Genetic Characterization of Periphyton Communities Associated with

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Genetic characterization of periphyton communities associated with selenium bioconcentration and trophic transfer in a simple food chain Vanessa Friesen, Lorne E. Doig, Blue E. Markwart, Monique Haakensen, Emily Tissier, and Karsten Liber Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on June 3, 2017

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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.

Enrichment Factor ( )

Trophic Transfer Factor

Environmental Science & Technology

Relative Taxonomic Composition

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Biofilm type

Biofilm type Selenite

Selenized Algae

Snail

ACS Paragon Plus Environment


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Genetic characterization of periphyton communities associated with selenium bioconcentration and trophic transfer in a simple food chain

3

Authors

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Vanessa Friesen1, Lorne E. Doig2,3, Blue E. Markwart2, Monique Haakensen1, Emily Tissier1,

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Karsten Liber*2,4

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1

Contango Strategies Limited, 15-410 Downey Road, Saskatoon, Saskatchewan

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2

Toxicology Center, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, S7N 5B3,

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Canada

9

3.

Global Institute for Water Security, University of Saskatchewan, 11 Innovation Boulevard,

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Saskatoon, SK, S7N 3H5, Canada

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4

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*Corresponding Author: [email protected]

Institute of Loess Plateau, Shanxi University, Taiyuan, Shanxi, PR China

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Abstract A major source of uncertainty in predicting selenium (Se) distribution in aquatic food

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webs lies in the enrichment factor (EF), the ratio of Se bioconcentration in primary

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producers and microorganisms relative to the concentration of Se in the surrounding water.

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It has been well demonstrated that EFs can vary dramatically among individual algal taxa,

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but data are lacking regarding the influence of periphyton community composition on EFs

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for a given geochemical form of Se. Therefore, the goals of this study were first to assess

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whether different periphyton communities could be established in aquaria with the same

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starting inoculum using different light and nutrient regimes, and second, to determine if the 1


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periphyton assemblage composition influences the uptake of waterborne Se (as selenite)

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and subsequent Se transfer to a model macroinvertebrate primary consumer. Periphyton

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biofilms were grown in aquaria containing filtered pond water (from Saskatoon, SK) spiked

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with approximately 20 µg Se/L (mean measured concentration 21.0 ± 1.2 µg Se/L), added

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as selenite. Five different light and nutrient regimes were applied to the aquaria (three

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replicates per treatment) to influence microbial community development. After six weeks of

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biofilm maturation, 40 to 80 immature cultured snails (Stagnicola elodes) were added to

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each aquarium. The bacterial and algal members of the periphyton community were

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characterized by targeted metagenomic analyses before and after addition of snails to

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ensure the snails themselves did not significantly alter the microbial community. Samples

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were collected for Se analysis of water, periphyton, and whole-body snail. The nutrient and

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light treatments resulted in substantially different compositions of the periphytic biofilms,

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with each being relatively consistent across replicates and throughout the study. Although

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the aqueous concentration of dissolved Se administered to treatments was constant, uptake

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by the different periphytic biofilms differed significantly. Both the low-light (61.8 ± 12.1 µg

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Se/g d.w.) and high-light (30.5 ± 4.7 µg Se/g d.w.) biofilms, which were found to have high

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proportions of Cyanobacteria, contained statistically higher concentrations of Se relative to

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the other treatments. Furthermore, the concentration of Se in bulk periphyton was

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predictive of Se bioaccumulation in grazing snails, but as an inverse relationship, opposite

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to expectations. The trophic transfer factor was inversely correlated with periphyton

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enrichment factor (r = -0.841). A number of different bacterial and algal taxa were

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correlated (either positively or negatively) with Se accumulation in periphyton biofilm and

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snails. Recent advancements in genetic methods make it possible to conduct detailed

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characterization of periphyton assemblages and begin to understand the influence that

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periphyton composition has on Se biodynamics in aquatic systems.

