DOI: 10.5061/dryad.3j9kd51rn
title: Data from: Impacts of weathered microplastic ingestion on gastrointestinal microbial communities and health endpoints in fathead minnows (Pimephales promelas)

description: Microplastics are a ubiquitous presence in the world’s aquatic
 environments and their threat to aquatic biota is poorly understood,
 especially in freshwater ecosystems. In the environment, microbial
 biofilms can form on the surface of microplastics, and these plastics have
 the potential to adsorb harmful toxins. Because lab-based studies on
 microplastics are often conducted with clean polymers, in ecologically
 unrealistic conditions and concentrations, the impact of these weathered
 microplastics on aquatic organisms in ecologically realistic conditions is
 still unclear. To help address the need for ecologically relevant
 microplastic exposure data, we incubated 500 μm polyethylene microplastic
 beads in Muskegon Lake, Michigan, USA and used them to conduct a 28-day
 ingestion study with male and female fathead minnows (Pimephales
 promelas). We examined the effects of microplastic ingestion on the fish
 gut microbial community along with hepatic gene expression and health
 parameters. We found that microplastic ingestion had statistically
 significant impacts on growth in male fathead minnows. Microplastic
 treatment did not significantly alter the beta diversity of the gut
 microbial community for either males or females, but there were clear
 differences between sexes and over time, indicating that these factors may
 outweigh the impacts of microplastic ingestion on beta diversity in the
 gut. The expression of immune response genes was not altered in males. It
 did, however, cause some changes to alpha diversity metrics in both sexes
 and there were several differentially abundant taxa among treatments.
 These data suggest that microplastic ingestion has health effects, but
 these effects may be sex specific across certain species and they are
 likely not being solely driven by changes in gut microbial communities.
description: Microplastic Incubation: We used 500-micron,
 blue, polyethylene beads (Cospheric, Inc., Santa Barbara, CA) for the
 incubation and ingestion study. First, a subsample of the new, clean
 microplastic beads were collected and stored at -80°C for 16S ribosomal
 RNA (rRNA) sequencing (‘pristine-MP’ sample). Then, the particles were
 secured in 16-micron nylon mesh bags attached to a PVC frame in Muskegon
 Lake, Michigan, USA at the Annis Water Resources Institute. The frame was
 secured at mid-depth in approximately 5 meters of water along the break
 wall from 14 June 2021 and incubated for a total of 56 days before final
 retrieval on 9 August 2021. Approximately every week during the
 incubation, the bags were inspected and gently rinsed and on 18 July 2021,
 after 34 days of incubation, we rotated the bags and examined them closely
 for wear, reinforcing seams as necessary. At final retrieval, the
 microplastics were removed gently from the incubation bags and combined in
 a composite container. We homogenized the weathered microplastics stored
 them in a 100 mL amber glass jar in Muskegon Lake surface water at 4°C for
 incorporation into the fish food for use in the ingestion study.
 Addition to Food: To prepare the fish food,
 individual 1.5 mm, slow-sinking, Finfish Starter pellets (Ziegler Bros
 Inc., Gardners, PA) were placed in wells of a standard 96-well PCR plate
 and moistened with laboratory grade deionized water. For treatment
 pellets, one microplastic bead was added to each well using fine-tipped
 forceps. For control pellets, no microplastic was added. We then pressed
 each softened pellet into the bottom of its well, embedding the
 microplastic particle into the treatment food pellets. As each plate was
 prepared, it was loosely covered by paper and placed in a dedicated
 refrigerator to dry overnight at 4°C. Pellets were divided into individual
 fish replicate meals in dedicated 5 mL tubes at the appropriate ratios for
 each treatment (outlined below). Pellets were prepared up to 4 days in
 advance and prepared meals stored at 4°C until use.
