and concentration. In H. sparna, there was a mild stimulation (34.9% increase) of Na uptake at the lowest Ag concentration

(Fig. 7B). Similarly, there was a 33.5% and 24.8% increase in Na uptake in A. abnormis at low and medium Ag concentrations, respectfully (Fig. 7A). Interestingly, M. pudicum exhibited an 18.8% decrease in Na uptake at the lowest Ag concentration (29.67 nM)

(p=0.0059). Across 6 aquatic insect species tested, Na uptake was inhibited at the highest Ag

concentration (741.65 nM) for 5 species (Fig. 7A-C; Fig. 8). Reductions ranged from 44.0% in Arctopsyche sp. (p=0.0145), 93.3% in M. pudicum (p< 0.0001), 94.0% in A. abnormis (p<

0.0001), 98.1% in Pteronarcys sp. (p< 0.0001), and 99.2% in Atherix sp. (p< 0.0001).

Conversely, increasing Cu concentrations elicited a stimulation of Na uptake rates in

3 of the species tested. Within A. abnormis, there was a 52.1% and 35.5% increase in Na uptake at medium (741.2 nM) (p=0.0028) and high Cu concentrations (1483 nM) (p=0.0272), respectively (Fig. 7A). Similarly, H. sparna exhibited an 81.7% and 104.8% increase at the medium (p= 0.0046) and high Cu concentrations (p=0.0003), respectively (Fig. 7B). M. pudicum showed a 21.7% increase (p=0.0099) at the low and 31.9% increase (p=0.0077) at the high Cu concentrations (Fig. 7C). Not all species showed an increase in Na uptake rates as Cu concentrations increased. Atherix sp. (p=0.0011) and Pteronarcys sp. (p=0.0142) exhibited a decrease in Na uptake at high Cu concentrations, with a 47.4% and 23.4% difference, respectively (Fig. 8). Together, results indicate that Cu and Ag at environmentally relevant concentrations generally do not act antagonistically on Na transport in aquatic insects, and in fact can stimulate increased Na uptake.

4.0 Discussion

A recent synopsis of sodium concentrations in freshwater habitats in USA surface waters suggests that the vast majority of freshwater systems contain Na concentrations between 0.04 and 8.7 mM (David Mount, US EPA, personal communication). Marine systems, which support relatively few insect species, typically range from 440 to 490 mM

Na, while hypersaline inland waters can be greater than 530 mM Na (Wright et al. 1995).

Surprisingly, we know more about osmoregulation in the few insect species that tolerate saline conditions (Bradley, 1987; Bradley et al. 2009; Bradley, 2013; Herbst, 2001; Herbst et al. 1988) than we do about the thousands of species that require freshwater. Saline tolerance in insects appears to be possible via the ability to produce concentrated urine (Bradley et al.

1975; Bradley et al. 1977a; Bradley et al. 1977b), the expression of compatible organic solutes (Patrick et al. 2000a; Patrick et al. 2000b), and the ability to osmoconform (Garrett et al. 1987; Patrick et al. 2001). Obligatorily freshwater insects are generally considered strict osmoregulators (Komnick, 1977; Stobbart, 1974) but remain poorly studied.

We observed that under a range of Na concentrations, the dominant anion could profoundly influence Na uptake rates. Sulfate as the major anion slowed Na uptake relative to chloride and bicarbonate. It is unclear whether this observation was driven by the anion influence on Na ionic activities, or was a physiological phenomenon (e.g. changes in electrochemical potential or effects on the Na transport system(s) in place). Such observations support studies demonstrating that water with similar conductivities but different ionic compositions could be differentially toxic in lab bioassays (Kunz et al. 2013).

Much more research is needed to better understand these processes.

Over a range of Na concentrations covering the vast majority of Na concentration found in U.S. freshwaters, the caddisfly H. sparna and the mayfly Maccaffertium sp. exhibit strikingly different patterns of Na uptake kinetics while maintaining relatively similar tissue concentrations. At the higher range of Na concentrations we worked with, Na transport is at or near saturation in H. sparna while it is still apparently far from saturation in

Maccaffertium sp. This finding is interesting because if acquisition of ions from freshwater

requires energy, it would imply that at higher Na concentrations, larvae are acquiring (and effluxing) Na at greater rates and apparently greater energetic cost than at lower Na concentrations. Thus, even though the concentration gradient between the larvae and the water is greatest under more dilute conditions, overall ion turnover rates (and likely the associated energy expenditure) are considerably smaller. It is unclear why feedback mechanisms are not in place under ion rich conditions, as is observed in saline tolerant mosquitos (Donini et al. 2007). Few insects thrive at the higher Na concentrations we worked with, yet Maccaffertium aggressively takes up Na under salinities that exclude most insect taxa.

Interestingly, we observe no change in oxygen consumption rates in salinity challenged aquatic insects (data not shown), which is consistent with several studies in aquatic insects

(Kolsch et al. 2011; Kapoor, 1979; Edwards, 1982). One view is that the energetic cost of osmoregulation is not substantially high enough to result in increased oxygen consumption

(Edwards, 1982). This would be possible if “exchange diffusion” occurs in these animals in order to maintain relatively stable internal ion concentrations (Evans, 1967; Motais et al.

1966). This process implies little energetic cost associated with the transport of ions.

However, it is not known if this process occurs in freshwater insects. Others suggest salinity challenge diverts resources from other functions (Kidder et al. 2006). The observation that exposure to elevated ion concentrations results in developmental delay (e.g. (Johnson et al.

2015)) when not directly lethal (e.g. (Kunz et al. 2013) supports the idea that salinity challenge is energetically costly to aquatic insects. Thus, there remains considerable uncertainty regarding the energetics of osmoregulation in freshwater insects.The caddisfly H.

sparna larvae held under a wide range (0.06 to 5.09 mM) of Na concentrations appeared to maintain consistent whole body tissue concentrations, though the larvae held at 5.09 mM Na were slightly elevated relative to the other treatment groups. Other studies suggest that very high (150 mM Na) sodium concentrations are required to disrupt hemolymph homeostasis in the freshwater obligate caddisfly Limnephilus affinis (Sutcliffe, 1961). In contrast, whole body Na appears to increase slightly with increasing Na concentration in Maccaffertium.

More detailed studies are needed to ascertain whether hemolymph Na is tightly regulated in this species (expected) or if is allowed to fluctuate with external medium (unlikely). The whole body Na values we obtained for H. sparna and Maccaffertium sp. are similar to those observed in the mayfly Stenonema femoratum (~260 μmol g-1 dry weight) but lower than values reported from the mayfly Leptophlebia cupida (~600 μmol g-1 dry weight) when held at 0.1 mM Na at pH 6.5 (Rowe et al. 1989).

The use of the buffer MOPS is potentially confounding when examining Na uptake rates.

