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A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

ABSTRACT: Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300-2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.

SUBMITTER: Fiedler S

PROVIDER: S-EPMC7725368 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

Fiedler Sonja S Wünnemann Hannah H Hofmann Isabel I Theobalt Natalie N Feuchtinger Annette A Walch Axel A Schwaiger Julia J Wanke Rüdiger R Blutke Andreas A

PloS one 20201209 12

Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the ex ...[more]

PMID: 33296424

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Project description:BackgroundGenetic maps characterizing the inheritance patterns of traits and markers have been developed for a wide range of species and used to study questions in biomedicine, agriculture, ecology and evolutionary biology. The status of rainbow trout genetic maps has progressed significantly over the last decade due to interest in this species in aquaculture and sport fisheries, and as a model research organism for studies related to carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. We constructed a second generation genetic map for rainbow trout using microsatellite markers to facilitate the identification of quantitative trait loci for traits affecting aquaculture production efficiency and the extraction of comparative information from the genome sequences of model fish species.ResultsA genetic map ordering 1124 microsatellite loci spanning a sex-averaged distance of 2927.10 cM (Kosambi) and having 2.6 cM resolution was constructed by genotyping 10 parents and 150 offspring from the National Center for Cool and Cold Water Aquaculture (NCCCWA) reference family mapping panel. Microsatellite markers, representing pairs of loci resulting from an evolutionarily recent whole genome duplication event, identified 180 duplicated regions within the rainbow trout genome. Microsatellites associated with genes through expressed sequence tags or bacterial artificial chromosomes produced comparative assignments with tetraodon, zebrafish, fugu, and medaka resulting in assignments of homology for 199 loci.ConclusionThe second generation NCCCWA genetic map provides an increased microsatellite marker density and quantifies differences in recombination rate between the sexes in outbred populations. It has the potential to integrate with cytogenetic and other physical maps, identifying paralogous regions of the rainbow trout genome arising from the evolutionarily recent genome duplication event, and anchoring a comparative map with the zebrafish, medaka, tetraodon, and fugu genomes. This resource will facilitate the identification of genes affecting traits of interest through fine mapping and positional cloning of candidate genes.

Project description:BackgroundMaternal transcripts are accumulated in the oocyte during oogenesis to provide for protein synthesis from oocyte maturation through early embryonic development, when nuclear transcription is silenced. The maternal mRNAs have short poly(A) tails after undergoing post-transcriptional processing necessary for stabilizing them for storage. The transcripts undergo cytoplasmic polyadenylation when they are to be translated. Transcriptome analyses comparing total mRNA and elongated poly(A) mRNA content among eggs of different quality can provide insight into molecular mechanisms affecting egg developmental competence in rainbow trout. The present study used RNA-seq to compare transcriptomes of unfertilized eggs of rainbow trout females yielding different eyeing rates, following rRNA removal and poly(A) retention for construction of the libraries.ResultsThe percentage of embryos to reach the 32-cell stage at 24 h post fertilization was significantly correlated to family eyeing rate, indicating that inviable embryos were developmentally compromised before zygotic genome activation. RNA sequencing identified 2 differentially expressed transcripts (DETs) from total mRNA sequencing comparing females with low-quality (< 5% eyeing), medium-quality (30-50% eyeing), and high-quality (> 80% eyeing) eggs. In contrast, RNA sequencing from poly(A) captured transcripts identified 945 DETs between low- and high-quality eggs, 1012 between low- and medium-quality eggs, and only 2 between medium- and high-quality eggs. The transcripts of mitochondrial genes were enriched with polyadenylated transcript sequencing and they were significantly reduced in low-quality eggs. Similarly, mitochondrial DNA was reduced in low-quality eggs compared with medium- and high-quality eggs. The functional gene analysis classified the 945 DETs between low- and high-quality eggs into 31 functional modules, many of which were related to ribosomal and mitochondrial functions. Other modules involved transcription, translation, cell division, apoptosis, and immune responses.ConclusionsOur results indicate that differences in egg quality may be derived from differences in maternal nuclear transcript activation and cytoplasmic polyadenylation before ovulation, as opposed to accumulation and storage of maternal nuclear transcripts during oogenesis. Transcriptome comparisons suggest low-quality eggs suffered from impaired oxidative phosphorylation and translation. The DETs identified in this study provide insight into developmental competence in rainbow trout eggs.