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Key words

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Periphyton, cyanobacteria, selenium, bioconcentration, enrichment factor, trophic

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transfer, metagenomics

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Introduction

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An essential micronutrient, selenium (Se) is also one of the most hazardous trace

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elements to waterfowl, fish, and egg laying aquatic vertebrates, with maternal transfer of Se

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causing deformities at dietary exposures of only 7-30 times above nutritionally required

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levels. 1,2 Reproductive failure and teratogenicity in fish and waterfowl are the main

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ecological concerns associated with environmental Se contamination. 3,4 Selenium is mainly

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accumulated in aquatic primary consumers through their diet 5, which is derived from the

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base of the food web (e.g., periphyton, biofilm, microbes, detritus). However, a major source

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of uncertainty in predicting Se distribution in aquatic food webs lies in the enrichment

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factor 6,7 (EF), the first step of the food web that involves Se bioconcentration from the

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abiotic environment (water) (e.g., to microorganisms and primary producers such as

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periphyton). EFs are known to range several orders of magnitude in planktonic marine

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algae and periphyton from a variety of ecosystems.6,8

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The composition of algal assemblages, including periphyton, varies greatly due to

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factors such as selective grazing pressure, water chemistry, flow rates, lighting and

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temperature.9 However, previous studies assessing the composition of primary producer 3


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communities have typically relied on microscopy to identify and count various taxa to a

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degree of resolution limited by visual differences, and cannot differentiate the microbial

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members of the community. Therefore, studies investigating Se bioconcentration in aquatic

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primary producers (e.g., algae) have typically involved either pure cultures in a laboratory

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environment, or limited investigations of the algal-microbial assemblage composition (e.g.,

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6,8,10,11,12 and

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microbial taxa associated with Se accumulation into periphyton through denaturing

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gradient gel electrophoresis 13; however, the resolution of the microbial population was

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limited due to the technique, and the 16S ribosomal RNA gene only targeting bacterial taxa.

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reviewed in 9). Genetic markers have previously been used to investigate

Recent developments in metagenomic analyses now provide powerful tools to

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determine the composition of algal and biofilm communities from different environments.

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Targeted metagenomic DNA sequencing can delineate the distribution of organisms in a

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sample by amplifying and genetically comparing sequences from a marker gene. Organisms

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can then be identified (named) if similar sequences are available in a reference database. A

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benefit of this technology is that regardless of their presence as named organisms in

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reference databases, DNA sequences allow the comparison of organisms to one another

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both within and across samples. The relative abundance of algae and bacteria from

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environmental samples can therefore be characterized and compared directly from genetic

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information, which also allows comparison to future samples (or reanalysis of communities

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as databases for naming become more robust).

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The goal of this study was to assess whether the composition of organisms within

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periphytic biofilms influences the enrichment of waterborne selenite to such biofilms, and

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subsequently influences the transfer of Se to macroinvertebrate primary consumers. This 4



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was assessed through a simple food chain experiment involving different

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periphyton/biofilm communities and the herbivorous freshwater snail, Stagnicola elodes, in

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combination with targeted metagenomic analyses. Both eukaryotic algae and cyanobacteria

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were targeted via plastid 23S rRNA genes 14,15, a marker that has conserved regions

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between both kingdoms and can therefore be used to compare the composition of

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periphyton among samples.

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Materials and Methods

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Setup

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Periphyton culture and subsequent snail exposures were conducted in a temperature controlled greenhouse ( average [± standard error] water temperature of 23.7 ± 0.4°C;

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12h:12h light:dark cycle). Three replicate aquaria were used per treatment. Aquaria (19-L

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volume) were established as flow-through systems fed from the same bulk-batch of influent

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(1000-L volume) containing a nominal concentration of 20 µg Se/L (measured

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concentration of influent was 21.0 ± 1.2 µg Se/L) as sodium selenite (Na2SeO3, Sigma-

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Aldrich St. Louis, MO, USA). Basic water chemistry variables for bulk influent is summarized

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in Table S1. The cultured periphyton communities were exposed to this influent for the

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duration of both the establishment phase and the 21-d snail exposure. Selenite was chosen

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because it is one common form of Se found in aquatic receiving environments and it is