Our results showed strikingly different effects of the use of MOPS depending upon the species tested and the concentration of Na in solution. Our results concur with other studies that have shown that MOPS can either increase (Brix et al. 2015) or decrease Na uptake

(Shih et al. 2012). It is unclear whether the influences of MOPS are the direct effects of the buffer itself or whether the use of KOH in buffer preparation results in altered Na uptake rates. Preliminary work in our lab (data not shown) suggests that K+ can have an inhibitory effect on Na uptake, depending upon the concentration of K and Na (i.e. Na:K ratio)

(Scheibener 2015, unpublished data). Further work is necessary to determine the effect of K+ on Na transport along with potential confounders such as buffers for pH control.

When we held various aquatic insect species under identical conditions (0.57 mM Na), uptake rates varied almost 18-fold across species and ranged from 39.8 nmol g-1 hr-1 (w.w.) in

Atherix sp. to 709 nmol g-1 hr-1 (w.w.) in Paraleptophlebia sp. There are few insect studies to compare our data to; Patrick et al. (2001) report Na uptake rates of ~600 and ~1250 nmol g-1 hr-1 (w.w.) in Culex quinquefasciatus and Culex tarsalis respectively. In other experiments, rainbow trout (Oncorhynchus mykiss) exhibit Na uptake at a rate of 523 nmol g-1 hr-1 (w.w.) under near ASW conditions (Na = 0.595 mM) (Laurén et al. 1985). Bury et al. (1999) found rainbow trout held under similar conditions to acquire Na at a rate of ~790 nmol g-1 hr-1

(w.w.). Daphnia magna exhibit Na uptake under similar water regimes (0.5 mM Na), at a rate of ~5500 nmol g-1 hr-1 (w.w.) (Stobbart et al. 1977).

The influence of body weight on Na uptake is complex. Taken on a per individual basis, it is apparent that larger species generally traffic more Na than do smaller species (about 61% of the variation in Na uptake rates across species in our dataset was explained by average body size). However, within individuals of a given species, differences in body weight had a surprisingly small influence on Na transport rates. This suggests that species specific physiological differences in Na transport rates are profound, even among species collected from the same location. On a weight specific basis, it is interesting that the taxa with the fastest Na uptake rate (Paraleptophlebia) is described as being particularly sensitive to elevated total dissolved solids (TDS) while that with the slowest uptake rate (Atherix) is described as being tolerant to elevated (TDS) (U.S.Environmental Protection Agency., 2011).

Whether there is a significant bioenergetics cost associated with excessive ion transport rates

under ion rich conditions, remains to be determined - particularly for insects adapted to dilute environments.

Although Na transport systems have been well characterized in some aquatic organisms, little is known about actual mechanisms of Na transport in aquatic insect species.

Comparative studies of apical ion transport have demonstrated that Na is typically actively transported via a V-type H+-ATPase (Potts, 1994) and basolaterally via a P-type Na+/K+-

ATPase (Skou, 1957). Suppression of the activity of this basolateral Na+/K+-ATPase is described as the target of copper and silver toxicity in a large number of aquatic organisms

(e.g. (Grosell M et al. 2002)). Studies on the mosquito Aedes aegypti have shown that they have similar Na transport systems as other aquatic organisms (Patrick et al. 2002), however, the specific transport systems of stream dwelling insects remain undescribed.

In our experiments, exposure to environmentally relevant concentrations of Cu and Ag did not inhibit Na uptake. In fact, we saw a stimulation of Na uptake in 3 species (A. abnormis, H. sparna, and M. pudicum) in response to Cu and in 2 species (A. abnormis and

H. sparna) in response to Ag. At extremely high concentrations of these metals, we did observe inhibition of Na transport (though not in Hydropsyche, a genus widely described as particularly metal tolerant in nature (Cain et al. 1992; Cain et al. 2006; Luoma et al. 2009).

In other studies, Cu and Ag suppress Na transport in fish (Morgan et al. 1997) and crustaceans (Bianchini et al. 2003; Bianchini et al. 2002). Our findings (stimulation rather than suppression) are similar to observations Ca transport in aquatic insects exposed to Cd and Zn (Poteat et al. 2014a). Together, these observations may help explain why aquatic insects are apparently insensitive to acute metal exposures in traditional toxicity tests (Brix et

al. 2005; Buchwalter et al. 2007; Poteat et al. 2014b). We speculate that the more proximal terrestrial ancestry of aquatic insects (Bradley et al. 2009; Kristensen, 1981; Misof et al.

2014) makes their osmoregulatory processes different from species with more proximal marine origin (e.g. fish and crustaceans).

Aquatic insects are critical players in freshwater ecosystems, and as such, are the most widely relied upon faunal group for assessing ecological conditions. Yet, our understanding of the physiology of this important group continues to be inadequate in the face of the pressures that humans impose on freshwater ecosystems. We suggest that a better understanding of fundamental physiological processes in this important faunal group will lead to improved interpretation of ecological monitoring data and reduce the uncertainty associated with setting environmental regulations.

Acknowledgements

We would like to thank Justin Conley (U.S. EPA) and Monica Poteat (Oak Ridge National

Laboratory) for their scientific guidance and methodology. We would also like to thank Allison

Camp (NCSU), Addie Harris (NCSU), anonymous reviewers, and editorial staff for valuable editorial comments. Shane Scheibener performed the experiments, data analysis, prepared and edited the publication. David Buchwalter developed the concepts and approach, assisted in experiments and data analysis, and prepared and edited the publication.

Figures

1 8 0 0

N aC l

. w

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Figure 1. Comparison of sodium salts (NaCl, NaHCO3, and Na2SO4) on Na transport dynamics in H. betteni. Estimated maximum transport rates (Vmax) are as followed: In

-1 -1 NaCl=2.194, In NaHCO3=1.39, and In Na2SO4= 0.784 µmol Na g hr w.w. Estimated affinity constants (Km) are as followed: In NaCl=7.71, In NaHCO3=3.321, and in

Na2SO4=2.106. Error bars represent mean ± S.E of 5 individuals.

A B

5 0 1 5 .2 2 m M 1 0 0 1 5.22 m M 5 .0 9 m M 5.09 m M 1 .7 0 m M 4 0 8 0 . 1.70 m M

.5 7 m M .

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Figure 2. Time course radiotracer study of sodium uptake at different dissolved Na concentrations versus time for the species H. sparna (Trichoptera: Hydropsychidae) (A) and

Maccaffertium sp. (Ephemeroptera: Heptageniidae) (B). Each point represents the mean ±

S.E. of 10-15 individuals.

1 0 0 0 M a c c a ffe rtiu m s p .

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Figure 3. Michaelis-Menten type kinetics for the species H. sparna and Maccaffertium sp.

-1 -1 Estimated maximum transport rates (Vmax) are as followed: H. sparna=521.3 nmol Na g hr w.w. Estimated affinity constants (Km) are as followed: H. sparna=3.664 mM Na. Vmax and

Km values could not be accurately quantified for Maccaffertium sp. Error bars represent mean

± S.E. of 10-15 individuals for H. sparna and 5 individuals for Maccaffertium sp.