 Ingestion Study Initiation: The study was
 conducted in a flow-through system using Muskegon Lake surface water at
 the Mesocosm Lab of Annis Water Resources Institute in Muskegon, Michigan,
 USA. The study organisms were approximately five-month-old, adult fathead
 minnows hatched 16-20 February 2021 (Aquatic BioSystems, Inc.; Boulder,
 Colorado). The fish were received 21 July 2021 and acclimatized to the
 study system for 28 days before test initiation in 10-gallon glass aquaria
 in groups of approximately 24 fish with an equal distribution of males and
 females. Test chambers were made from bisected 10-gallon aquaria divided
 with plexiglass and opaque coverings between chambers. Each pair of
 chambers consisted of 1 male and 1 female of the same treatment. Chambers
 for each treatment group were randomly distributed throughout the test set
 up which consisted of three heavy-duty wire racks with two shelves
 each. Fish were randomly distributed to individual test
 chambers four days prior to test initiation, on 7 August 2021 (day -4).
 Each treatment consisted of 14 individuals (7 males and 7 females).
 Initial measurements for standard length (SL), total length (TL), and mass
 were taken for each fish as they were distributed to their respective
 chamber. For the next three days leading up to the test initiation (days
 -3 to -1), pre-test fecal pellets were collected from the bottom of each
 chamber within 4 hours of being fed (‘before fecal’ samples). Each of
 these samples was gently centrifuged at 1000 rpm for approximately 30
 seconds, the overlying water removed, and the fecal pellet frozen at -80°C
 for future DNA extraction for 16S rRNA sequencing of the before fecal
 microbial community. The exposure test began on 11 August 2021 (day 0).
 Each replicate was fed once daily at a rate of 3% by mass of the average
 body weight of all the test fish, which equaled an average of 16 pellets
 per day per fish. The test treatments were control, low, and high. Control
 replicates received 16 control pellets; low replicates received 4
 microplastic-laden pellets and 12 control pellets; and high replicates
 received 16 microplastic-laden pellets each meal.
 Chamber water level was maintained at approximately 10 liters
 with 2-4 daily water volume replacements maintained with adjustable flow
 valves on each chamber. Temperature was maintained at 23°C ± 2°C and
 dissolved oxygen (DO) was maintained at ≥ 6.0 mg/L. Temperature and DO
 were monitored in a subset of chambers daily while pH and nitrogen levels
 were monitored in the incoming water weekly. Feces, uneaten food, and
 other particulate was removed from each chamber by syphoning and disposed
 of daily before feeding and the entire system was scrubbed and flushed
 weekly to maintain water quality. No mortality was observed over the
 course of the study. <em> </em>Test
 Termination: For the three days leading up to test
 termination (days 25-27), post-test fecal samples were collected from the
 bottom of each tank within four hours of the daily feedings (‘after fecal’
 samples); samples were processed the same way as the pre-test fecal
 samples. The test was terminated on 8 September 2021 (day 28) with a
 random order for replicate processing. Each fish was measured for TL, SL,
 and mass in the same procedure as the pre-test measurements before being
 placed in a 500 mg/L solution of tricaine methanesulfonate (MS-222),
 adjusted to a pH between 7 and 8. After at least 5 minutes and complete
 anesthesia, each fish was taken from the solution, the caudle peduncle
 removed with a sterile scalpel and blood collected with a heparinized
 hematocrit tube. Each tube was plugged with clay and held vertically until
 centrifugation and measurement. Each fish was then
 decapitated, and the liver was immediately removed, placed in  RNA-later
 (Invitrogen, Waltham, MA) and flash frozen in liquid nitrogen for future
 qPCR analysis of hepatic stress gene expression. The gastrointestinal
 tract from just posterior to the stomach to the anal pore was removed and
 the gut contents were expressed and placed into a 1.5 mL microcentrifuge
 tube (‘internal fecal’ samples) and the gut tissue was placed in another
 tube (‘gut tissue’ samples). Both the internal fecal and gut tissue
 samples were flash frozen with liquid nitrogen. All tools were flame
 sterilized and wiped down with RNA away (Thermo Scientific, Waltham, MA)
 between replicates. After each third of the test was taken down (14
 replicates), all of samples were removed to the -80°C freezer and the
 hematocrit samples processed. Blood samples were centrifuged with a
 Micro-Hematocrit Centrifuge (Unico, Franksville, WI) at 11,500 rpm for 5
 minutes. Readings were taken manually with calipers, measuring the length
 of the packed red blood cells and the whole blood.