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readily bioavailable.9,16

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The average flow rate through each aquarium over the course of the experiment was 2.08 ± 0.30 L/day, with no significant difference among treatments (p < 0.05). Each 5


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aquarium had a separate inflow, constant aeration, a glass cover to limit evaporation, and an

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outflow covered with plastic mesh to prevent the snails from escaping. A 15-L water level

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was constant in each aquarium, maintained by the level of the outflow spout. Basic water

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chemistry (conductivity, hardness, and alkalinity), temperature, pH and DO concentration

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were measured on days 0 and 21 in three replicates of each treatment. These data are

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summarized in Table S2 in Supporting Information (SI).

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Establishing periphyton communities

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Periphyton were grown in aquaria under five different light and nutrient regimes prior

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to the addition of S. elodes. Immediately upon set-up, all aquaria were augmented with 2.5

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mL (0.25 g d.w.) of Nutrafin® fish food slurry (Hagen, West Yorkshire, UK) providing

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nutrients for algal growth. Each aquarium received a 100 mL inoculum of pond water

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containing local algae, filtered with Nytex mesh (35µm) to remove zooplankton. The

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inoculum was collected from ponds on the University of Saskatchewan grounds (Saskatoon,

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SK, Canada). Three weeks after setup, an additional 5 mL (0.5 g d.w.) of Nutrafin® slurry

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were added to each aquarium to provide further nutrients to the biofilms. The aquaria were

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exposed to their respective treatments during the establishment phase (described in the

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following section). Periphyton communities were allowed to establish for 6 weeks prior to

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addition of S. elodes, to allow sufficient biomass to develop to support their growth.

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Treatments

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Different nutrient content and light exposure conditions were used to encourage the

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growth of different periphyton communities in the different treatments. Treatments

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included: 6



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1. Low light (LL) treatment: Aquaria covered in shade-cloth, limiting light intensity

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below 5,000 lux. This treatment was maintained throughout the periphyton

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establishment and snail exposure periods.

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2. High light (HL) treatment: Aquaria exposed to ambient greenhouse lighting, which

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exceeded the range of the light meter (20,000 lux), throughout the entire periphyton

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establishment and snail exposure periods.

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3. Nitrogen amended (N) treatment: 15 mL of slow-release urea fertilizer (polymer

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coated urea 44-0-0, Agrium Inc., CA, USA) per aquarium. The nitrogen fertilizer was

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replaced when the majority of the slow-release beads had visibly burst (approximately

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2 weeks). Nutrient addition was terminated six days prior to the addition of snails.

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4. Nitrogen and silica amended (NSi) treatment: 0.9 g of dissolved sodium metasilicate

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nonahydrate was added per aquarium upon set-up and on day 20 of the periphyton

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establishment period. Additionally, nitrogen was added three times as described for the

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N treatment. Nutrient addition was terminated six days prior to the addition of snails.

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5. Nitrogen, phosphorus and potassium amended (NPK) treatment: Water-soluble NPK

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fertilizer (5 g) was added per aquarium upon set-up. From weeks two through four, an

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additional 0.5 g was added twice weekly. This treatment resulted in an abundance of

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planktonic algae; therefore, nutrient addition was stopped 12 days prior to addition of

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snails, making it possible to see into the aquaria for future snail sampling.

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6. Control: On day 0 of the snail exposure, three aquaria were set up as a control, with

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aeration and a glass cover. Control snails were fed the same food-source (Aquasmart, 7


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Diamond V Mills, Cedar Rapids, IA, USA) used for culturing. The Se content of the culture

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food-source was below the limit of quantification (1, with amphipods representing the exception (approximately 0.9). No studies

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were found that sampled and characterized co-occurring snails and periphyton in

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contaminated systems either in the field or under laboratory conditions. Our data suggest

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that snails have Se TTFs that extend the lower range of the reported values for freshwater

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invertebrates.

