Metamorphosis Affects Metal Concentrations and Isotopic Signatures

Jan 12, 2017 - Metamorphosis Affects Metal Concentrations and Isotopic. Signatures in a Mayfly (Baetis tricaudatus): Implications for the. Aquatic-Ter...
0 downloads 9 Views 992KB Size
Subscriber access provided by HACETTEPE UNIVERSITESI KUTUPHANESI

Article

Metamorphosis affects metal concentrations and isotopic signatures in a mayfly (Baetis tricaudatus): Implications for the aquatic-terrestrial transfer of metals Jeff S. Wesner, David M Walters, Travis S Schmidt, Johanna M. Kraus, Craig A. Stricker, William H. Clements, and Ruth E. Wolf Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05471 • Publication Date (Web): 12 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Environmental Science & Technology

1

Metamorphosis affects metal concentrations and isotopic signatures in a mayfly (Baetis

2

tricaudatus): Implications for the aquatic-terrestrial transfer of metals

3 4

Jeff S. Wesner*,1, David M. Walters2, Travis S. Schmidt3, Johanna M. Kraus2, Craig A. Stricker2,

5

William H. Clements,4 Ruth E. Wolf5

6 7

1

Department of Biology, University of South Dakota, Vermillion, SD 57069, USA

8

2

U. S. Geological Survey, Fort Collins Science Center, Fort Collins, CO 80526, USA

9

3

U. S. Geological Survey, Colorado Water Science Center, Denver, CO 80225, USA

10

4

Department of Fish, Wildlife & Conservation Biology and Graduate Degree Program in

11

Ecology, Colorado State University, Fort Collins, CO 80523, USA

12

5

13

*Corresponding Author: 605-677-6178, Email: [email protected]

14

word count: ~6875

15

Abstract

Perkin Elmer, Inc. San Jose, CA 95134

16

Insect metamorphosis often results in substantial chemical changes that can alter

17

contaminant concentrations and fractionate isotopes. We exposed larval mayflies (Baetis

18

tricaudatus) and their food (periphyton) to an aqueous zinc gradient (3-340 µg Zn/l) and

19

measured zinc concentrations at different stages of metamorphosis: larval, subimago, and imago.

20

We also measured changes in stable isotopes (δ15N and δ13C) in unexposed mayflies. Larval zinc

21

concentrations were positively related to aqueous zinc, increasing 9-fold across the exposure

22

gradient. Adult zinc concentrations were also positively related to aqueous zinc, but were 7-fold

23

lower than larvae. This relationship varied according to adult substage and sex. Tissue

1 ACS Paragon Plus Environment

Environmental Science & Technology

24

concentrations in female imagoes were not related to exposure concentrations, but the converse

25

was true for all other stage-by-sex combinations. Metamorphosis also increased δ15N by ~0.8‰,

26

but not δ13C. Thus, the main effects of metamorphosis on insect chemistry were large declines in

27

zinc concentrations coupled with increased δ15N signatures. For zinc, this change was largely

28

consistent across the aqueous exposure gradient. However, differences among sexes and stages

29

suggest that caution is warranted when using nitrogen isotopes or metal concentrations measured

30

in one insect stage (e.g. larvae) to assess risk to wildlife that feed on subsequent life stages (e.g.

31

adults).

32 33 34

Introduction Aquatic insects are nearly ubiquitous in freshwater ecosystems, where they form key

35

links between aquatic and terrestrial food webs when emerging adults enter terrestrial food webs

36

via consumer1-3 or detrital pathways4. In addition to transferring nutrients and energy to

37

terrestrial ecosystems3, 5, adult aquatic insects may also transfer aquatic-derived contaminants6-11.

38

For example, tetragnathid spiders that fed on emerging chironomids from a lake contaminated

39

with polychlorinated biphenyls (PCBs) had concentrations that exceeded wildlife values for

40

arachnivorous birds12. While there is clear potential for adult aquatic insects to transfer

41

contaminants from water to land, not all are transferred equally. For example, contaminants that

42

biomagnify in food chains (e.g. organochlorines and methyl mercury) tend to be retained during

43

metamorphosis, while those that do not biomagnify (e.g. metals) are typically lost during

44

metamorphosis13-15. Maternal transfer of metals to eggs represents another elimination

45

mechanism. Kim et al.13 found that gravid mayflies (Neocloeon triangulifer) had ~27% higher

46

zinc body burdens than postpartum females, indicating substantial transfer of zinc to eggs.

2 ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33

47

Environmental Science & Technology

There is evidence that the loss of contaminants is dependent on exposure concentrations.

48

In a meta-analysis, Kraus et al.14 found that organisms exposed to high concentrations of

49

contaminants lost proportionally more contaminants during metamorphosis than insects exposed

50

to lower concentrations. However, this result was based on categorical comparisons between

51

“high” versus “low” concentrations, rather than a formal test across an experimental contaminant

52

gradient. Such tests are needed to understand how metal loss in insects varies across

53

environmentally relevant exposure conditions. In addition to contaminant loss, stables isotopes

54

can fractionate during metamorphosis14, 16-18. In particular, δ15N typically increases in adults

55

relative to larvae due to protein metabolism during metamorphosis and the subsequent loss of

56

metabolic waste that is depleted in 15N14, 18. This finding is not universal, however, and δ15N can

57

also be reduced in adults relative to larval insects in some cases16. δ13C, which is used as a diet

58

tracer, can also fractionate during metamorphosis, potentially affecting the estimation of carbon

59

sources in food webs16.

