Metamorphosis Affects Metal Concentrations and Isotopic Signatures

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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

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Environmental Science & Technology

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

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

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

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William H. Clements,4 Ruth E. Wolf5

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

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

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

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

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

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

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word count: ~6875

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Abstract

Perkin Elmer, Inc. San Jose, CA 95134

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Insect metamorphosis often results in substantial chemical changes that can alter

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

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

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

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

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

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

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

1 ACS Paragon Plus Environment


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

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

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

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

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

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

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

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adults).

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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contaminants lost proportionally more contaminants during metamorphosis than insects exposed

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

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

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

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

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

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

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

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

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

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sources in food webs16.

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

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

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

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

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

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

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

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

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

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



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

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

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trophic level14.

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

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

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

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

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

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

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

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

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

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

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

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

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

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that fractionation occurs between larvae, subimagoes, and imagoes.

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Methods

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Experimental design

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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above each bucket to capture emerging insects.

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Water quality

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

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

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

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

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

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

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

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

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

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boxplots revealed no difference in any response variable between blocks.

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

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

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

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

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

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

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Insect and algal sampling


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

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

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

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

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

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

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

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

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to the nearest 0.01 mg.

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Stable isotope and metals analysis

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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minimal effects.

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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fit.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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subsequent relationship between exposure concentrations and tissue concentrations varied as a

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

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



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

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and female subimagoes, but not in female imagoes.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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


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

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

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

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

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

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

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

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

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

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The mechanism explaining the reduction in metal concentrations between larvae and

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

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

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

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

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

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

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this critical life-history event.

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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



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

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

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

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

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

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mechanism is speculative.

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

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

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

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

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additional fractionation between subimagoes and imagoes indicates that nitrogen stable isotope

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

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

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

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

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

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

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riparian wildlife that rely on aquatic-terrestrial subsidies14.

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

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

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

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

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


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

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

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

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

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treatments, though we did not measure this directly.

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

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

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

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

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

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

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

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different relationships between zinc in adult insects and exposure concentrations.

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Acknowledgements

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

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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.



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

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

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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

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

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

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528

38.

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

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

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534 535


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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


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


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).


Page 26 of 33

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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



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)


Larvae , Pr = 0.98 Adults, Pr = 0.92

Page 28 of 33

Page 29 of 33


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


a)

Aqueous zinc (µg/L) 590 591

Figure 2



Figure 3

a) male subimagoes


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)


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


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



166x141mm (150 x 150 DPI)

ACS Paragon Plus Environment

Page 32 of 33

Page 33 of 33


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


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


15

9.5

10

15Nobserved


survivalobserved

20

d)

larvae

10

25

-31.8

ACS Paragon Plus Environment

10.5 15Npredicted

11

Metamorphosis Affects Metal Concentrations and Isotopic Signatures

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