Hydropsyche sparna Maccaffertium sp. Na Uptake Whole Body % New Na uptake Whole Body % New [Na] rate Na Daily Na rate Na Daily Na mM (nmol g-1 hr-1 (µmol Na (nmol g-1 (µmol Na wet wt.) g-1 wet wt.) hr-1 wet g-1 wet wt.) wt.) 0.06 12.4±1.0 45.5±2.7 0.7 5.7±0.5 49.7±1.6 0.2

0.19 26.1±2.3 37.6±1.1 1.7 20.7±1.8 44.59±3.8 (0.9-1.4)

0.57 83.7±8.0 43.7±3.6 (3.3-5.1) 50.9±3.7 44.92±1.3 (2.6-2.9) 153±16 (9.6-14.0) 1.70 39.2±2.8 137±10 43.8±8.0 (7.0-8.4) 116±17 - 305±22 (16.6-20.9) 5.09 52.4±2.5 277±40 54.68±14.1 (9.7-18.8) 216±25 - 15.22 419±18 - - 716±133 41.3±6.1 (33.5- 66.9)

A B

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Figure 4. The influence of ambient Na concentration and prior exposure history on the loss rates of previously acquired 22Na. Raw efflux data (mean ± S.E.) versus time for the species

H. sparna, where initial Na uptake was performed in 0.06mM Na (A) or 5.09mM Na (B).

Efflux was either performed in .06mM Na (represented by the circles) or performed in

5.09mM Na (represented by the squares).

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Figure 5. Comparison of Na uptake in the presence or absence of MOPS. Na uptake rates across species (A) and in the species H. betteni under various ambient Na concentrations (B).

Error bars represent mean ± S.E of 5 individuals.

A B

8 0 0 2 5

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0 0 0 5 0 1 0 0 1 5 0 0 5 0 1 0 0 1 5 0 w e t w e ig h t (m g ) w e t w e ig h t (m g )

Figure 6. Sodium uptake rates (mean± S.E.) on a per mass basis (A) or per individual basis

(B) plotted against body weight (mean ± S.E.) for 9 species of aquatic insects. Symbols represent the following species:  A. irrorata,  Atherix sp.,  H. sparna,  Diplectrona sp.,  P. immarginata,  M. pudicum,  Paraleptophlebia sp.,  Pteronarcys sp., and 

Tipula sp.

3 5 0 A c ro n e u ria a b n o rm is

3 0 0 *

. * w

. 2 5 0

- r

h 2 0 0

a 1 5 0

l o

m 1 0 0 n

5 0 *

3 5 0 H y d ro p s y c h e s p a rn a *

. 3 0 0 *

w .

w 2 5 0

r h

2 0 0

- g

1 5 0

o 1 0 0 m n 5 0

1 4 0 0 M a c c a ffe rtiu m p u d ic u m

. 1 2 0 0 *

w .

w 1 0 0 0 *

r h

8 0 0 1

- * g

6 0 0

o 4 0 0

m n 2 0 0 * 0 l o g g g u u u tr A A A C C C n M M M M M M o n n n n n n C .7 .3 .7 .9 .2 .2 9 8 1 7 1 3 2 4 4 4 4 8 1 7 1 7 4 1

Figure 7. The influence of dissolved metal concentrations on sodium uptake rates in three species of aquatic insects.

1 5 0

A r c to p s y c h e ir ro r a ta .

A th e r ix s p . w . *

w P te ro n a rc y s s p . 1

- 1 0 0

a * * N

5 0

o m n * * * 0

l g u l g u l g u ro ro ro t A C t A C t A C n n n o M M o M M o M M C n n C n n C n n 5 8 5 8 5 8 .6 .1 .6 .1 .6 .1 1 3 1 3 1 3 4 8 4 8 4 8 7 4 7 4 7 4 1 1 1

Figure 8. The influence of dissolved metal concentrations on sodium uptake rates in either no presence of metal (control), 741.65 nM Ag, or 1483.18 nM Cu for three species of aquatic insects.

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CHAPTER 2

SULFATE TRANSPORT KINETICS AND TOXICITY ARE MODULATED BY SODIUM IN AQUATIC INSECTS

Shane Scheibenera, Justin M. Conleya,b and David Buchwalter*a a Department of Biological Sciences, North Carolina State University, Raleigh, NC, USA b Current address: United States Environmental Protection Agency, Durham, NC, USA * Corresponding author: [email protected] Department of Biological Sciences Box 7633 North Carolina State University Raleigh, NC 27695

Submitted to Aquatic Toxicology (2017)

Abstract

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 can dominate freshwater invertebrate diversity, 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 10.9 mM improved 96-hr survivorship associated with 20.8 mM SO4 from 44% to 73% in a concentration dependent manner (SO4:Na from 30:1 to 2:1). However, when Na was increased to 21.8 mM (SO4:Na = ~1:1), 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. This finding has implications for toxicity testing, as Na2SO4 is the primary salt used to evaluate

SO4 toxicity. Overall, it is important that we develop a better understanding of major ion toxicity to effectively mitigate and protect freshwater biodiversity from salinization as not all conductivities are equally toxic.

Keywords: Aquatic insects, Major Ions, Physiology, Osmoregulation, Toxicity, Sulfate

1. Introduction

Freshwater salinization has emerged as a topic of global ecological concern (Cañedo-

Argüelles et al., 2016, 2013; Kaushal et al., 2005) and results from human activities such as de-icing of roads (Karraker et al., 2008), mining practices (Pond et al., 2008), hydraulic fracturing (Entrekin et al., 2011) and irrigation of arid lands (Williams, 2001a, 2001b).

Depending on local land uses/activities and geology, the total dissolved solids (TDS)/ionic composition of salinized waters can vary spatially. For example, waters impacted by road de- icing may have elevated Na+ and Cl- in some areas (Ramakrishna and Viraraghavan, 2005), or elevated Mg2+ and Cl- in others (Lewis, 1999), whereas streams impacted by mountaintop

2+ 2+ - 2- coal mining operations are typically enriched in Ca , Mg , HCO3 and SO4 (Bryant et al.,

2002; Pond et al., 2008). Biodiversity loss and alterations in ecosystem function are commonly associated with salinity increases (Cañedo-Argüelles et al., 2013; Kefford et al.,

2012; Pond et al., 2008), however our understanding of how and why different species respond to different ionic matrices remains limited.

The ecological consequences of freshwater salinization are typically observed via biological surveys of insect dominated systems (Pond, 2010; Pond et al., 2008) and are commonly manifested as loss of apparently sensitive taxa (e.g. mayflies). However, whole effluent toxicological evaluations using traditional crustacean bioassay organisms often fail to identify high conductivity waters as toxic (Echols et al., 2010; Kennedy et al., 2004). To address the issue of bioassay organism relevance and to reduce the possibility that toxicity was due to other contaminants (e.g., trace elements), Kunz et al. (2013) compared major ion toxicity in three re-constituted waters. The study compared two standard crustacean bioassay

organisms, the amphipod (Hyalella azteca) and the cladoceran (Ceriodaphnia dubia), and two lesser used taxa for toxicity testing, the mussel (Lampsilis siliquoidea) and the mayfly

(Neocloeon triangulifer). That work indicated aquatic invertebrates vary in susceptibility to

TDS toxicity (the mayfly and mussel were more sensitive) and that toxicity in the mayfly was not driven by conductivity alone. Different ionic compositions produced differential toxicity at comparable conductivities (Fig. 1). That work highlights the need to better understand how ions interact to modulate toxicity in sensitive aquatic organisms.