60

Understanding how metamorphosis alters food web tracers like stable isotopes and the

61

relationship between contaminant loss and exposure concentration is critical for predicting the

62

flux of contaminants from water to land and for assessing risks to wildlife that feed on adult

63

aquatic insects14, 19, 20. For example, if the magnitude of contaminant loss is consistent across

64

exposure concentrations (assuming an element is not limiting), then it should be relatively

65

straightforward to predict adult concentrations and the risk to predators of adults if larval

66

concentrations are known, and vice versa. Likewise, isotope fractionation during metamorphosis

67

can alter source information, predictions of trophic position, and estimates of bioaccumulation

68

for organisms that eat larval (e.g. fish) versus adult (e.g. birds) insects14, 16, 18. For example, since

69

δ15N is typically increased in adults versus larvae, the trophic level of a hypothetical organism

3 ACS Paragon Plus Environment

Environmental Science & Technology

70

that eats only adult aquatic insects would be underestimated if it was determined from larval

71

values of δ15N14. Such underestimation could in turn alter estimates of biomagnification based on

72

trophic level14.

73

To experimentally test for concentration-dependent loss of zinc during metamorphosis,

74

we exposed the mayfly Baetis tricaudatus to a gradient of aqueous zinc in the lab. Mayflies are

75

hemimetabolous insects that do not have a pupal form, but rather transform directly from larvae

76

to adult (i.e. incomplete metamorphosis). The first adult life stage, the subimago, is immature,

77

and must complete an additional molt to enter the sexually mature, or imago, adult stage. We

78

measured zinc concentrations in insects at each of these life-stages (larva, subimago, imago) to

79

test the hypothesis that metal loss during metamorphosis changes as a linear function of exposure

80

concentrations. Specifically, we created an exposure gradient of dissolved aqueous zinc that

81

varied over two orders of magnitude at environmentally relevant concentrations. We then tested

82

1) whether tissue concentrations in larvae, subimagos, and imagos were positively related to

83

exposure concentrations, 2) whether the magnitude of zinc lost during metamorphosis varied

84

across the exposure gradient, and 3) whether these relationships differed for male and female

85

insects. We also measured stable nitrogen and carbon isotopes in insects to test the hypothesis

86

that fractionation occurs between larvae, subimagoes, and imagoes.

87 88

Methods

89

Experimental design

90

Three days prior to the start of the experiment, we collected ~800 late-instar B.

91

tricaudatus larvae and ~70 periphyton-covered rocks from Spring Creek in Fort Collins, CO,

92

USA. We identified late instars as mayflies that were relatively large and similarly sized, but

4 ACS Paragon Plus Environment

Page 4 of 33

Page 5 of 33

Environmental Science & Technology

93

without wing pads (mayflies in the final instar have visible wing pads). Aqueous zinc

94

concentration at Spring Creek at the time of collection was 6.2 µg/L and hardness was 147 mg/L.

95

Spring creek is a cold water urban stream with cobble substrates. Larvae and rocks were

96

transported to holding tanks at the Aquatic Experimental Laboratory (AXL) at the Fort Collins

97

Science Center (U.S. Geological Survey (USGS), Fort Collins, CO). Holding tanks consisted of

98

20 1L plastic cups filled with dechlorinated tap water and suspended in a water bath within a

99

Living Stream™ recirculating tank (model LS-900) equipped with a water chiller (model D1-33)

100

supplied by Frigid Units, Inc. (Toledo, OH). Water in the living stream was maintained at a

101

temperature of ~15ºC with a 16:8 light:dark cycle. Mayflies began emerging from the holding

102

tanks after 1-2 days, and we used the emerging individuals and larvae collected on the same days

103

from the holding tanks for stable isotope analyses. Sample sizes for δ15N were 10 larvae, 6

104

subimagoes, and 8 imagoes. Samples sizes for δ13C were 11 larvae, 8 subimagoes, and 9

105

imagoes. The remaining larvae were used in the zinc experiment described below.

106

After three days of acclimation, 40 larvae and 5 rocks were placed in each of 12 replicate

107

flow-through microcosms (Fig. S1). A microcosm consisted of a 15 L bucket submerged in a

108

water bath in two Living Streams (6 buckets per stream) to which we added 5 L of soft water

109

reconstituted from reverse-osmosis filtered water. Flow-through conditions were created by a

110

mesh-covered PCV pipe, which allowed surface water to flow out of the bucket. Clean water

111

(reconstituted as above) was continuously gravity-fed to each bucket from a common head tank

112

above each living stream, creating a flow-through rate of 5 L/day. Stream temperature was held

113

at a constant 16.2 +/- 0.2℃ (mean +/- sd) within the microcosms. Flow was maintained via

114

magnetic pumps attached to the bottom of the bucket with PVC. The pumps only circulated

5 ACS Paragon Plus Environment

Environmental Science & Technology

115

water within each bucket and did not introduce water from the water bath. We fit mesh netting

116

above each bucket to capture emerging insects.

117 118

Water quality

119

On the same day that larvae were added (day 0) we created a zinc gradient by adding

120

dissolved ZnSO4 at nominal concentrations ranging from 0 to 567 µg Zn/L via constant drip

121

from twelve 20L Mariotte bottles positioned 1.5 m above each microcosm (Fig. S1). This

122

gradient was intended to span the range of Zn concentrations typical of nearby streams, while

123

also spanning below and above the U.S. EPA aquatic life criterion for zinc (120 µg Zn/L).

124

Microcosms were randomly assigned a nominal zinc concentration within each living stream,

125

which were treated as a block. Because zinc concentrations were not replicated, we alternated the

126

assignment of each successive concentration among blocks to ensure that blocks contained a

127

similar range of zinc concentrations. We did not include block in the final analyses because

128

boxplots revealed no difference in any response variable between blocks.

129

We sampled aqueous zinc concentrations on days 1, 6, and 18. For each sample, we

130

filtered 50 mL of water through a 0.45 µm Acrodisc syringe filter with Supor membrane into a

131

50mL Falcon™ tube. Water samples were acidified with ultrapure HNO3 and analyzed as

132

described below. We used the mean of these three measurements (day 1, 6, and 18) as the basis

133

for the data analysis below. Temperature and conductivity were measured on days 2 and 6; pH

134

was measured on day 6 (HQ40, Hach Company, Loveland, CO).