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Periphyton assemblage composition

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Despite having the same starting inoculum, periphyton communities were successfully

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developed into different assemblages. Furthermore, significant differences among

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treatments in Se accumulation, both in periphyton and in snails, suggested that factors

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other than total dissolved Se concentration in the water influence Se accumulation in

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periphyton and the subsequent transfer to a higher trophic level, including composition of

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the periphyton and water chemistry (e.g., phosphate). 15


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MRPP analyses of the periphyton composition confirmed that significantly different

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communities were obtained for each treatment (MRPP: A = 0.4981, p = 0.001). Ordination

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(Figure 5) demonstrated that the LL, N, and NPK periphyton communities were relatively

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stable from day 0 to day 21, however, the HL and NSi treatments had noticeable but

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consistent shifts in assemblage composition between day 0 and day 21 across all replicates

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within each treatment (Figure 5). Therefore, the average assemblage composition from day

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0 and day 21 for each treatment (unless otherwise noted), was taken to best represent the

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periphyton assemblage during the entire exposure period.

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Targeted metagenomic analyses provide the distribution as a relative abundance (or

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proportion/percentage) of the community. The periphyton populations of the HL, LL and N

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treatments were similar in that they were comprised (range; mean) mostly of cyanobacteria

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(89.91-99.97%; mean 98.65%) with a low relative abundance of eukaryotic algae (0.03-

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11.09%; mean 1.35%). In contrast, the NSi assemblage had a higher relative abundance of

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eukaryotic algae (33.07-99.69%; mean 72.48%), compared to cyanobacteria (0.31-66.93%;

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mean 27.52%). The NPK treatment resulted in an assemblage with a mean of 27.74% (5.74-

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68.85%) eukaryotic algae and 72.26% (31.15-94.26%) cyanobacteria. These general

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distributions can be seen in Figure 5, where the left side of the plot shows each sample as a

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single point (i.e., comparison of population in each sample, where points that are closer

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together indicate more similar communities). The right plot shows the composition based

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on kingdom level classification, to demonstrate the dominance of bacterial or eukaryotic

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algae in samples. This shows that a single starting inoculum can generate distinctly different

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periphyton community compositions with simple modification of environmental conditions,

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such as light and nutrients. This type of shift in community is also expected to happen in

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natural environments where environmental conditions change significantly.

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Operational taxonomic units (OTUs) are groupings of organisms, in this case, based on a

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97% identity threshold for the plastid 23S rRNA region sequenced (Figure 6, Table 1). The

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plastid 23S rRNA gene was used for this study as it simultaneously detects photosynthetic

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eukaryotic and bacterial organisms, allowing for relative abundances of OTUs to be

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compared within and across samples. However, the plastid 23S rRNA gene is less studied

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than other target regions that have been used for targeted metagenomic studies (e.g., 16S or

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18S rRNA genes), and so identifying 23S rRNA genes to lower taxonomic levels (e.g., genus,

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species) is currently not possible for all 23S rRNA sequences encountered. Therefore, some

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OTUs were classified only to “Cyanobacteria”, while others were classified to species,

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depending on the contents of the reference database (and the accuracy of classifications in

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such databases). The following text focuses on OTUs (rather than grouping by taxonomic

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classification such as genus or family, for example) and their potential role in Se

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bioconcentration. Where names are included, they should be interpreted with caution as

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they may be revised or refined in the future as databases are improved, and further

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targeted metagenomic studies are performed with plastid 23S rRNA genes.

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Periphyton composition and selenium biodynamics

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Factors other than assemblage composition can influence the enrichment of trace

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elements in primary producers. 35 For example, bloom dilution can occur in aquatic systems

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having a finite amount of trace element available for uptake. However, this present study

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had a surplus of selenite flowing through the system at all times, and would not be affected

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by growth dilution as could occur in a lentic environment with limited concentrations of a 17


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trace element. Additionally, a study assessing selenite uptake at environmentally relevant

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Se concentrations into 14 marine algal species found that Se uptake was generally constant

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for algae in the log phase of growth for the majority of species. 8 However, algal Se content

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varied for some species with phase of growth, with Se content increasing as cells

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approached the stationary phase. Similarly, Se content in the marine phytoplankton

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Isochrysis galbana (Haptophyta) was found to be inversely related to algal growth rate. 29