Relatively little research has been conducted regarding basic osmoregulatory processes in aquatic insects and how they relate to toxicity. Insects may differ from other freshwater species (e.g. crustaceans) with a more proximate marine origin because they are derived from terrestrial ancestors that invaded freshwater habitats numerous times from land

(Bradley et al., 2009; Kristensen, 1981). The consensus seems to be that freshwater insects are strict osmoregulators (Komnick, 1977; Stobbart and Shaw, 1974) and maintain hemolymph osmolalities via the continuous turnover of ions. Uptake of ions occurs through mitochondrial-rich structures such as ionocytes, chloride epithelia or anal papillae (Komnick,

1977), with hemolymph maintenance and excretion occurring in the Malpighian tubules and hindgut (Bradley, 1987). Previous work has demonstrated that Ca (Poteat and Buchwalter,

2014) and Na (Scheibener et al., 2016) uptake rates vary widely among aquatic insects and are influenced by body size and phylogeny. Sulfate uptake remains relatively unexplored in freshwater insects (but see (Maddrell and Phillips, 1978, 1975)) and is surprisingly understudied in aquatic organisms in general (Gerencser et al., 2001).

In this study, we used a radiotracer approach to determine if ionic interactions might help explain observations described above by Kunz et al. (2013). Briefly, they found that synthetic waters with low Ca:Mg ratios and high SO4:Na ratios were toxic to mayflies, whereas waters with higher Ca:Mg ratios and lower SO4:Na ratios were not toxic at comparable conductivities. We first asked if Mg was antagonistic to Ca uptake in four aquatic insect taxa. We then characterized Michalis-Menten type SO4 uptake kinetics for 5 mayfly species to understand the extent to which SO4 transport varies across taxa. We further assessed ionic interactions in relation to SO4 uptake and tested the hypothesis that SO4 toxicity could be modulated by other major ions. We discuss the implications of this work in relation to promotion of field-based benchmarks (U.S. EPA, 2011) based on conductivity alone.

2. Methods

2.1 Sample Collection and Preparation

Aquatic insect larvae were collected from either Basin Creek, NC or the Eno River,

NC using a D-frame kick-net and transported to the laboratory using coolers filled with aerated stream water, cold packs and mesh substrate. The larvae were acclimated to room temperature conditions (21-23°C, 12 h: 12 h light:dark photoperiod) for a minimum of 48 hrs. N. triangulifer larvae (Stroud Water Research Center, Clone WCC-2) were raised in laboratory conditions and were used for radiotracer studies when individuals reached late instar (3-4 week old). Larvae were not fed prior to or during any radiotracer experiment.

American Society for Testing and Materials (ASTM) artificial soft water (ASW) was used for acclimation and as the base water for the sulfate kinetics in mayflies and also to

determine if Mg influenced Ca uptake (see below). Major ion concentrations (mM) for

ASTM ASW were: 0.57 NaHCO3, 0.17 CaSO4·2H2O, 0.25 MgSO4·7H2O and 0.03 KCl; pH=7.8 ± 0.02. For all other experiments, a modified ASW was used for acclimation and as a base water to better reflect soft water conditions found in US surface waters (David Mount,

US EPA personal communication). Major ion concentrations (mM) for modified ASW were:

0.69 NaHCO3, 0.10 CaSO4·2H2O, 0.20 CaCl2, 0.14 MgSO4·7H2O and 0.03 KHCO3; pH=7.8

± 0.02. Insects were weighed wet (unless explicitly stated) on a Sartorius™ analytical scale

(model: CP 124 S) to the nearest 0.01mg. Water samples (15mL) were collected prior to the start of each experiment to verify major ion concentrations (ICP-AES; North Carolina State

University Environmental and Agricultural Testing Service Lab, Raleigh, NC). All measurements were within 5% of nominal concentrations and instrument check standards were within 2.5% of the expected concentrations.

2.2 Radioactivity Measurement

35 35 The β-emitting isotope S was obtained as Na2 SO4, the γ-emitting (and β-emitting)

22 22 45 45 isotope Na was obtained as NaCl and the β-emitting isotope Ca was obtained as CaCl2

(PerkinElmer, Billerica, Ma, USA). Each isotope was diluted in deionized water to make working stock solutions. For all experiments, working solutions ranged from 156 to 260 Bq mL−1. Measurement of 22Na, 45Ca and 35S in working solutions (1 mL subsamples) and in insect larvae was performed on a Beckman LS6500 Multipurpose Scintillation Counter. For

22 35 Na and SO4 experiments, larvae were rinsed prior to measurement in a rich Na2SO4

22 35 solution (0.35M) containing stable Na and SO4 to displace loosely adsorbed Na and SO4 from the exoskeleton. For Ca experiments, a 0.5M EDTA rinse was used to remove loosely

adsorbed 45Ca (see Poteat and Buchwalter, 2014 for methods). Insect samples were digested with 1mL soluene (2-3mL for larger individuals) for 2-3 days (depending upon the size of the individuals). Larvae were subsequently counted in 20 mL glass scintillation vials with 16 mL of scintillation cocktail (Perkin Elmer Ultima Gold uLLT). For dual-labeled 22Na and 35S experiments, the measurement protocol included verification against single isotope samples and corrections for spill-over narrowing the energy windows. All samples were counted for 3 min and had counting errors and lumex values < 5%.

2.3 Assessing effects of Mg on Ca uptake

To determine if [Mg] influenced Ca uptake, 45Ca uptake rates were compared among field-collected taxa. Information for species tested, average wet weights, # of replicates per treatment, ions manipulated, base water to which ions were added and exposure duration for this and the following experiments are provided in Table 1. Control water was unaltered

ASW (Ca:Mg=2.2:1) and the treatment water was an ASW base with Mg added (as MgSO4) to a final concentration of 2.88 mM (Ca:Mg=1:10). 45Ca was added to 1.5 L bulk solutions for each treatment. A replicate consisted of a single larva in an aerated high-density polyethylene (HDPE) beaker (100 mL) with 80mL of the treatment solution. For this experiment and all other experiments, a Teflon square substrate was added for the larvae and a Parafilm™ cover was placed to reduce evaporative loss.

2.4 Sulfate uptake kinetics in 5 mayfly species

To characterize SO4 uptake variability in mayfly taxa across different SO4 concentrations, radiotracer experiments were performed in lab-reared and field-collected taxa. Nominal SO4 concentrations in solution included a range of values that are typically

observed in surface waters throughout the continental U.S. (0.1, 0.3, 0.7, 1.6, 4.3, 10.5 and

15.6 mM). SO4 was added as Na2SO4 for the field-collected larvae and as an equal molar addition of CaSO4*2H2O and MgSO4*7H2O for N. triangulifer. All salt additions were added to premade ASTM ASW base water. SO4 uptake was determined for only 6 concentrations of

35 SO4 in N. triangulifer based on available larvae. SO4 was added to 400 mL bulk solutions for each SO4 concentration. A replicate consisted of a single larva in an aerated HDPE beaker

(100mL) with 80mL of the treatment solution. All insects were dried overnight at 60°C and weighed on a Sartorius™ analytical scale (model: CPA2P) to the nearest 0.001 mg prior to digestion for liquid scintillation analysis.