135 136

Insect and algal sampling

6 ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33

137

Environmental Science & Technology

Each morning during the experiment, all nets were inspected for recently emerged adults,

138

noting their developmental stage (subimago or imago) and sex at the time of collection. Adults

139

collected on day one were excluded from analysis to ensure that all insects had at least 24 hours

140

of exposure (Fig. S2). We collected between 1 and 4 larvae from each microcosm on days 4 and

141

12. The experiment ended on day 19, following three days with no emergence. On this day, we

142

collected algal samples by scraping algae into a centrifuge tube from at least two rocks per

143

microcosm. All samples were placed in individually labeled, factory-clean centrifuge tubes (2.5

144

mL) and stored frozen at -20°C. All samples were later dried at 60°C for > 48 hours and weighed

145

to the nearest 0.01 mg.

146 147

Stable isotope and metals analysis

148

Samples for stable isotopes were oven dried, ground to a fine powder, and massed into 4

149

x 6 mm tin capsules. Insects were pooled when necessary to achieve a target mass of ~1 mg dry

150

mass, and were analyzed for δ15N and δ13C by continuous flow isotope ratio mass spectrometry

151

using a Carlo Erba NC2500 interfaced to a Micromass Optima mass spectrometer21. Isotopic data

152

were normalized to air and V-PDB with USGS 40 (δ15N = -4.52 ‰, δ13C = -26.24 ‰) and USGS

153

41 (δ15N = 47.57 ‰, δ13C = 37.76 ‰). Quality control and accuracy were assessed using internal

154

standards and primary standards respectively; precision was ±0.2 ‰ for both isotopes.

155

For metals analysis, insect samples were pooled across collection dates to achieve a target

156

mass of 1 mg dry mass per sample. Composites contained a mix of 3-6 insects collected between

157

days 4-12 (larvae) or days 2-10 (subimagoes and imagoes) (Fig. S2), with the exception of one

158

treatment (567 µg/l nominal zinc) in which larvae were only collected on day 4. Larvae from this

159

treatment were not included in the analysis to ensure that all larval samples had similar lengths of

7 ACS Paragon Plus Environment

Environmental Science & Technology

160

exposure. Prior to analysis we identified two clear outliers. One was a larval composite with

161

24,200 µg Zn/g. The other was a subimago composite with 4,270 µg Zn/g. These values were an

162

order of magnitude higher than any other larval or subimago samples, respectively (Fig. S3). We

163

assumed they were contaminated or mislabeled (e.g. a larval sample mislabeled as an adult

164

sample) and excluded them from the analysis. All other samples were used to estimate the

165

average concentration of zinc in insects or algae for each microcosm.

166

We measured zinc concentrations in larval (n = 11), subimago and imago (n = 12 each)

167

samples at the end of the experiment. These estimates represent means of 2-6 composite samples

168

per microcosm. Because most of our samples contained larvae and adults pooled across different

169

dates, we were unable to reliably track concentration changes over time. However, for emerging

170

mayflies, the mean exposure time varied from 2 to 8 days among samples (mean 3.7 days). That

171

is, a composite sample with two imagoes collected on days two and four would represent three

172

days of exposure on average, and a sample of two imagoes collected on days seven and nine

173

would represent a mean of eight days of exposure. However, we found no evidence of an

174

interaction between mean length of exposure and aqueous zinc concentrations in explaining adult

175

tissue concentrations (exposure length x aqueous Zn interaction, mean and (95% credible

176

intervals): 0.0003 (-0.00088, 0.00095, Table S1), indicating that pooling across time likely had

177

minimal effects.

178

Most of our imago composites (46/55 composites) contained either all females or all

179

males. We used these samples to test whether the relationship between zinc tissue concentrations

180

and aqueous zinc differed between sexes for both subimagoes and imagoes. Not all combinations

181

were present in each microcosm, so the analyses are based on the following sample sizes: male

8 ACS Paragon Plus Environment

Page 8 of 33

Page 9 of 33

Environmental Science & Technology

182

subimagoes (n = 8 microcosms), female subimagoes (n = 7), male imagoes (n = 11), and female

183

imagoes (n = 9). Composited samples were acid digested using a microwave digester (MarsXpress; CEM

184 185

Corporation, Matthews, NC, USA) and a mixture of 4mL HNO3, 2 mL H2O2, and 4 mL H2O.

186

The resultant solutions, along with water samples, were analyzed for total zinc by inductively

187

coupled plasma-mass spectrometry (ICP-MS) at the Crustal Geophysics and Geochemical

188

Science Center (USGS, Denver, CO). Measured zinc concentrations were normalized to the dry

189

mass of insects used in each digestion. All zinc tissue concentrations are reported in units of raw

190

or log-transformed µg Zn/g dry mass. Quality analysis and control samples included blanks and

191

Standard Reference Material (50 mg). Blanks for tissues consisted of nitric acid, DI water, and

192

peroxide run through the digestion procedure exclusive of tissue. In order to compare digestion

193

blank values to tissue concentrations directly the blank values were converted to µg/g using a

194

maximum sample weight of 4 mg. Tissue blanks (n = 8) contained 55 ± 43 µg Zn/g (mean ± sd,

195

range: 8-108 µg/g), representing < 3% of mean insect tissue concentrations (~1900 µg/g).

196

Recovery of zinc in standard reference material for metals in biological tissue (DOLT3 –

197

National Research Council Canada) was 125% of reported values. For water analyses, blanks (DI

198

only) were below detection (99% probability that the slope was >0 (Table S2, Fig. 1a; slope (95%

298

CrI): 0.62 (0.42, 0.8)). There was a positive relationship between predicted values generated

299

from the posterior distribution and observed values (r2 = 0.87, Fig. S5b), indicating good model

300

fit.

