Overview
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- in the Food Chain (CONTAM), 2012). median muscle mercury content. Additionally, we compared the gut
- specific factors (Bolnick et al., 2014; Fuess et al., 2021; Hahn et al., 2022; Calboli et al. (2021) (Supplementary Table S1). Briefly, 25 three-spined
- converted to ng.g−1 DW. Mean and median mercury concentrations prokaryotic 16S rRNA gene between 314F (5’-CCTACGG
- in Supplementary Table S1. ATCC-3′) spanning the hypervariable V3-V4 regions (Klindworth
- populations from four sites with the lowest median muscle mercury 1 min, and a final step elongation at 72 °C for 7 min (Siriyappagouder
- 45.13 ng.g−1; Mombeek (mom): 47.5 ng.g−1; Laakbeek (lak): 50.98 ng. separating the PCR product on a 1.5% w/v agarose gel followed by
- g−1) and four sites with the highest median muscle mercury content excision of positive bands (~550 bp) and gel purification using the
- current study (Figure 1; Supplementary Table S1; all values presented To obtain the final amplicon libraries, the purified PCR product
- 80% in fish tissue, these median dry weight concentrations correspond primers (Illumina Inc., San Diego, CA, United States), followed by
- present study, four locations marked in red with the highest median mercury concentrations (VEL: 195.71 ng·g−1; MOT: 218.23 ng·g−1; LED:
- 302.70 ng·g−1; and MLB: 326.634 ng·g−1) and four marked in black with the lowest median mercury concentrations (ABE: 21.56 ng·g−1; MDB:
- on the name of the location (see Supplementary Table S1). The main course of the Scheldt and Maas are marked in blue.
- 2.6 Statistical analyses Analysis of Deviance tables using the car v3.1–1 package (Fox and
- average of 24,957 reads per individual (Supplementary Table S2).
- The alpha diversity of the iteratively rarefied microbiota were removed for being either singletons (n = 311), unassigned ASVs
- abundances was calculated using Chao1 diversity (estimated species (n = 21), or belonging to mitochondria (n = 24), chloroplasts
- (n = 199), Archaea (n = 19), or Euryarchaeota (n = 5). Interestingly, Of the 4,421 ASVs unique to hosts with low mercury content, 2,551
- the ASVs assigned as chloroplasts appeared to be most abundant in (30.2% of total ASVs) were unique to females, 1,540 (18.2% of total
- the Laakbeek (lak) population. However, their inclusion or removal ASVs) were unique to males, and a mere 330 (3.9% of total ASVs)
- trimmed and ASV-filtered libraries had a total read count ranging unique to hosts with high muscle mercury content, 1,267 (15.0% of
- between 5,206 and 47,217, which equated to a 9-fold difference in total ASVs) were unique to females, 1,417 (16.8% of total ASVs) were
- sampling depth (Supplementary Table S2). Rarefaction to an even unique to males, and 150 (1.8% of total ASVs) were shared among
- (abe: n = 2; lak: n = 2; mom: n = 2) with low sequencing coverage. than the number of ASVs unique to the mercury content level but
- individuals with higher read counts. A total of 8,843 different ASVs majority (788 ASVs or 14.8% of total ASVs) were also shared across
- remained after rarefaction to 9,500 reads (Supplementary Table S2). the sexes (Figure 2). The overall sex ratios (female: male) for
- The gut microbiota of fish with high and low levels of mercury these, the two most abundant phyla accounted for about 60 to 80% of
- (Figure 2). Three-spined stickleback with a high muscle mercury population, mercury content level, or sex (77.7% read total; Figure 3).