2.5 Effects of ionic composition on Na and SO4 uptake

To assess how ionic composition affected both Na and SO4 uptake, we compared uptake rates using different SO4 salts (MgSO4, CaSO4, K2SO4 and Na2SO4) at three different

SO4 concentrations (0.24, 1.45 and 5mM) for each salt. Salt additions were added to a premade modified ASW base water. For the Na2SO4 salt treatment, we only evaluated SO4 uptake at 5mM due to availability of larvae. Larvae were acclimated for at least 48 hrs in

35 22 treatment waters prior to use in radiotracer experiments. SO4 and Na were added to 150 mL bulk solutions for each SO4 concentration and for each salt treatment. A replicate consisted of a single larva in an aerated HDPE beaker (50mL) with 25mL of the treatment solution.

2.6 Influence of [Na] on SO4 and Na uptake

To examine the influence of Na on SO4 uptake, we first examined the effect of Na on

SO4 and Na uptake at two Na concentrations: low (0.69 mM) and high (5 mM) in field-

collected taxa. Two separate batch solutions (350 mL) were produced with a modified ASW base, with one solution containing 0.69 mM Na and the other solution containing 5 mM Na

(as NaCl). Additionally, MgSO4 was added to each treatment at a final concentration of 5

35 22 mM SO4. SO4 and Na were added to each 350 mL batch solution. For H. betteni, only

SO4 uptake was determined. A replicate consisted of a single larva in an aerated HDPE beaker (50mL) with 25mL of the treatment solution.

To further investigate the effect of Na concentration on ion uptake, we quantified SO4 and Na uptake across six Na concentrations at elevated SO4. Six 500 mL solutions were produced at increasing Na concentrations (0.65, 1.4, 2.8, 5.4, 10.9, 21.7 mM NaCl). Base water was modified ASW and MgSO4 was added to each treatment to a final concentration of

35 22 5mM SO4. SO4 and Na were added to each batch solution. A replicate consisted of a single larva in an aerated HDPE beaker (50 mL) with 25mL of the treatment solution.

35 We assessed potential anion influence by comparing SO4 uptake in the presence of equimolar (10.9 mM) NaCl and NaHCO3. Two 150mL batch waters were produced with a modified ASW base water with SO4 additions as MgSO4 (SO4 = 5 mM). The pH was controlled using 0.1M KOH or HNO3 to maintain pH at 7.8 ± 0.02. A replicate consisted of a single larva in an aerated HDPE beaker (50 mL) with 25mL of the treatment solution.

2.7 Can Na ameliorate SO4 toxicity in N. triangulifer?

To assess the influence of Na concentrations on SO4 toxicity in larvae of the mayfly

N. triangulifer were reared from hatching in 150 mL Pyrex™ evaporating dishes containing

125 mL aerated modified ASW until they were 10 days old. Prior to testing, larvae were fed lab-cultured diatom Navicula sp. (see Soucek et al., 2013 for culturing methods) at ambient 66

lab temperature (23° C). Reagent grade NaCl was serially added to six separate 1 L solutions

(final concentrations: 0.69, 1.4, 2.7, 5.4, 10.9 and 21.8 mM Na). Each solution had a modified ASW base water and 20.8 mM SO4 was further added to each treatment solution

(11.6 mM CaSO4*2H2O and 9.2 mM MgSO4*7H2O). Control water was ASW (described above). The treatment waters were aerated for 24 hours prior to the start of the assay. Glass beakers (50 mL) were used for testing and filled with 30 mL treatment water. Each treatment had 5 replicates with 10 individuals placed in each beaker (10 day old larvae). Each replicate was provisioned with 10 µL of suspended Navicula sp. at the 0h, 24h and 48h time points.

All beakers were covered with Parafilm™ to reduce evaporative loss. Beakers were not aerated during the experiment. Each replicate was checked daily to ensure vitality and after

96 hours, the surviving nymphs were counted.

2.8 Data Analysis

Data analysis was performed using GraphPad Prism (v6.07). Unidirectional Ca, Na and SO4 uptake rates were determined by taking the average of individual larvae uptake rates. Unpaired student’s t-tests were performed to determine the effect of Mg additions on

Ca uptake. For [SO4] kinetics experiments in Isonychia sp., Maccaffertium sp., Ephemerella sp. and Drunella sp. and N. triangulifer, nonlinear regression curves were fit using a one-site specific binding equation to estimate Vmax and Km. One-way ANOVA was performed using

Tukey’s multiple comparison test to determine the effect of the cation on SO4 uptake at different SO4 concentrations. Unpaired Student’s t-tests were performed to determine the effect of Na concentration on SO4 uptake. An unpaired Student’s t-test was performed to determine the effect of the anion on SO4 uptake. To evaluate the effect of [Na] on SO4

uptake, a one phase decay equation was used to model the association between SO4 uptake and Na concentration. Na uptake relationships in the same dual-label experiments were fit using a one-site specific binding equation to estimate Vmax and Km. All mass specific rates of

Ca, Na or SO4 uptake are reported on a wet weight basis with the exception that the SO4

Michaelis-Menten type experiments were reported on a dry weight basis. One-way ANOVA was performed using Tukey’s multiple comparison test to determine the effect of Na additions on SO4 toxicity in the mayfly N. triangulifer. Results were significant when P ≤

0.05. Error bars represent ± SEM throughout.

3. Results

3.1 Assessing effects of Mg on Ca uptake

Ca uptake rates were not affected by the addition of Mg in Pteronarcys sp.,

Paraleptophlebia sp. and Acroneuria sp. when the ratio of Ca:Mg was altered from 2.2:1 to

1:10 (Table 2). Ca uptake was reduced by 47% in Isonychia sp. when exposed to elevated

Mg (p=0.0006). When comparing average Ca uptake rates between species in ASW, rates varied from 12.6 ± 2.5 nmol g-1 day-1 in Pteronarcys sp. to 780.5 ± 29.2 nmol g-1 day-1 in

Isonychia sp. The differences in Ca uptake between species may have been heavily influenced by body weight. Species with smaller wet weight averages (Paraleptophlebia sp. and Isonychia sp.) had higher uptake rates than species with larger wet weights (Acroneuria sp. and Pteronarcys sp.) (see Table 2).