301

Larval zinc concentrations averaged 3027 µg Zn/g (95% CrI: 2185-4104 µg Zn/g), and

302

were positively related to aqueous zinc concentrations with a 98% probability that the slope was

303

>0 (Table S2, Fig. 1b). Zinc concentrations in adults (subimagoes + imagoes) declined by >7-

304

fold relative to larvae, averaging 417 (CrI: 332-510 µg Zn/g), but were still positively related to

305

aqueous zinc (Fig. 1b). However, the positive relationship between adult zinc and aqueous zinc

306

differed between subimagoes and imagoes. Subimago zinc concentrations were positively related

307

to aqueous zinc (Fig. 2b), with a slope that was nearly identical to that of larvae [subimago slope:

308

0.38 (0.06, 0.69), larva slope: 0.39 (0.06, 0.72); Table S2; Fig. 3)]. In contrast, imago zinc

309

concentrations were unrelated to aqueous zinc [slope: 0.08 (-0.42, 0.69)] with only a 62%

310

probability that the slope was > 0 (Table S2, Fig. 2c).

311

Changes in the relationship between zinc in insects and aqueous zinc during

312

metamorphosis are reflected in the fold-change of zinc between life-stages. Adults had 2-17 fold

313

lower zinc concentrations than larvae, but this change was not related to aqueous zinc

314

concentrations for either larvae vs adults (Fig. 2d) or larvae vs. subimagoes (Fig. 2e), with only a

315

68 or 65% probability, respectively, that the slope was negative. In contrast, the fold-change

316

between subimagoes and imagoes ranged from -2.3 to 2.9 and was negatively related to aqueous

14 ACS Paragon Plus Environment

Page 14 of 33

Page 15 of 33

Environmental Science & Technology

317

zinc with a probability of 99%, indicating that imagoes exposed to higher aqueous zinc

318

continued to lose zinc relative to subimagoes (Fig. 2f).

319

Male and female subimagoes (Fig. 3a and 3b) and male imagoes (Fig. 3c) were positively

320

related to aqueous zinc, and the model slopes (0.36, 0.45, and 0.42, respectively; Table S2) were

321

similar to those for larvae [0.39 (0.12, 0.70), Table S2]. In contrast, there was no relationship

322

between zinc in female imagoes and aqueous zinc [slope: -0.05 (-0.35, 0.25); Table S2, Fig. 3d],

323

with a probability of 99% that the slopes between male and female imagoes differed (Table S2).

324 325 326

Isotopes Both subimagoes and imagoes were higher in δ15N relative to larvae. The mean increase

327

was 0.8‰ (0.3-1.3) for subimagoes and 0.9‰ (0.4-1.3) for imagoes with a >99% probability that

328

these changes were >0 (Table S2; Fig. 4). In contrast, while there was a 92% probability that

329

δ13C differed between larva and subimago, the mean difference between these stages was small

330

and may not be ecologically important [0.5‰ (-0.2, 1.2); Fig. 4]. There was only a 66%

331

probability that δ13C differed between imagoes and subimagoes, with imagoes declining by

332

0.2‰ (0.9, -0.6) (Fig. 4), which is within analytical uncertainty (0.2 per mil).

333 334 335

Discussion The change in zinc concentrations during metamorphosis in B. tricaudatus, and the

336

subsequent relationship between exposure concentrations and tissue concentrations varied as a

337

function of adult stage (subimago vs imago) and sex (male vs female). Despite a 7-fold reduction

338

in zinc concentrations of adults compared to larvae, there was still a positive relationship to

15 ACS Paragon Plus Environment

Environmental Science & Technology

339

aqueous zinc concentrations. This relationship was retained in male subimagoes, male imagoes,

340

and female subimagoes, but not in female imagoes.

341

In field studies (and most laboratory studies), insects are typically characterized only as

342

larvae or adults, rather than by adult substage or sex. In that sense, our finding that adult mayflies

343

overall retained a positive relationship between zinc in tissues and zinc in the water are

344

consistent with findings from a broad field survey in which zinc concentrations in adult mayflies

345

(but not most other insects) were positively correlated to aqueous zinc concentrations9.

346

Moreover, zinc concentrations in riparian tetragnathid spiders were positively correlated with

347

concentrations in their mayfly prey, particularly Baetis spp., in a field study.9 However, Kraus et

348

al.9 did not measure zinc concentrations in larval insects and did not distinguish between

349

subimagoes and imagoes or by sex. While our study shows that distinguishing subimagoes and

350

imagoes in mayflies can reveal important differences in zinc concentrations, it is limited to

351

mayflies, which are the only aquatic insect that molt as adults, while other aquatic insect orders

352

have only a single adult stage28. Thus, the differences between males and females in this study

353

are likely more generalizable to other taxa than differences in adult substage (mayflies only), and

354

deserve further study. For example, our results, along with those of Kim et al.13, suggest that

355

using zinc concentrations of adult aquatic insects as proxies for measuring aquatic contamination

356

is possible, but only after accounting for zinc loss in females. This is supported by the striking

357

similarity in the mean slopes of aqueous concentrations and tissue concentrations for larvae,

358

male subimagoes, female subimagoes, and male imagoes (0.39, 0.36, 0.45, 0.42, respectively;

359

Table S2), compared to female imagoes (-0.05)

360 361

Regardless of whether adult insect concentrations reflect aqueous concentrations, it is clear that adult insects lose the majority of their zinc during metamorphosis from larva to

16 ACS Paragon Plus Environment

Page 16 of 33

Page 17 of 33

Environmental Science & Technology

362

subimago. However, the amount of zinc lost appears variable among taxa, which may simply be

363

attributable to variation in the initial amount contained in larvae. For example, in a meta-

364

analysis, Kraus et al.14 found that adult insects from multiple taxa (which includes results from B.