- content hosted 2,834 unique ASVs and shared 1,200 ASVs with hosts The two most abundant phyla were Proteobacteria (53.5% of read
- with low mercury accumulation. The latter group hosted a 55.9% total) and Firmicutes (24.17% of read total; Figure 3). These phyla
- greater number of unique ASVs (i.e., 4,421), despite the lower number were, in turn, dominated by the classes Gammaproteobacteria (38.0%
- of stickleback individuals in this group (57 vs. 64; of read total), Clostridia (21.9%), and Alphaproteobacteria (15.5% of
- Supplementary Table S3; Figure 2). The proportion of all ASVs unique read total). The most common genera were Clostridium sensu stricto
- Notably, ASVs of the genus Clostridium sensu stricto 1 ranged between Host populations with a high and low mercury content showed
- a Log2 Fold Change of −17.01 and 15.82, meaning that the abundance several differences in gut microbiome composition. Permutational
- mercury content level explained the most variation in gut microbial p-value = 0.010; Table 1). In other words, the differences in gut microbial
- Table 1). The level of mercury accumulation (high vs. low) in the muscle Although clustering by total mercury content was more diffuse than
- of the stickleback individuals itself explained 1.5 to 2.4% of the variation by host population, there was some separation between the bacterial
- 0.0001; Table 1). PERMANOVAs of two distance metrics also indicated values (ranging from 0.12 to 0.17) suggest a usable fit.
- that sex explained about 1% of the variation in gut microbial community
- diversity between sexes (Table 1). However, between 5 and 7% of the To test for the effect of mercury on the diversity and composition
- quartiles, and whiskers extend to the most extreme value within 1.5*IQR (interquartile range). Significant pairwise differences (PHSD < 0.05) in alpha
- TABLE 1 Permutational ANOVA results (10,000 permutations). The proportion of variance in the Bray-Curtis, Jaccard, unweighted UniFrac, and
Methods (brief)
- may play a crucial role in both the reabsorption and depuration of Three-spined stickleback individuals were collected from locations at
- mercury (Li et al., 2019; Tan et al., 2022). eight rivers and streams across the lower catchments of the Maas River
- Smith et al., 2015; Rennison et al., 2019). Three-spined sticklebacks are stickleback individuals were collected at each site using dip nets (mesh
- tissue of the collected individuals is described in detail in Calboli et al. GmbH, Germany). The quality and quantity of the total DNA extract
- (2021). In short, the muscle tissue samples were vacuum freeze-dried were assessed with microvolume UV spectrophotometry and
- (HR-ICP-MS, Thermo Scientific Finnigan element 2, United States). PCR amplification was conducted using bacteria-specific
- to 4 to 10 ng.g−1 wet weight for the low mercury populations and 39 clean-up using Mag-Bind TotalPure NGS magnetic beads (sample
- from Scheldt-E (mlb, mom, vel, lak), and one location from the Maas of the template using the following conditions: initial denaturation
- The frozen intestinal tissue samples from the eight host quality of the pooled libraries were assessed on the 4150 TapeStation ®
- on the name of the location (see Supplementary Table S1). The main course of the Scheldt and Maas are marked in blue.
- their corresponding sample IDs at the Norwegian Sequencing Centre. ASVs, and ASVs assigned to Archaea, Euryarchaeota, mitochondria,
- Initial sequence quality was also assessed and visualized using fastQC and chloroplasts. Then, only samples with a minimum of 5 different
- and summarized with multiQC (Andrews, 2010; Ewels et al., 2016). ASVs per sample were selected for downstream analyses. Rarefaction
- v2022.11.2 built into the QIIME2 toolkit v2022.11.1 with the following Differences in the number of reads between samples were then
- eliminated this difference but necessitated the removal of 6 samples the mercury content level and sex was between 4.5 to 9.5-fold greater
- abundance of two others were significantly enhanced. The et al. (2019) argue that the retrieval of Ca. Megaira from samples
- reported shifts in the abundance of these ASVs suggest an active such as the current intestinal samples is likely uninformative of
- and intestinal samples and has probiotic effects in mammalian gut microbiota may originate from the host’s aquatic environment,
Implications
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Update history
The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git. When DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.