Dataset Information

The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass (Micropterus dolomieu).

ABSTRACT: There is an increasing emphasis on effects-based monitoring to document responses associated with exposure to complex mixtures of chemicals, climate change, pathogens, parasites and other environmental stressors in fish populations. For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology. Consequently, formalin-fixed, paraffin-embedded (FFPE) tissue can be an important reservoir of nucleic acids as technologies emerge that utilize molecular endpoints. Despite the cross-linking effects of formalin, its impact on nucleic acid quality and concentration, amplification, and sequencing are not well described. While fresh-frozen tissue is optimal for working with nucleic acids, FFPE samples have been shown to be conducive for molecular studies. Laser capture microdissection (LCM) is one technology which allows for collection of specific regions or cell populations from fresh or preserved specimens with pathological alterations, pathogens, or parasites. In this study, smallmouth bass (Micropterus dolomieu) liver was preserved in three different fixatives, including 10% neutral buffered formalin (NBF), Z-Fix® (ZF), and PAXgene® (PG) for four time periods (24 hr, 48 hr, seven days, and 14 days). Controls consisted of pieces of liver preserved in RNALater® or 95% ethanol. Smallmouth bass were chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors. Small liver sections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality. Sanger sequencing and the NanoString nCounter® technology were used to assess the suitability of these samples in downstream molecular techniques. The results revealed that of the formalin fixatives, NBF samples fixed for 24 and 48 hr were superior to ZF samples for both Sanger sequencing and the Nanostring nCounter®. The non-formalin PAXgene® samples were equally successful and they showed greater stability in nucleic acid quality and concentration over longer fixation times. This study demonstrated that small quantities of preserved tissue from smallmouth bass can be utilized in downstream molecular techniques; however, future studies will need to optimize the methods presented here for different tissue types, fish species, and pathological conditions.

SUBMITTER: Walsh HL

PROVIDER: S-EPMC7416921 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass (Micropterus dolomieu).

Walsh Heather L HL Sperry Adam J AJ Blazer Vicki S VS

PloS one 20200810 8

There is an increasing emphasis on effects-based monitoring to document responses associated with exposure to complex mixtures of chemicals, climate change, pathogens, parasites and other environmental stressors in fish populations. For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology. Consequently, formalin-fixed, paraffin-embedded (FFPE) tissue can be an important reservoir of nucleic acids as technologies emerge that utilize molec ...[more]

PMID: 32776939

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Project description:BackgroundMany animals appear to preferentially renest in proximity to a site they previously occupied. Evidence of nest fidelity is often inferred from a right skewed distribution of distances between the nests of individuals that breed in two consecutive reproduction episodes, where many individuals nest some arbitrarily close distance to their prior nest and others, in the extended right tail of the distribution, nest far from the nest they previously occupied. Because right skewed distributions of inter-nest distances can arise even when individuals choose nest locations randomly, however, such inferences are prone to error. The importance of null models-used to generate patterns of individual inter-nest distances by processes that do not involve site attachment-for inferences about site fidelity has been known for decades but is still often unappreciated or ignored.MethodsThe right skewed distributions of inter-nest distances observed in two earlier studies of male smallmouth bass (Micropterus dolomieu) suggest prima facie that males exhibit nest site fidelity between annual reproduction episodes, but patterns of inter-nest distances have yet to be compared to an adequate null model. Here, we evaluate the nest site fidelity of marked male M. dolomieu in a decade-long dataset, where we apply a randomization procedure based on the rencontre probability problem to generate null models. Eight observed distributions of individual, annual inter-nest distances are compared to a year-specific null model to determine whether random processes are sufficient to explain the observed distributions of inter-nest distances.ResultsThrough contrasts between observed annual inter-nest distances and results derived from null models that imposed realistic constraints on behavior, we show that some males were undoubtedly nest-site faithful. To reinforce the utility of null models and to make these kinds of models more accessible, we also provide a supplemental tutorial. The tutorial illustrates how random site choices, subject to common ecological and behavioral constraints, and even how distance is measured, can produce patterns of inter-nest distances that falsely imply nest site fidelity, or a lack of fidelity. The R code needed to reproduce these null models is included. The inference errors evident in our examples generalize to other forms of site fidelity, such as the apparent patch fidelity of certain sea bird foragers.ConclusionsThe comparisons of observed distributions of inter-nest distances with those generated by null models imply that, as suggested in prior studies, male M. dolomieu indeed exhibit annual nest site fidelity. Procedures like those we apply are necessary first steps in analyses when distributions of distances between the nests of individuals in consecutive reproduction episodes are used to infer nest-site fidelity. Why male M. dolomieu are site faithful is a question yet to be answered.

Project description:The observation of testicular oocytes in male fishes has been utilized as a biomarker of estrogenic endocrine disruption. A reconnaissance project led in the Northeastern United States (US) during the period of 2008-2010 identified a high prevalence of intersex smallmouth bass on or near US Fish & Wildlife Service National Wildlife Refuges that included the observation of 100% prevalence in smallmouth bass males collected from the Wallkill River, NJ, USA. To better assess the prevalence of intersex smallmouth bass across the state of New Jersey, a tiered reconnaissance approach was initiated during the fall of 2016. Surface water samples were collected from 101 (85 river, 16 lake/reservoir) sites across the state at base-flow conditions for estrogenicity bioassay screening. Detectable estrogenicity was observed at 90% of the sites and 64% were above the US Environmental Protection Agency trigger level of 1 ng/L. Median surface water estrogenicity was 1.8 ng/L and a maximum of 6.9 ng/L E2EqBLYES was observed. Adult smallmouth bass were collected from nine sites, pre-spawn during the spring of 2017. Intersex was identified in fish at all sites, and the composite intersex prevalence was 93.8%. Prevalence across sites ranged from 70.6% to 100%. In addition to intersex, there was detectable plasma vitellogenin in males at all sites. Total estrogenicity in surface water was determined at these fish collection sites, and notable change over time was observed. Correlation analysis indicated significant positive correlations between land use (altered land; urban + agriculture) and surface water estrogenicity. There were no clear associations between land use and organismal metrics of estrogenic endocrine disruption (intersex or vitellogenin). This work establishes a baseline prevalence of intersex in male smallmouth bass in the state of New Jersey at a limited number of locations and identifies a number of waterbodies with estrogenic activity above an effects-based threshold.

Dataset Information

The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass (Micropterus dolomieu).

Publications

The effects of tissue fixation on sequencing and transcript abundance of nucleic acids from microdissected liver samples of smallmouth bass (Micropterus dolomieu).

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