3.2 Sulfate uptake kinetics in 5 mayfly species

Radiotracer studies with N. triangulifer, Drunella sp., Ephemerella sp., Isonychia sp. and Maccaffertium sp. demonstrated that SO4 uptake is concentration dependent (Fig. 2). For

-1 -1 example in Maccaffertium sp., SO4 uptake rates ranged from 16.8 ± 2.2 µmol g day (dry

-1 -1 weight) at 0.1 mM SO4 up to 130.7 ± 12.5 µmol g day (dry weight) at 12.7 mM SO4. All species exhibited saturation kinetics across the SO4 concentrations tested and apparent differences existed between maximum uptake rate (Vmax) and affinity (Km) constants when

-1 comparing across species. The Vmax and Km were lowest for Ephemerella sp. (20.3 µmol g d-1 and 0.7 mM, respectively) and highest for Maccaffertium sp. (145.4 µmol g-1 d-1 and 3.9 mM, respectively). If we compare uptake rates at a single SO4 concentration (3.3-4.3 mM),

-1 -1 Maccaffertium sp. had an average SO4 uptake rate of 61.3 µmol g day , whereas Isonychia

-1 -1 sp. had an average SO4 uptake rate of 23.9 µmol g day . These two species had similar average body weights (see above), thus species-specific differences in SO4 influx are only partially driven by body weight and osmoregulatory physiologies among taxa impart strong control over SO4 uptake. Note that different water chemistry used for N. triangulifer radiotracer studies (see methods) preclude direct comparison with the other four species

Drunella sp., Ephemerella sp., Isonychia sp. and Maccaffertium sp. However, it is likely that the small body size of N. triangulifer contributes to the relatively rapid SO4 uptake kinetics

-1 -1 observed (Vmax = 229.3 ± 21.0 µmol g day , Km=4.1 ± 1.1 mM).

3.3 Effects of ionic composition on Na and SO4 uptake

We assessed the influence of cations on sulfate uptake by exposing H. betteni to three

SO4 salts (MgSO4, CaSO4 and K2SO4) at three different SO4 concentrations (0.24, 1.45 or 5 mM SO4). SO4 uptake in H. betteni increased with concentration (Fig. 3). No difference in

uptake occurred at a particular SO4 concentration (0.24, 1.45 or 5 mM SO4) when comparing these three salts to each other. Similar results were seen in P. biloba at 0.24 and 5 mM SO4

(data not shown). However, Na2SO4 significantly inhibited SO4 uptake in H. betteni at 5 mM

SO4 relative to all other salt treatments at equimolar concentrations (p≤0.05). Average SO4 uptake rates at 5 mM SO4 for MgSO4, CaSO4 and K2SO4 were 20.3 ± 4.5, 25.0 ± 4.6 and

-1 -1 24.6 ± 5.7 µmol g day , respectively, whereas average SO4 uptake at 5mM Na2SO4 was 9.6

± 0.31 µmol g-1 day-1.

Because this was a dual labeled experiment, we were able to assess whether Na uptake was influenced by the associated cation of the different SO4 salts (CaSO4, MgSO4 or

K2SO4) at each treatment concentration (data not shown). There was no statistical difference

(unpaired Student’s t-test) in Na uptake rates between any SO4 salt (Ca, Mg or K) for any

SO4 concentration. The pooled average Na uptake rate across all treatments was 3.1 ± 0.2

µmol g-1 day-1. Body weights were not significantly different in any treatment.

3.4 Influence of [Na] on SO4 uptake

To assess the influence of Na concentration on SO4 uptake, we performed radiotracer experiments at elevated SO4 (5 mM) and at low (0.7 mM) or high (5 mM) Na. Elevated Na (5 mM) reduced SO4 uptake compared to low Na treatments (0.7 mM) for P. biloba, H. betteni and E. vitreus (p≤0.05) (Fig. 4). For example, average SO4 uptake in P. biloba at low Na was

16.3 ± 2.5 µmol g-1 day-1 and reduced to 4.9 ± 0.8 µmol g-1 day-1 at high Na (~70% reduction). Similarly, SO4 uptake was inhibited in H. betteni and N. triangulifer along an increasing Na gradient (0.7-21.7 mM Na) (Fig. 5A). For example, average SO4 uptake in N. triangulifer at low Na (0.7 mM) was 52.5 ± 4.5 µmol g-1 day-1 and decreased exponentially to

8.7 ± 0.2 µmol g-1 day-1 at high Na (5 mM). These data were well-fit by a one-phase decay model for both N. triangulifer and H. betteni (R2=0.81 and 0.59, respectively).

Na uptake increased with Na concentration for both H. betteni and N. triangulifer

(Fig. 5B). These results were well fit by Michaelis-Menten model with estimated constants

-1 -1 2 (Vmax and Km) as follows: H. betteni = 21.5 µmol Na g day and 2.6 mM Na (R =0.68), N. triangulifer = 19.9 µmol g-1 day-1 and 3.1 mM Na (R2=0.76). Interestingly, Na uptake did not increase in the elevated Na treatment for both P. biloba and E. vitreus (data not shown).

Cumulative Na uptake (average of both treatments combined) for P. biloba and E. vitreus was 3.4 ± 0.5 µmol g-1 day-1 and 28.6 ± 9.6 µmol g-1 day-1, respectively. Body weights were not significantly different in any treatment for a given species.

To examine the possibility that the anion affected SO4 uptake, we compared SO4 uptake rates in H. betteni at constant SO4 (5 mM) and elevated Na (10.9 mM as NaCl or

NaHCO3). There was no significant difference in SO4 uptake when exposed to either Na salt

-1 -1 (Fig. 5A inset). Average SO4 uptake rates were 6.7 ± 0.4 µmol g day in the presence of 5

-1 -1 mM NaCl and 6.5 ± 1.3 µmol g day in the presence of 5 mM NaHCO3.

3.5 Can Na ameliorate SO4 toxicity in N. triangulifer?

We performed a 96-hr lethality bioassay with N. triangulifer to test the hypothesis that Na could possibly protect against SO4 toxicity. Control survival for N. triangulifer was

100% for all replicates (Fig. 6). The addition of 20.8 mM SO4 effectively reduced survival to

44% ± 8.1% (p≤0.0001) at a Na concentration of 0.7 mM. When we held SO4 constant (20.8 mM) and increased Na (as NaCl), we observed a significant (p=0.04) increase in survivorship from 44 ± 8% at 0.7 mM Na to 73 ± 9% at 10.9 mM Na. Intermediate Na treatments

increased survivorship in a concentration dependent manner, but were not statistically different from the 0.7 mM Na treatment (Fig. 6). However, when Na was increased to 21.8 mM, survivorship significantly decreased (p=0.02) to 16 ± 5% relative to the 0.7 mM Na treatment.

4. Discussion

The emergence of freshwater salinization as a major ecological issue (Cañedo-

Argüelles et al., 2016, 2013; Kaushal et al., 2005) requires that we better understand basic physiological processes across the diversity of freshwater species. One could make the argument that we currently know more about the physiology of the relatively few insect species that tolerate saline habitats (Bradley, 1987; T. J. Bradley et al., 2009; Herbst, 2001;

Herbst et al., 1988) than we do about obligate freshwater species. This study builds upon previous work (Kunz et al., 2013) demonstrating that major ion toxicity to the mayfly N. triangulifer was apparently due to the ionic composition of water rather than specific conductance alone. 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.