365

tricaudatus in this study) had ~2-fold lower average zinc concentrations than larvae,

366

considerably smaller than the 7-fold reduction in our study. However, final average zinc

367

concentrations in adults (~450 µg/g) in this study were similar to averages of the five mayfly

368

species in Kraus et al.14 (~380 µg/g). In contrast, larval B. tricaudatus averaged 3632 µg/g in this

369

study, which is more than 2-fold higher than the five other mayfly species analyzed in Kraus et

370

al. (2014b), only one of which contained larval concentrations >1000 µg/g.

371

The mechanism explaining the reduction in metal concentrations between larvae and

372

adults is unclear. One route of metal loss for insects is through the exuvium, which is shed

373

during molts. However, metal loss in the exuvium is often a small fraction (50% of metal loss. However, while the

378

meconium seems to be a likely pathway of metal loss in our study, we are unaware of studies

379

documenting the presence of meconium in aquatic insects. Nor have we observed obvious signs

380

of it during our collections. Resolving the mechanisms behind metal loss during metamorphosis

381

in aquatic insects remains a critical next step in understanding how metals are removed during

382

this critical life-history event.

383 384

In addition to changes in metal concentration during metamorphosis, we also found differences between male and female imagoes, in which males retained a positive relationship

17 ACS Paragon Plus Environment

Environmental Science & Technology

385

between aqueous zinc and zinc tissue concentrations, but females did not. A likely explanation

386

for this difference is that excess zinc in females was transferred to eggs and lost during

387

oviposition. Kim et al.13 found that eggs contained ~27% of the total zinc concentration in

388

imagoes of the parthenogenetic mayfly, Neocloeon triangulifer. We did not collect eggs nor

389

distinguish between pre-partum and post-partum females in our experiment, however, so this

390

mechanism is speculative.

391

Fractionation of stable nitrogen isotopes occurred entirely during the larva to subimago

392

molt. The ~0.8‰ increase in δ15N is consistent with other studies showing higher δ15N in adults

393

relative to larvae16, 18, 30. It is also ecologically relevant, representing ~1/4 of estimated

394

fractionation during trophic transfer (i.e. trophic enrichment factor = 3.4‰31). The lack of

395

additional fractionation between subimagoes and imagoes indicates that nitrogen stable isotope

396

signatures in B. tricaudatus adults, and perhaps other mayflies, can reliably be used regardless of

397

whether subimagoes or imagoes are consumed. When combined with large changes in metal

398

concentration, it is clear that estimates of trophic magnification and exposure risk for predators

399

of adult aquatic insects need to be estimated directly from adults or corrected for changes in

400

nitrogen isotopes and metal concentrations during metamorphosis from larva to adult14, 16.

401

Failure to do so can result in misleading estimates of trophic position and exposure risk for

402

riparian wildlife that rely on aquatic-terrestrial subsidies14.

403

Survival to emergence was low overall, but unrelated to aqueous zinc. This was

404

somewhat surprising, given that Baetis tricaudatus are often among the most sensitive aquatic

405

insects in mesocosms studies of metal exposure32-35, even though they persist or quickly recover

406

in response to metal contamination in field studies36. In a previous experiment with the baetid

407

mayfly Neocloeon triangulifer, Wesner et al.37 found that survival to emergence was reduced at

18 ACS Paragon Plus Environment

Page 18 of 33

Page 19 of 33

Environmental Science & Technology

408

aqueous exposures below the EPA chronic criterion (85 µg Zn/l – hardness adjusted) due to

409

mortality during metamorphosis. In the current experiment, the hardness-adjusted criterion was

410

56 µg Zn/l. Average zinc concentrations in 10 of our 12 treatments in this experiment exceeded

411

that level, so it is possible mortality during metamorphosis affected insects in most of our

412

treatments, though we did not measure this directly.

413

Metamorphosis is a critical period in insect life-cycles that alters survival and insect

414

chemistry14, 37, 38, with strong implications for linked freshwater-terrestrial food webs39. Recent

415

work indicates that metamorphosis can alter survival estimates37, contaminant concentrations14,

416

and isotopic signatures14, 16, 18 of organisms exposed to contaminants. Our study adds to this

417

knowledge by showing that a) the majority of metal loss and stable isotope change in mayflies

418

occurs during the molt from larva to adult, b) the amount of metal lost in this molt is not

419

concentration-dependent, and c) sex-specific differences in metal concentrations can lead to

420

different relationships between zinc in adult insects and exposure concentrations.

421 422

Acknowledgements

423

We thank John Simon for help in the construction and maintenance of the artificial streams,

424

Robert Zuellig for help in locating and identifying B. tricaudatus, and Lauren Hargis for

425

sampling help. Statistical analyses were improved by travel support for JSW to attend a Bayesian

426

workshop at Colorado State University (Training in Bayesian Modeling for Practicing

427

Ecologists, NSF Award #1145200). Primary funding for the experiments was from the U.S.

428

Geological Survey through a research grant to WHC. This research was subjected to USGS

429

review and approved for publication. Any use of trade, product, or firm names is for descriptive

430

purposes only and does not imply endorsement by the U.S. Government.

19 ACS Paragon Plus Environment

Environmental Science & Technology

431

Supporting Information

432

Description of Bayesian analysis, figures with experimental details and statistical results, and a

433

table with full model results. The data used in analyses are available at this link:

434

https://dx.doi.org/10.5066/F72V2D85. This information is available free of charge via the

435

Internet at http://pubs.acs.org.

436 437

References

438

1.

439

invertebrate prey link streams and riparian zones. Freshw. Biol. 2005, 50, (2), 201-220.

440

2.