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The periphytic biofilms cultured in the present study were grown for six weeks prior to the

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introduction of snails. Therefore, it was likely that all biofilms were past the log-phase of

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growth and were minimally influenced by changes in growth rate (and hence possible

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changes to Se content associated with a change in growth rate) during the subsequent 21

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days of snail exposure phase. The influence of growth phase on Se content in periphyton

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biofilms was not part of this study. Nevertheless, it is recommended that the influence of

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growth phase on Se uptake in periphyton (with infinite Se resupply to avoid bloom dilution)

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be more thoroughly investigated to better address uncertainty surrounding this potential

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modifying factor.

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Various water quality characteristics have been assessed for their influence on Se

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uptake in Chlamydomonas reinhardtii.36 For example, selenite uptake was significantly

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reduced with increasing phosphate concentration. Therefore, our NPK treatment could

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possibly be confounded by the two additions of phosphate during periphyton culture. The

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lower concentrations of Se in periphyton in this treatment could be a partial artifact of

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phosphate addition. However, the addition of phosphate to this treatment ceased prior to

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the 21-d snail exposure period and Se concentration in this treatment did not increase

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during the 21-d period. The absence of an increase of Se content in the periphyton during 18



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the exposure phase suggests that phosphate addition during periphyton incubation did not

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modify Se content in the periphyton. Nevertheless, the phosphate treatment (NPK) was

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removed from the final correlation of EFs and TTFs (Figure 4), and instead shown for

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comparative purpose only, although its inclusion did not substantially change the

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significance of the inverse relationship shown.

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There were significant positive and negative correlations with the relative abundance of

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bacterial or eukaryotic algae to the EF, respectively, with the opposite direction of

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correlation present with the TTF (i.e., negative correlation with relative abundance of

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cyanobacteria and positive with eukaryotic algae) (Table 2). It was of interest to determine

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if this was consistent at other taxonomic classification levels (e.g., all dominant

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cyanobacteria OTUs maintain this trend). To assess whether certain organisms in the

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biofilm were associated with Se enrichment or elevated/reduced TTFs, the relative

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abundances of OTUs were correlated with the EF or TTF values for each treatment replicate.

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While multiple statistically significant correlations were found (Table 2), only OTUs with

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average relative abundances > 5% in at least one treatment were analyzed, since an OTU

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needs to comprise a significant proportion of the periphyton community to influence the

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bulk Se content, or comprise a significant proportion of the snail diet. Of the OTUs having an

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average of >5% abundance, four were strongly positively correlated with EFs (Spearman

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correlation coefficient > 0.75, p < 0.01), while three OTUs were negatively correlated with

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EFs (r < -0.70 and p < 0.01). Of the OTUs present in greater abundance, only cyanobacteria

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OTUs were positively correlated with EFs, whereas negative correlations were found for

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OTUs of both eukaryotic algae and Cyanobacteria (Table 2). When assessing the

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relationship between periphyton assemblage composition and Se accumulation in snails, 19


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four OTUs (all Cyanobacteria) with high relative abundances were negatively correlated

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with the TTF (coefficient < -0.80, p < 0.05; Table 2), while two OTUs were strongly

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positively correlated with the TTF (denovo4264 Scenedesmus and denovo4698

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Synechococcus sp., coefficient > 0.90, p < 0.01; Table 2).

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Although correlations between OTUs and the EF or TTF are provided for discussion

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purposes, it is emphasized that the results are not meant to explain which organisms may

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drive Se EFs and TTFs in the various treatments. Instead, correlations are provided to

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demonstrate that there is a relationship between periphyton assemblage composition and

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overall periphyton Se bioconcentration thus influencing the EF. Furthermore, the TTF to

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snails appears to be dependent upon the types of organisms comprising the periphyton

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(with respect to dietary preference). These relationships appear to be more complex than

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simply determining the relative abundance of Cyanobacteria and eukaryotic algae. Rather,

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different periphyton organisms appear to have a positive or negative correlation to the

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overall enrichment of Se in the periphyton assemblage from water and subsequent transfer

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of Se to snails. The direction of correlation is opposite for eukaryotic algae between EF

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(negative) and TTF (positive), and the response was uniform (Table 2). This could be

438

interpreted as comparatively less uptake of Se by periphyton dominated by eukaryotic taxa

439

compared to cyanobacteria, followed by preferential consumption of these taxa by snails. In

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contrast, correlations of cyanobacteria OTUs with EF and TTF appear to be highly variable,

441

with some groups being highly positively correlated and others being strongly negatively

442

correlated with either factor.