To test the hypothesis that elevated Mg could pose physiological problems in aquatic insects by being antagonistic to Ca uptake, we compared Ca uptake at Ca:Mg ratios of 2.2:1 and 1:10. Reference stream Ca:Mg ratios in surface waters can vary greatly due to local geology but typically range from 1:1 to 5:1 (Naddy et al., 2002). However, waters impacted by mountaintop coal mining can have Ca:Mg ratios ranging from 1:1 to 1:4 (Kunz et al.,

2013; Pond et al., 2008). Thus, our 1:10 Ca:Mg ratio represents an extreme situation. We

show that Ca:Mg of 1:10 generally did not affect Ca uptake in three of the four species tested. In Isonychia sp. however, exposure to this extreme ratio did decrease Ca uptake by

47%. Taken together, these results did not compel us to explore Mg antagonism of Ca further. However, it is important to note that these tests were performed only in low ionic strength matrices with relatively low [Ca], so our observations are specific to Ca transport systems operational under such conditions.

We next characterized SO4 uptake kinetics as a function of SO4 concentration in 5 mayfly species. We show that estimates of Vmax and Km vary substantially across species and

SO4 uptake increased with concentration to different extents dependent upon the taxa. There is remarkably little data for us to compare our results to. We were only able to find research on insect SO4 transport in salt-water mosquitoes Aedes campestris and Aedes taeniorhynchus

(Maddrell and Phillips, 1978, 1975). Those species drink/absorb copious amounts of SO4 and their internal hemolymph concentrations remained hypotonic to their surroundings even at highly elevated SO4 concentrations. The authors indicate that these aquatic insects must have an active SO4 excretory system in order to maintain internal hemolymph concentrations. The authors also observed that SO4 uptake increased with concentration, however uptake rates from those studies cannot be compared to current findings because the authors did not explicitly state insect weights, and insect weight has been shown to strongly influence transport rates (Poteat and Buchwalter, 2014; Scheibener et al., 2016). Little is known about sulfate transport in freshwater organisms, and some have even questioned whether sulfate transporters occur in apical ionocytes or chloride cells (Griffith, 2017). Our work provides evidence that apical sulfate transport occurs in mayflies.

We observed concentration dependent sulfate uptake rates in the caddisfly H. betteni and compared uptake of different sulfate salts to determine if major cations influenced sulfate uptake. We tested sulfate uptake with different sulfate salts (MgSO4, CaSO4 and K2SO4) and found no influence of these cations at various SO4 concentrations (0.24, 1.45 or 5mM SO4).

However, we observed a considerable decrease in sulfate uptake when SO4 was delivered as

Na2SO4 (at 5 mM SO4). This unexpected finding prompted us to examine potential Na effects on SO4 transport in other taxa, a stonefly (P. biloba) and a mayfly (E. vitreus), in addition to more experiments with the caddisfly (H. betteni). Results across taxa were remarkably consistent, with increasing Na markedly decreasing SO4 uptake. We found no evidence for cations (Mg, Ca, K) or sulfate affecting Na transport.

The observation that SO4 uptake was decreased by NaSO4, NaCl and NaHCO3 leads us to believe that Na is likely antagonistic to SO4 transport in the aquatic insects we tested.

We have no mechanistic understanding of how this might work. The literature on sulfate transport in aquatic organisms is surprisingly sparse. There is well-documented evidence of an apical membrane Na/SO4 cotransporter located in the renal proximal tubule cells of mammals (Busch et al., 1994; Markovich et al., 1993) and eels (Nakada, 2005). However, studies in flounder (Paralichthys lethostigma) provide no evidence for the occurrence of a

Na/SO4 cotransport system (Renfro and Pritchard, 1982), but there is evidence for a

SO4/anion exchange process (Renfro and Pritchard, 1983). Research on SO4 transport in crustaceans provides evidence for a SO4/Cl exchange process in lobsters (Homarus americanus) that is modulated by pH (Cattey et al., 1992). Previous studies of SO4 transport in aquatic insects are limited to the Maddrell and Phillips papers described above. No studies

to date have yet characterized SO4 transport systems in aquatic insects. It is possible that Na antagonism of SO4 transport occurs in other species, but it is also possible that they have unique SO4 transport systems.

To better characterize the influence of Na on SO4 transport, we performed a dual label experiment in which we held sulfate constant and manipulated Na (as NaCl) at concentrations ranging from 0.7 to 21.7 mM Na in the caddisfly H. betteni and in the mayfly

N. triangulifer. While SO4 transport rates were generally higher in N. triangulifer than in H. betteni, the effect of Na was strong in both species. It was unclear if there was any link between SO4 uptake and toxicity in aquatic insects. Since SO4 uptake was reduced at elevated Na, our hypothesis was that the addition of Na could protect against SO4 toxicity.

To test this hypothesis in N. triangulifer, we used data from Soucek and Dickinson

(2015) to select 20.8 mM SO4 as a concentration that could produce acute (96-hr) toxicity in this species (Soucek and Dickinson, 2015). Our 96-hr bioassay reduced survivorship below

50% at 20.8 mM SO4 (added as CaSO4 and MgSO4). Soucek and Dickinson report a 96-hr

LC50 of 12.8 mM SO4 (added as Na2SO4). We show that adding additional Na to the exposure solutions (as NaCl) decreased toxicity in a concentration dependent manner between 0.7 and 10.9 mM Na. However, when ambient Na reached 21.8 mM, we see an increase in toxicity overall. Interestingly, Soucek and Dickenson report a 96-hr LC50 of 29.7 mM for NaCl (converted to molar units) in this species, suggesting that the toxicity we observed at 20.8 mM SO4 and 21.8 mM NaCl is likely the result of the combined effects of the salts. It should be noted that it is still generally unclear whether salinity toxicity results

from (1) total ionic concentration, (2) the concentration of ions above a certain threshold or

(3) the relative ratio of ions to each other.

In streams affected by mountaintop mining, SO4 concentrations are reported as high as 62 mM (Cormier et al., 2013). Importantly, Na is generally low in these systems; unmined

= 0.1 mM, mined = 0.5 mM (Pond et al., 2008). Using NaSO4 to test SO4 toxicity in insects

(and maybe other taxa too) will likely underestimate toxicity. We note that it is typical to use

Na2SO4 to evaluate SO4 toxicity and a survey of EPA’s ECOTOX database showed that

~75% of SO4 toxicity tests are performed as Na2SO4 for aquatic species. Further, the results for N. triangulifer presented in Kunz et al. (2013) and in this paper should caution us against the use of conductivity alone as a predictor of toxicity. Other studies show that the salt used

(NaCl, MgSO4 or NaHCO3) can alter mayfly drift, abundance and community metabolism to different degrees (Clements and Kotalik, 2016). Field based benchmarks (U.S. EPA, 2011) are based on powerful associations between conductivity and species occurrence. However, it is apparent that ionic composition is tremendously important to species performance, and there is much to be learned about the fundamental osmoregulatory physiology of aquatic organisms.