 <em> </em>Sample Processing:
 Gastrointestinal Microbial Community: DNA was
 extracted from all 16S rRNA samples using PowerFecal Pro column extraction
 kits (Qiagen, Germantown, MA) according to the manufacturer protocol. A
 positive microbial community (ZymoBIOMICS Microbial Community Standards -
 cat. D6305, Zymo Research, Irvine, CA) and extraction blank were also
 extracted. Extracted samples were run on a 1% aragose gel for 60 minutes
 to verify genomic DNA was successfully extracted. Once all samples were
 extracted, the library was prepared according to the Illumina MiSeq 16S
 Metagenomic Sequencing Library Preparation protocol, with the
 amplification targeting the V4 region using the 515F and 806R primers
 (Caporaso et al., 2012). Samples were indexed using Nextera XT Index
 Primers (Illumina, Inc., San Diego, CA). After the final clean up step
 using the AMPure XP beads (Beckman Coulter, Brea, CA), each sample was
 quantified on a Qubit 3.0 Fluorometer using a dsDNA HS Assay kit
 (Invitrogen) to determine DNA concentration. Each sample was also run on a
 2100 Bioanalyzer using a High Sensitivity DNA kit (Agilent, Santa Clara,
 CA) to determine target amplicon length and quality of the sample. Samples
 showing degradation were excluded from normalization and pooling.
 Acceptable samples were each normalized to 4 nM and pooled together. The
 sample library was denatured with freshly prepared 0.2N NaOH and diluted
 to 7 pM. PhiX was diluted to a concentration of 7 pM, denatured, and a 15%
 spike-in was included with the final sequencing library. Sequencing was
 performed on an Illumina MiSeq sequencer using a 2 x 250 bp
 format. Gene Expression: Liver RNA
 was extracted from male fathead minnows with a modified TRIzol protocol
 (Invitrogen). Each sample was thawed at room temperature until the liver
 tissue could be removed from the RNA later. The liver was then blotted dry
 and placed in a safe-lock microcentrifuge tube containing two 5 mm steel
 beads and placed in liquid nitrogen. The samples were then removed from
 the liquid nitrogen to allow the tissue to soften at room temperature for
 3 minutes and homogenized in a TissueLyzer LT (Qiagen, Hilden, Germany) at
 50 oscillations/s for 2 minutes. After phase separation with
 TRIzol/chloroform, and precipitation with isopropanol, the pellet was
 washed twice with 75% ethanol and resuspended in 100 μL of DEPC treated
 nuclease-free water. The raw extracted RNA was
 quantified on a NanoDrop spectrophotometer (ThermoFisher, Waltham, MA) and
 the samples diluted to a target of 150 ng/μL at 100 μL, which was split
 into two 50 μL aliquots. One of these aliquots was cleaned using a Turbo
 DNA-free Kit (Invitrogen) and total RNA concentration, 260/280, and
 260/230 ratios were assessed by NanoDrop after cleaning. Each sample was
 run on the 2100 Bioanalyzer using a Pico RNA kit (Agilent), to assess the
 quality of the extracted RNA, and quantified on a Qubit 3.0 Fluorimeter
 using an RNA HS/BR Assay Kit (Invitrogen) . Five samples from each
 treatment were selected for qPCR analysis based on quality as assessed
 through the bioanalyzer and these were diluted to 10 ng/μL.