441

habitats of a tallgrass prairie stream. Am. Midl. Nat 1993, 129, 288-300.

442

3.

443

terrestrial and aquatic food webs. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, (1), 166-170.

444

4.

445

subsidies: experimental addition of aquatic insects increases terrestrial arthropod densities.

446

Ecology 2011, 92, (11), 2063-2072.

447

5.

448

bird by altering aquatic‐insect subsidies. Ecology 2010, 91, (8), 2406-2415.

449

6.

450

emergent insect-mediated flux of methyl mercury across a gradient of contamination. Environ.

451

Sci. Technol. 2013, 47, (3), 1614-1619.

452

7.

453

from an aquatic insect to terrestrial insect predators. PloS ONE 2013, 8, (6), e67817.

Baxter, C. V.; Fausch, K. D.; Carl Saunders, W. Tangled webs: reciprocal flows of

Gray, L. J. Response of insectivorous birds to emerging aquatic insects in riparian

Nakano, S.; Murakami, M. Reciprocal subsidies: dynamic interdependence between

Hoekman, D.; Dreyer, J.; Jackson, R. D.; Townsend, P. A.; Gratton, C. Lake to land

Epanchin, P. N.; Knapp, R. A.; Lawler, S. P. Nonnative trout impact an alpine‐nesting

Tweedy, B. N.; Drenner, R. W.; Chumchal, M. M.; Kennedy, J. H. Effects of fish on

Mogren, C. L.; Walton, W. E.; Parker, D. R.; Trumble, J. T. Trophic transfer of arsenic

20 ACS Paragon Plus Environment

Page 20 of 33

Page 21 of 33

Environmental Science & Technology

454

8.

Cristol, D. A.; Brasso, R. L.; Condon, A. M.; Fovargue, R. E.; Friedman, S. L.; Hallinger,

455

K. K.; Monroe, A. P.; White, A. E. The movement of aquatic mercury through terrestrial food

456

webs. Science 2008, 320, (5874), 335-335.

457

9.

458

Cross‐ecosystem impacts of stream pollution reduce resource and contaminant flux to riparian

459

food webs. Ecol. Appl. 2014, 24, (2), 235-243.

460

10.

461

cross‐system subsidy: Chronic stream pollution controls riparian spider populations. Ecology

462

2011, 92, (9), 1711-1716.

463

11.

464

export organic contaminants to riparian predators. Ecol. Appl. 2008, 18, (8), 1835-1841.

465

12.

466

from Contaminated Sediments to Terrestrial Ecosystems and Potential Risks to Arachnivorous

467

Birds†. Environ. Sci. Technol. 2009, 44, (8), 2849-2856.

468

13.

469

dynamics in the mayfly Centroptilum triangulifer. Ecotoxicology 2012, 21, (8), 2288-2296.

470

14.

471

Metamorphosis alters contaminants and chemical tracers in insects: Implications for food webs.

472

Environ. Sci. Technol. 2014, 48, (18), 10957-10965.

473

15.

474

chironomids (Diptera, Chironomidae). Environ. Pollut. 1989, 62, (1), 73-85.

Kraus, J. M.; Schmidt, T. S.; Walters, D. M.; Wanty, R. B.; Zuellig, R. E.; Wolf, R. E.

Paetzold, A.; Smith, M.; Warren, P. H.; Maltby, L. Environmental impact propagated by

Walters, D. M.; Fritz, K. M.; Otter, R. R. The dark side of subsidies: adult stream insects

Walters, D. M.; Mills, M. A.; Fritz, K. M.; Raikow, D. F. Spider-Mediated Flux of PCBs

Kim, K.; Funk, D.; Buchwalter, D. Dietary (periphyton) and aqueous Zn bioaccumulation

Kraus, J. M.; Walters, D. M.; Wesner, J. S.; Stricker, C. A.; Schmidt, T. S.; Zuellig, R. E.

Timmermans, K. R.; Walker, P. A. The fate of trace metals during the metamorphosis of

21 ACS Paragon Plus Environment

Environmental Science & Technology

475

16.

Alp, M.; Peckarsky, B. L.; Bernasconi, S. M.; Robinson, C. T. Shifts in isotopic

476

signatures of animals with complex life-cycles can complicate conclusions on cross-boundary

477

trophic links. Aquat. Sci. 2013, 75, (4), 595-606.

478

17.

479

level and metamorphosis on discrimination of hydrogen isotopes in a plant-herbivore system.

480

PLoS ONE 2012, 7, (3), e32744.

481

18.

482

diet: an ontogenetic shift in δ15N during insect metamorphosis. Funct. Ecol. 2008, 22, (1), 109-

483

113.

484

19.

485

transport to terrestrial arthropod consumers in a multiuse river system. Environ. Pollut. 2016,

486

213, 53-62.

487

20.

488

tree swallows: implications for trace-element exposure after habitat remediation. Arch. Environ.

489

Contam. Toxicol. 2013, 65, (3), 575-587.

490

21.

491

coupled. delta. 13C and. delta. 15N measurements. Anal. Chem. 1992, 64, (3), 288-291.

492

22.

Team, R. C. R: A language and environment for statistical computing. 2013.

493

23.

McElreath, R. Statistical Rethinking: A Bayesian Course with Examples in R and Stan.

494

CRC Press: Boca Raton, U.S.A. 2016; Vol. 122.

495

24.

496

Princeton University Press: Princeton, U.S.A., 2015.

497

25.

Peters, J. M.; Wolf, N.; Stricker, C. A.; Collier, T. R.; del Rio, C. M. Effects of trophic

Tibbets, T. M.; Wheeless, L. A.; Del Rio, C. M. Isotopic enrichment without change in

Alberts, J. M.; Sullivan, S. M. P. Factors influencing aquatic-to-terrestrial contaminant

Beck, M. L.; Hopkins, W. A.; Jackson, B. P. Spatial and temporal variation in the diet of

Fry, B.; Brand, W.; Mersch, F.; Tholke, K.; Garritt, R. Automated analysis system for

Hobbs, N. T.; Hooten, M. B. Bayesian Models: a Statistical Primer for Ecologists.