It is unclear if Na affects SO4 toxicity in other taxa or if this phenomenon is insect specific. Other aspects of ion transport appear to be unique to insects. For example, we know that Cd and Zn can alter Ca uptake in fish, crustaceans, mollusks and crabs as they are believed to share a Ca transport system (Bjerregaard and Depledge, 1994; Hogstrand et al.,

1996; Tan and Wang, 2008; Wright, 1997). Recent research has shown that this does not hold true for aquatic insects. For example, in Ephemerella invaria, Ephemerella catawba,

Hydropsyche sparna and Drunella modesta, Ca uptake was not reduced, but in fact stimulated in the presence of Cd and Zn. These findings were similar to research in the dipteran Chironomus riparius (Gillis and Wood, 2008). Additionally, Cu and Ag can reduce

Na uptake in fish (Morgan et al., 1997) and crustaceans (Bianchini et al., 2002; Bianchini and

Wood, 2003). Recent work has shown that Cu can actually increase Na uptake in aquatic insects and that it takes unrealistically high concentrations of Ag to reduce Na uptake, indicating fundamental physiological differences compared to other aquatic taxa (Scheibener et al., 2016).

This paper is an initial step in understanding osmoregulatory physiology in this diverse faunal group. The complexity of freshwater salinization demands that we develop deeper insight into why many insect taxa are so vulnerable to different forms of freshwater salinization. This knowledge is essential to strengthen the interpretive power of ecological monitoring data and strengthen the scientific basis for establishing regulatory protections on freshwater ecosystems.

Acknowledgements

We would like to thank David Funk for the supply and use of periphyton assemblages. Shane

Scheibener performed the experiments, data analysis, prepared and edited the publication.

Justin Conley assisted in experiments and data analysis, and prepared and edited the publication. David Buchwalter developed the concepts and approach, assisted in experiments and data analysis, and prepared and edited the publication.

Figures

1 0 0 L o w C a :M g , h ig h S O 4:Na

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Table 1. Summary of experimental descriptions including species tested, average weights*, # of replicates per treatment, ions manipulated, base water to which ions were added and the duration of the experiment. Average # of replicates Base Exposure Experiment Species Tested Weight Ions Manipulated per treatment water Duration (mg ± SEM) (2.3) Maccaffertium sp. 8.9 ± 0.7 Paraleptophlebia sp. 3.4 ± 0.2 Ca:Mg = 2.2:1 or 1:10 ASTM Effect of Mg on 5 72 hrs Acroneuria sp. 165.8 ± 20.9 Mg added as MgSO4 ASW Ca uptake Isonychia sp. 9.9 ± 1.0 Isonychia sp. 2.2 ± 0.2* SO as Na SO Maccaffertium sp. 2.4 ± 0.1* 4 2 4 [SO ] = 0.1, 0.3, 0.7, 1.6, 4.3, (2.4) Ephemerella sp. 3.9 ± 0.2* 4 10.5 or 15.6 mM ASTM Sulfate uptake Drunella sp. 1.4 ± 0.1* 5 24 hrs ASW kinetics SO4 as MgSO4 and CaSO4 Neocloeon triangulifer 0.5 ± 0.03* [SO4] = 0.1, 0.3, 0.7, 1.6, 10.5 or 15.6 mM (2.5) SO salts: MgSO , CaSO , 4 4 4 Modified Effect of cation on Hydropsyche betteni 18.1 ± 0.9 5 K2SO4 and Na2SO4 24 hrs ASW SO4 uptake [SO4] = 0.24, 1.45 or 5 mM (2.6) Pteronarcys biloba 25 ± 2.1 SO = 5 mM as MgSO , Modified Influence of [Na] Hydropsyche betteni 19.3 ± 2.4 5 4 4 24 hrs Na = 0.69 or 5 mM as NaCl ASW on SO4 uptake Epeorus vitreus 9.8 ± 1.3

(2.6) Hydropsyche betteni 13.1 ± 0.8 SO4 = 5 mM as MgSO4, Modified Influence of [Na] 5 Na = 0.65, 1.4, 2.8, 5.4, 10.9 24 hrs ASW on SO4 uptake Neocloeon triangulifer 3.3 ± 0.1 or 21.7 mM as NaCl (2.7) SO = 5 mM as MgSO , 4 4 Modified Influence of anion Hydropsyche betteni 14.1 ± 1.8 5 Na = 10.9 mM as NaHCO3, 24 hrs ASW on SO4 uptake Na2SO4 or NaCl

(2.8) SO4 = 20.8 mM as CaSO4 and 5 MgSO combo Modified Influence of Na Neocloeon triangulifer N/A 4 96 hrs (10 larvae/replicate) Na = 0.69, 1.4, 2.7, 5.4, 10.9 ASW on SO4 toxicity or 21.8 mM as NaCl *Indicates dry weights, all others are wet weights

Table 2. Influence of Mg on Ca uptake rates (nmol g-1 day-1 (wet wt.)) at two different Ca:Mg ratios (Ca:Mg = 2.2:1 or 1:10) for Pteronarcys sp., Paraleptophlebia sp., Acroneuria sp. and Isonychia sp. Ca uptake rates are means of 5 individuals ± SEM for each treatment. An asterisk (*) represents a species with significantly different Ca uptake rates (p<0.001). Ca Uptake in ASW Ca Uptake at elevated [Mg] Species (nmol g-1 day-1) (nmol g-1 day-1) (Ca:Mg= 2.2:1) (Ca:Mg = 1:10) Pteronarcys sp. 12.6 ± 2.5 11.4 ± 2.3 Paraleptophlebia sp. 708.2 ± 128.4 620.4 ± 100.9 Acroneuria sp. 43.4 ± 11.8 35.9 ± 11.6 Isonychia sp.* 780.5 ± 29.2 414.2 ± 60.7

1 V K Species max m R2 (µmol g-1 d-1) (mM) Neocloeon triangulifer 229.3±21.0 4.1±1.1 0.89 Drunella sp. 67.0±11.3 6.4±2.6 0.79 2 5 0 Ephemerella sp. 20.3±3.1 0.7±0.4 0.28

) Isonychia sp. 39.1±3.0 2.0±0.5 0.84 .

Maccaffertium sp. 145.4±17.7 3.9±1.2 0.80

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-1 -1 Figure 3. Influence of the cation on SO4 uptake rates (µmol SO4 g d (wet wt.)) for a caddisfly H. betteni. Four different sulfate salts (MgSO4, CaSO4, K2SO4 and Na2SO4) were used at 3 different concentrations. SO4 uptake rates were obtained at only 5 mM for Na2SO4 due to availability of larvae. Values are means ± 1 SEM (n=5, * represents cation treatment differences (p≤0.05, One-way ANOVA)) for each bar.

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S O 4 :N a

Figure 6. Influence of ambient Na on SO4 toxicity in the mayfly N. triangulifer. All SO4 treatments (shaded bars) were held constant at 20.8 mM as a mixture of MgSO4 and CaSO4. Control treatment (white bar) was a modified artificial soft water. Na (as NaCl) was added to treatment groups to adjust the SO4:Na ratio. An apparent protective effect of Na is observed up to a SO4:Na of 2:1, but toxicity is exacerbated at a 1:1 ratio. Bars represent mean ± 1 SEM (n=5 replicates; 10 individuals/replicate, * represents SO4 treatment differences (p≤0.05, One-way ANOVA) relative to the 0.7mM Na treatment).

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