443 444

The question addressed by this study was whether or not the taxonomic composition of a periphyton community affects the bioconcentration of Se in that periphyton and 20



445

subsequently Se transfer to macroinvertebrate grazers. The results presented here

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demonstrate that periphyton assemblage composition does affect transfer of selenite from

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water to bulk periphyton, and from bulk periphyton to macroinvertebrate primary

448

consumers (snails). Without details regarding periphyton composition, Se concentration in

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water (as selenite) is not necessarily predictive of Se content in periphyton. Additionally,

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periphyton EFs in this study (6.7-fold difference among all treatments) were far less

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variable than what has been found for individual phytoplankton species (several orders of

452

magnitude difference, e.g., 8) suggesting that a mixed assemblage may provide some

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normalization of Se concentration (as some microbes accumulate large quantities and

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others take up very little). Similarly, Se content in bulk periphyton is not necessarily

455

predictive of transfer to primary consumers. Rather, this information must be considered in

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the context of the specific organisms present in the periphyton, which can strongly

457

influence the trophic transfer of Se either through differential accumulation of Se among

458

biofilm taxa or through preferential grazing by primary consumers. In the present study,

459

certain organisms within the periphyton communities demonstrated strong correlations,

460

positive or negative, with EF or TTF. The direction of correlation was dependent upon the

461

trophic level (EF or TTF) and type of organism. This study also contributed to other current

462

areas of Se research, such as the influence of lentic versus lotic systems on Se

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bioconcentration 37, as it may be that differences in primary producer assemblages in these

464

systems (i.e., periphyton) could account for some of the observed differences in Se TTFs.

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Using a metagenomic approach to augment standard assessment protocols, it is

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recommended that the influence of the periphyton and phytoplankton assemblage

467

composition on Se biodynamics in lotic versus lentic systems be more thoroughly assessed

468

to refine predictive modelling. 21


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Acknowledgements

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The authors would like to thank Colleen Carter for technical support, and Xiaofeng Wang for ICP-MS

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analysis.

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References

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Table 1 – Mean Se concentrations in periphyton and snails (µg/g dw), snail weight (mg per animal dw) and survival, and number of

575

eukaryotic algae and cyanobacteria OTUs per treatment.

Periphyton Se μg/g dw (±SD)

Treatment HL LL N NPK

576 577 578 579 580 581 582

Day 0

Day 21

Mean

31.62 (4.22) 82.01 (22.28) 16.29 (2.04) 12.00 (4.64) 9.40 (2.50)

29.45 (5.39) 41.56 (4.21) 24.90 (2.64) 14.80 (4.54) 22.26 (7.31)

30.54 (4.68) 61.79 (12.09) 20.60 (2.22) 13.40 (4.1) 15.83 (4.87)

6.58 (1.10) 1.79 (0.50)

Mean snail weight day 21 (±SD) mg d.w. 3.73 (3.12) 0.53 (0.11)

5.47 2

0.34 2

Mean Se snails day 21 (±SD) μg/g d.w.

Average snail recovery (percent±1 SD) as of day 21 70.0 ± 8.8 69.2 ± 6.9 54.2 ± 3.8

# OTUs 1 Euk.

Cyano.

31

190

18

286

10

109

6.48 6.55 69.6 ± 11.4 135 142 (0.75) (3.84) NSi 8.39 6.02 60.8 ± 11.4 84 66 (2.95) (2.70) 75.8 ± 3.1 Control 8.97 0.93 3 ‒ ‒ ‒ ‒

Genetic Characterization of Periphyton Communities Associated with

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