Team, S. D. Stan: a C++ library for probability and sampling, version 1.3. 2013.

22 ACS Paragon Plus Environment

Page 22 of 33

Page 23 of 33

Environmental Science & Technology

498

26.

Buerkner, P. brms: Bayesian Regression Models using Stan. R package version 0.6. 0

499

2015.

500

27.

501

P. Bayesian Data Analysis in Ecology Using Linear Models with R, BUGS, and Stan. Academic

502

Press: Cambridge, U.S.A. 2015.

503

28.

Lancaster, J.; Downes, B. J. Aquatic Entomology. OUP Oxford: Oxford, U.K. 2013.

504

29.

Yasunobu, A.; Suzuki, K. T. Excretion of cadmium and change in the relative ratio of

505

iso-cadmium-binding proteins during metamorphosis of fleshfly (Sarcophaga peregrina). Comp.

506

Biochem. Physiol. Part C: Comparative Pharmacology 1984, 78, (2), 315-317.

507

30.

508

and gut analysis. Amer. Midl. Nat. 1994, 131, (1), 146-155.

509

31.

510

assumptions. Ecology 2002, 83, (3), 703-718.

511

32.

512

Ginés, M. a. J. s. The use of field and mesocosm experiments to quantify effects of physical and

513

chemical stressors in mining-contaminated streams. Environ. Sci. Technol. 2016, 50, (14), 7825-

514

7833.

515

33.

516

in stream microcosms: understanding differences between single species tests and field

517

responses. Environ. Sci. Technol. 2013, 47, (13), 7506-7513.

518

34.

519

assemblage from a Rocky Mountain stream in experimental microcosms. J. North Am.

520

Benthological Soc. 1994, 13, (4), 511-523.

Korner-Nievergelt, F.; Roth, T.; von Felten, S.; Guélat, J.; Almasi, B.; Korner-Nievergelt,

Mihuc, T.; Toetz, D. Determination of diets of alpine aquatic insects using stable isotopes

Post, D. M. Using stable isotopes to estimate trophic position: models, methods, and

Cadmus, P.; Clements, W. H.; Williamson, J. L.; Ranville, J. F.; Meyer, J. S.; Gutiérrez

Clements, W. H.; Cadmus, P.; Brinkman, S. F. Responses of aquatic insects to Cu and Zn

Kiffney, P. M.; Clements, W. H. Effects of heavy metals on a macroinvertebrate

23 ACS Paragon Plus Environment

Environmental Science & Technology

521

35.

Mebane, C. A.; Schmidt, T. S.; Balistrieri, L. S. Larval aquatic insect responses to

522

cadmium and zinc in experimental streams. Environ. Toxicol. Chem. 2016.

523

36.

524

stream ecosystem. Elementa: Science of the Anthropocene 2015, 3, (1), 000042.

525

37.

526

Metamorphosis enhances the effects of metal exposure on the mayfly, Centroptilum triangulifer.

527

Environ. Sci. Technol. 2014, 48, (17), 10415-10422.

528

38.

529

between larval stress, adult asymmetry and individual quality. Funct. Ecol. 2008, 22, (2), 271-

530

277.

531

39.

532

disproportionately to larval density along a stream metals gradient. Environ. Sci. Technol. 2013,

533

47, (15), 8784-8792.

Mebane, C. A.; Eakins, R. J.; Fraser, B. G.; Adams, W. J. Recovery of a mining-damaged

Wesner, J. S.; Kraus, J. M.; Schmidt, T. S.; Walters, D. M.; Clements, W. H.

Campero, M.; De Block, M.; Ollevier, F.; Stoks, R. Metamorphosis offsets the link

Schmidt, T. S.; Kraus, J. M.; Walters, D. M.; Wanty, R. B. Emergence flux declines

534 535

24 ACS Paragon Plus Environment

Page 24 of 33

Page 25 of 33

Environmental Science & Technology

536

Table 1. Water quality in each microcosm. Mean Zn, Ca, Mg, and Hardenss are averaged over three measurements (Day 1, Day 6, and Day 18), conductivity and temperature (°C) are averaged over two measurements, and pH is a single measurement on day 6. Nominal Mean Zn Zn (µg/l) (µg/l) 0 52 103 155 206 258 309 361 412 464 515 567

3 24 59 78 75 141 100 199 340 248 264 264

Day 1 (µg/l)

Day 6 (µg/l)

Day 18 (µg/l)

3 17 13 38 27 32 29 8 135 38 102 60

2 20 57 89 64 141 117 257 306 279 300 282

3 34 106 108 135 251 155 332 579 426 390 450

pH Cond Temp.

6.7 6.7 6.7 6.65 6.64 6.62 6.59 6.56 6.6 6.69 6.58 6.57

150 160 154 160 153 160 164 147 149 158 160 153

16 16.3 16.2 16.5 16 16.2 16.4 16 15.9 16.5 16.7 16.3

537

25 ACS Paragon Plus Environment

Ca Mg Hardness (mg/l) (mg/l) (mg/L) 7.6 8.1 7.5 7.8 8.3 6.1 7.6 8.8 8.7 8.8 8.9 8.1

5.5 5.4 5.5 5.4 5.3 5.4 5.6 5.6 5.9 5.5 5.5 5.7

41.5 45.9 42.7 44.8 42.6 44.7 44.6 37.4 41.3 42.2 43.5 41.7

Environmental Science & Technology

538

Figure Captions

539

Figure 1. Relationship between aqueous zinc exposure (x-axis) and zinc tissue concentrations (y-

540

axis) in a) algae and b) larval and adult insects. Circles are raw data. Lines show the regression

541

line (straight line) and 95% credible intervals (curved lines). Pr is the probability that the slope is

542

greater than 0. Values for the mean and 95% credible intervals of the slope are as follows: a)

543

0.61 (0.42, 0.8), b) Larvae: 0.39 (0.02, 0.77), Adults: 0.25 (-0.12, 0.59).