 Primers and probes were developed in house using the IDT
 PrimerQuest Tool (https://www.idtdna.com/pages/tools/primerquest) and
 sequences obtained from GenBank (Supplementary Table 1). Amplified
 products from each primer pair were sequenced on a 3500 Genetic Analyzer
 (Applied Biosystems, Waltham, MA) to verify correct target amplification
 before further validation. Validation included running a no reverse
 transcriptase control with each gene to verify the lack of DNA
 contamination remaining in the sample, testing the housekeeping gene
 across treatments to assess its constancy in expression, and calculating
 efficiency for each gene with a standard curve. Hepatic
 gene expression was assessed with RT-qPCR on an Applied Biosystems
 OneStepPlus instrument. Gene targets were <em>cyp1a</em>,
 <em>gst</em>, and <em>ncf2</em>, which were
 normalized against <em>elfa1</em> as the internal housekeeping
 gene. Relative gene expression was assessed using a
 ∆∆C<sub>t</sub> protocol (Livak and Schmittgen, 2001;
 Schmittgen and Livak, 2008) on a OneStepPlus (Applied Biosystems) using
 Luna® Universal Probe One-Step RT-qPCR Kit (New England BioSystems). We
 used 2-μL of RNA template per well with each sample and control run in
 triplicate. Each reaction volume was 20 μL consisting of 10 μL of 2x Luna
 Universal Probe One-Step Reaction Mix, 1 μL of 20x Luna WarmStart RT
 Enzyme Mix, 0.4 uM final concentration of the  forward and reverse
 primers, 0.2 μM final concentration of the probe (FAM labeled with a
 Zen/Iowa BlackTM FQ double quencher), and the template RNA. The
 thermocycle for all validation and experimental plates for the gene
 expression analysis included an initial hold at 55°C for 10 min., followed
 by a denaturing step at 95°C for 1 min., then 40 cycles of 95°C for 10
 sec., and 55°C for 1 min.
 <em> </em>Statistical Analysis:
 Health Endpoints: Change in mass was
 calculated as final mass (g) minus initial mass (g) for each replicate.
 Fulton’s Condition Factor (FCF) was calculated as the mass (g) divided by
 cubic length (cm). Change in FCF was calculated as final FCF minus initial
 FCF for each replicate. The hematocrit was calculated as the ratio of the
 red blood cells to the whole blood after centrifugation (Shiver &
 Grove, 2011). Exploratory data analysis showed interaction for all three
 of these endpoints between the sex and treatment factors and so growth,
 change in FCF, and hematocrit were analyzed for males and females
 separately. Shapiro-Wilk tests were used to test for normal distributions
 and Levene’s tests for equal variances. If all three treatments were
 normally distributed and had equal variances, they were compared by
 one-way ANOVA tests with TukeyHSD post-hoc. If any of the treatments were
 not normally distributed or had unequal variances, they were compared with
 Kruskal-Wallis tests with Wilcoxon post-hoc with Bonferroni adjustment.
 All analyses for health endpoints were conducted in R, version 4.2.0 (R
 Core Team, 2021). Gastrointestinal Microbial
 Community Sequence data were processed using the dada2
 package, version 1.24.0, in R to generate amplicon sequence variants
 (ASVs) (Callahan et al., 2016). Briefly, sequences were filtered by
 quality scores, the sequencing error was ‘denoised’ by modeling error in
 the sequencing reads, chimeras were removed, and then the pairs merged.
 Sequences were aligned against the SILVA 16S reference database (Yilmaz et
 al., 2014). The resultant ASV and taxonomy tables were then analyzed in R
 using the phyloseq, version 1.40.0 (McMurdie and Holmes, 2013), vegan,
 version 2.6.2 (Oksanen et al., 2019), and DESeq2, version 1.36.0 (Love et
 al., 2014) packages. Phyloseq was used to compile taxonomy, ASV, and
 sample data into a single experiment-level object. Exploratory data
 analysis on all samples included assessing family level composition with
 stacked bar charts and unconstrained ordinations with principal coordinate
 analysis (PCoA). Samples were split into internal
 fecal, gut tissue, before fecal, and after fecal sample types and then
 each of those was subset into males and females for independent analysis.