Project description:Purpose: a transcriptomic analysis was performed to extend our understanding on the immune response picture of rainbow trout exposed to I. multifiliis. Methods: Gill samples were collected from fish in each tank (control and infected group) at day 8 after infection. Total RNA was extracted using RTN350 (Sigma-Aldrich), according to the manufacturer’s instruction and subsequently, DNase treated with DNase I (Thermo Scientific, USA). Quality and integrity of the total RNA were checked on an Agilent Bioanalyzer 2100 total RNA Nano series II chip (Agilent, Amstelveen, Netherlands). Illumina RNAseq libraries were prepared from 500 ng total RNA using the Illumina TruSeqTM Stranded mRNA LT Sample Prep Kit according to the manufacturer’s instructions (Illumina Inc. San Diego, CA, USA). All RNAseq libraries (150-750 bp inserts) were sequenced on an Illumina HiSeq2500 sequencer as 1 x 50 nucleotides single-end reads according to the manufacturer’s protocol. Image analysis and base calling were done using the Illumina pipeline. Reads were aligned to the Rainbow trout reference genome (http://www.genoscope.cns.fr/trout/data/) using TopHat (version 2.0.5) and on average 53.4% of the RNAseq reads could be mapped. The resulting files were filtered using SAMtools (version 0.1.18) to exclude secondary alignment of reads. For statistical comparison of gene expression levels between groups, aligned fragments per predicted gene were counted from SAM alignment files using the Python package HTSeq (version 0.5.3p9). To make comparisons across samples possible, these fragment counts were corrected for the total amount of sequencing performed for each sample. As a correction scalling factor, we employed library size estimates determined using the R/Bioconductor (release 2.11) package DESeq. Read counts were normalized by dividing the raw counts obtained from HTSeq by its scale factor. Correction for false positives is included in the statistical analysis of gene expression through DESeq. The cut-off for significance was set to adjusted p<0.05 and at least 2-fold change. Results: a transcriptomic analysis was performed on infected rainbow trout gills and it showed that a total of 3,352 (7.2%) out of 46,585 identified genes were revealed significantly expressed after parasite infection. Of differentially expressed genes, 1.796 genes were up-regulated and 1.556 genes down-regulated. These were classified into 61 Gene Ontology (GO) terms and mapped to 282 reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Infection of I. multifiliis induced a clear differential expression of immune genes, related to both innate and adaptive immunity. A total of 268 (6.86%) regulated genes was known to take part in 16 immune-related pathways. These involved pathways related to the innate immune system such as Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration. Conclusion: a transcriptomic profile of rainbow trout gills exposed to the parasitic I. multifiliis was reported for the first time. A total of 3,355 differentially expressed unigenes were identified. Of these were 1,184 unigenes (mapped to 952 genes) annotated 282 KEGG pathways and 268 unigenes were associated with 16 immune pathways. Most unigenes were related to innate immune system pathways (Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration) although a number of unigenes was related to adaptive responses (antigen processing and presentation, T and B cell receptor signaling pathway). The present study gave a far better resolution of the immune response of rainbow trout exposed to a parasitic protozoan than has ever been presented previously. The identification of a series of immune genes involved in several but important was useful for understanding of immune mechanism of the rainbow trout responding to the parasite I. multifiliis. Our results provide tools to link innate and adaptive immune elements in the process and present basic information which will be useful in the future studies related to immunoprophylaxis.

Project description:BackgroundMicroRNAs (miRNAs) are small regulatory molecules which post-transcriptionally regulate mRNA stability and translation. Several microRNAs have received attention due to their role as key metabolic regulators. In spite of the high evolutionary conservation of several miRNAs, the role of miRNAs in lower taxa of vertebrates has not been studied with regard to metabolism. The liver-specific and highly abundant miRNA-122 is one of the most widely studied miRNA in mammals, where it has been implicated in the control of hepatic lipid metabolism. Following our identification of acute postprandial, nutritional and endocrine regulation of hepatic miRNA-122 isomiRNA expression in rainbow trout, we used complementary in silico and in vivo approaches to study the role of miRNA-122 in rainbow trout metabolism. We hypothesized that the role of miRNA-122 in regulating lipid metabolism in rainbow trout is conserved to that in mammals and that modulation of miRNA-122 function would result in altered lipid homeostasis and secondarily altered glucose homeostasis, since lipogenesis has been suggested to act as glucose sink in trout.ResultsOur results show that miRNA-122 was functionally inhibited in vivo in the liver. Postprandial glucose concentrations increased significantly in rainbow trout injected with a miRNA-122 inhibitor, and this effect correlated with decreases in hepatic FAS protein abundance, indicative of altered lipogenic potential. Additionally, miRNA-122 inhibition resulted in a 20% decrease in plasma cholesterol concentration, an effect associated with increased expression of genes involved in cholesterol degradation and excretion.ConclusionsOverall evidence suggests that miRNA-122 may have evolved in early vertebrates to support liver-specific metabolic functions. Nevertheless, our data also indicate that metabolic consequences of miRNA-122 inhibition may differ quantitatively between vertebrate species and that distinct direct molecular targets of miRNA-122 may mediate metabolic effects between vertebrate species, indicating that miRNA-122 - mRNA target relationships may have undergone species-specific evolutionary changes.

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A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

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A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies.

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