544 545

Figure 2. Relationship between aqueous zinc exposure (x-axis) and zinc tissue concentrations

546

(top row) for B. tricaudatus sampled as a) larvae, b) subimagoes, and c) imagoes. The bottom

547

row shows the fold change in concentrations between d) larvae and all adults (subimagoes +

548

imagoes), e) larvae and only subimagoes, and f) subimagoes and imagoes. Circles are raw data.

549

Lines show the regression line (straight line) and 95% credible intervals (curved lines). Pr is the

550

probability that the slope is greater than 0 (for zinc concentration) or less than 0 (for fold

551

change). Values for the mean and 95% credible intervals of the slope are as follows: a) 0.39

552

(0.06, 0.72), b) 0.38 (0.06, 0.69), c) 0.08 (-0.42, 0.69), d) -0.75 (-4.29, 2.8), e) 0.58 (-2.85, 4.22),

553

f) -1.16 (-2.13, -0.21).

554 555

Figure 3. Relationship between aqueous zinc exposure (x-axis) and zinc tissue concentrations (y-

556

axis) for B. tricaudatus males and females sampled as subimagoes or imagoes. Circles are raw

557

data. Lines show the regression line (straight line) and 95% credible intervals (curved lines). Pr

558

is the probability that the slope is greater than 0. Values for the mean and 95% credible intervals

559

of the slope are as follows: a) 0.36 (0, 0.71), b) 0.45 (-0.07, 0.97), c) 0.42 (0.12, 0.7), d) -0.05, (-

560

0.35, 0.25).

26 ACS Paragon Plus Environment

Page 26 of 33

Page 27 of 33

Environmental Science & Technology

561 562

Figure 4. Fractionation of δ15N and δ13C during metamorphosis in the mayfly, Baetis tricaudatus.

563

Open circles are the raw values of individual insects. Black circles and error bars are the

564

posterior mean and 95% credible intervals.

565 566 567

27 ACS Paragon Plus Environment

Environmental Science & Technology

Figure 1 a) Algae, Pr = 1

b)

Zinc concentration (µg Zn/g dry mass)

568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589

Aqueous zinc (µg/L)

28 ACS Paragon Plus Environment

Larvae , Pr = 0.98 Adults, Pr = 0.92

Page 28 of 33

Page 29 of 33

Environmental Science & Technology

Pr = 0.99

b)

Pr = 0.99

c)

Pr = 0.62

d)

Pr = 0.68

e)

Pr = 0.65

f)

Pr = 0.99

Fold-change in tissue zinc concentrations

Zinc concentration (µg Zn/g dry mass)

a)

Aqueous zinc (µg/L) 590 591

Figure 2

29 ACS Paragon Plus Environment

Environmental Science & Technology

Figure 3

a) male subimagoes

Zinc concentration (µg Zn/g dry mass)

592 593 594 595 596 597 598 599 600 601 602 603 604 605

b) female subimagoes

Pr = 0.97

Pr = 0.96

c) male imagoes

d) female imagoes

Pr = 0.36

Pr = 1

Aqueous zinc (µg/L)

30 ACS Paragon Plus Environment

Page 30 of 33

Page 31 of 33

Figure 4 12

-30 -31

11 δ13C

-32 δ15N

606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626

Environmental Science & Technology

10

-33 9

8

-34 -35

0.5 larvae 1 1.5subimagoes 2 2.5 imagoes 3 3.5

0.5 larvae 1 1.5subimagoes 2 2.5 imagoes 3 3.5

627

31 ACS Paragon Plus Environment

Environmental Science & Technology

166x141mm (150 x 150 DPI)

ACS Paragon Plus Environment

Page 32 of 33

Page 33 of 33

Environmental Science & Technology

c)

b)

a)

10 R² = 0.1306

9 8.5 8 7.5

e)

10 R² = 0.8425

fold-change larvae to adultsobserved

ln (µg Zn/g)observed

7

9 8 7 6 5

-12 -17 -11

-9 fold-change larvae to adultspredicted

R² = 0.4286 -2

0

2

larvae

subimagos

-7

13Cobserved

6 5

3

7 ln (µg Zn/g)predicted

R² = 0.0149

-7 -12 -17

larvae

subimagos

-7

imagos

11 10 9 R² = 0.4141

8 10

5 5.5 6 6.5 ln (µg Zn/g)predicted

-9 -8 fold-change larvae to subimagospredicted

12

R² = 0.4017

5

9

-2

i)

7

imagos

-31 -32 -33 -34

R² = 0.0332 -35 -32.2 -32 13Cpredicted

7

-10

female imagos male imagos female subimagos male subimagos

9

fold-change subimago to imagopredicted

-30

-7

7

8

f)

-2

h)

4 3 2 1 0 -1 -2 -3

9

10

3 R² = 0.0276

9

ln (µg Zn/g)observed

fold-change subimago to imagoobserved

g)

7 ln (µg Zn/g)predicted

8 9 ln (µg Zn/g)predicted

adults

R² = 0.8425

5

16

fold-change larvae to subimagosobserved

13 14 15 survivalpredicted

5

j)

R² = 0.868

7

5 12

ln (µg Zn/g)observed

15

9.5

10

15Nobserved

ln (µg Zn/g)observed

survivalobserved

20

d)

larvae

10

25

-31.8

ACS Paragon Plus Environment

10.5 15Npredicted

11