 To reiterate, ‘internal fecal’ samples consisted of the fecal material
 removed from the gut at test termination, ‘gut tissue’ samples consisted
 of the remaining gastrointestinal tract after feces was removed at test
 termination, ‘before fecal’ samples consisted of fecal material sampled
 from the bottom of the tank after initial acclimation but prior to the
 start of the ingestion study, and ‘after fecal’ samples consisted of fecal
 material collected from the bottom of the tank at the end of the ingestion
 study. We used the control group of the internal fecal samples for males
 and females as a representative subset of samples to assess sex
 differences. We assessed the community composition at
 the beginning of the experiment using the before fecal samples in each sex
 and expected no differences between treatments since all individuals were
 on the same standardized diet prior to the beginning of the study.  To
 investigate whether the gut microbial community changed over the course of
 the ingestion period, we compared the before fecal and after fecal samples
 within each treatment. We used the internal fecal, gut tissue, and after
 fecal samples to assess the differences between treatments after the
 microplastic exposure period. For diversity metrics,
 each group was rarefied to the lowest sample read depth in each group
 (Supplemental Table 2). For Chao1 richness estimates and Shannon diversity
 estimates, Shapiro-Wilk tests were used to test for normal distributions
 and Levene’s tests for equal variances. If all levels were normally
 distributed and had equal variances, they were compared by one-way ANOVA
 tests with TukeyHSD post-hoc. If any of the treatments were not normally
 distributed or had unequal variances, they were compared with
 Kruskal-Wallis tests with Wilcoxon post-hoc with Bonferroni adjustment or,
 in the case of the fecal control group, the Wilcoxon rank sum test. Chao1
 and Shannon estimates were also calculated and compared for the before and
 after samples together to assess changes over time in each treatment. As
 these are paired data, they were tested with either paired T-tests or
 Wilcoxon signed-rank tests. Beta diversity was explored by generating PCoA
 ordinations based on a Bray-Curtis distance matrices within each sample
 type. Permutational multivariate analysis of variance (PERMANOVA) tests
 were performed with the adonis function in the vegan package for each sex
 in each sample type. As the before and after fecal samples assessed
 together violated the assumption of independence, these were not tested by
 PERMANOVA. Differential abundance was assessed on non-rarefied count data
 using DESeq2 which assumes a negative binomial distribution of the
 abundance count data. Data was independently filtered to include only
 those taxa present at least three times and in at least 10% of the
 samples. The DESeq2 results were then subject to log fold change shrinking
 with the lfcShrink function to account for effect sizes. Taxa were
 considered biologically significant in their differential abundances if
 they had a log2 fold change value greater than 0.25 and a p-value less
 than 0.05. This threshold translates to approximately +/- 20% in abundance
 and allowed us to focus on those taxa with the greatest differences among
 treatment types; those which may have had impacts on the microbial
 community function. Raw sequencing data has been deposited on NCBI’s
 Sequence Read Archive (SRA) with accession number SUB14107689.
 Gene Expression The coefficient of variation
 (CV) was calculated for the housekeeping gene, <em>ef1a</em>,
 using transformed C<sub>t</sub> values () for all
 study replicates. For each target gene (<em>cyp1a</em>,
 <em>gsta</em>, and <em>ncf2</em>) the coefficient
 of variation was also calculated for the controls using the transformed
 ∆C<sub>t</sub> values (), as calculated in Equation
 1 in manuscript. As there was high variation in the
 controls for <em>gsta</em> in particular, we used the
 geometric mean to calculate the ∆∆C<sub>t</sub> values. Gene
 expression levels for each of the three target genes were normalized
 against the housekeeping gene <em>efla1</em> using the
 ∆∆C<sub>t</sub> method, Equation 2 (in manuscript)
 These values were then transformed into gene fold, Equation 3 (in
 manuscript) These gene fold values were log-transformed
 then compared across treatment groups with one-way ANOVA for each
 gene.
description: # Data from: Impacts of weathered microplastic ingestion on
 gastrointestinal microbial communities and health endpoints in fathead
 minnows (Pimephales promelas)
 [https://doi.org/10.5061/dryad.3j9kd51rn](https://doi.org/10.5061/dryad.3j9kd51rn) This study broadly addressed three main areas: 1. health endpoints (growth, condition factor, and hematocrit), 2. gastrointestinal microbial community, and 3. hepatic gene expression. For each of these, respectively, this dataset includes the 1. health measurement data and R code, 2. the namefile, dada2 R code, and downstream ASV analysis R code, and 3. the RT-qPCR data and R code. The raw 16S sequence data for the microbial community analysis has been deposited on the NCBI Sequence Read Archive with the Accession Number PRJNA1088468.  ## Description of the data and file structure * Health endpoints: * fhm_health.csv * NA explanations: * fhm_01 was removed from test on day 1, no further data was collected on this fish * fhm_34, technician failed to record final mass and sample collection times * all hematocrit NAs were due to insufficient blood volumes for the EZ card reader and/or manual measurement * initiation_order: order in which fish were randomly selected from stock to add to test, randomizing placement within treatments * fish_id: individual identification based on initiation order, unique to each fish * treatment: microplastic treatment group; low = 4 microplastic particles/day, high = 16 microplastic particles/day, control = no microplastic particles/day * treatment_number: each treatment had 14 replicates, treatment number refers to which replicate within each treatment * replicate: combination of treatment and treatment_number for a unique replicate identification within the entire test * chamber_number: identification of physical chamber * chamber: naming scheme with ch_# for each chamber * sex: male (m) or female (f) * pre_tank: refers to which acclimatization stock tank the fish were held in before test initiation * test_date: date of initiation (introduction into test system) for each fish * test_day: which day of the 28 day exposure test each fish was introduced to system * initial_mass_g : weighed in grams on date of initiation for each fish * initial_tl_mm: total length nose to end of tail in millimeters on date of initiation for each fish * initial_sl_mm: standard length nose to end of spine (excluding caudal fin) in millimeters on date of initiation for each fish * deceased: whether fish died during test, yes or no * replaced: whether fish was replaced during test, only fish fhm_01 in low_12 was replaced on test day 1 due to poor condition * replaced_date: date any replaced fish was replaced * test_days: total number of days each fish was in test * termination_order: randomized order for termination of test fish (taking measurements, sacrificing, and collecting samples, etc.) * final_mass_g: weighed in grams on date of termination for each fish * final_tl_mm: total length nose to end of tail in millimeters on date of termination for each fish * final_sl_mm: standard length nose to end of spine (excluding caudal fin) in millimeters on date of termination for each fish * diff_mass_g: initial minus final mass in grams for each fish * diff_tl_mm: initial minus final total length in millimeters for each fish * diff_sl_mm: initial minus final standard length in millimeters for each fish * hem_percent: hematocrit measured using EZ Reader hematocrit reader card after centrifugation * hem_packed_mm: length of packed red blood cells in capillary tube in millimeters after centrifugation * hem_total_mm: length of total blood sample in capillary tube in millimeters after centrifugation * calc_hem: hematocrit calculation of packed length over total length x 100 * ms_222_time: time each fish was submerged in MS-222 to euthanize before sample collection * liv_nitro_time: time from removal of each fish from MS-222 until liver was collected and placed in liquid nitrogen (in RNA later) * gut_nitro_time: time from removal of each fish from MS-222 until gut content and tissue samples were placed in liquid nitrogen * fhm_health.R * Microbial community: * mp_fhm_namefile.csv * Sex: male or female * Treatment: low (4 microplastics/day), high (16 microplastics/day), control (no microplastics/day) * Type: microbial sample type -- * before (B) = fecal sample collected from the bottom of the tank for three days preceding test initiation * after (A) = fecal sample collected from the bottom of the tank for three days preceding test termination * fecal (F) = fecal sample collected directly from gut contents at test termination * gut (G) = gut tissue collected after emptying contents at test termination * Sample_ID: combination of Type, Treatment, and Treatment Replicate; refers to specific sample collected * Fish: Treatment Replicate; refers to individual fish from which several samples were collected * Replicate: Treatment Replicate number * mp_fhm_dada2_manuscript.R * Ran with the raw sequence files off the Illumina MiSeq (SRA Accession Number PRJNA1088468) * mp_fhm_ASV_manuscript.R * Downstream community analysis and visualizations. * Gene expression: * fhm_ddct.csv * gene: abbreviation of target or housekeeping gene name * ddct_trans: final transformed delta delta Ct value used in statistical analysis, see paper for full calculation description * treatment: Control (no microplastics/day), Low (4 microplastics/day), High (16 microplastics/day) * sample: liver sample collected from each fish, named combining treatment and treatment replicate number * gene_expression.R * .TXT files * Sanger sequences for each target gene