MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets.

The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25-3, Let-7a, miR-103, miR-99-5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25-3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99-5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.

Bacillus subtilis KM0 Impacts gut Microbiota Profile and Transcription of Genes Related to Transcellular Transport in Zebrafish (Danio rerio).

It is known that probiotic microorganisms play important roles in the composition of the intestinal microbiota. Also, probiotics can affect the paracellular and transcellular transport mechanisms performed by intestinal cells. The aim of this work was to evaluate the effect of the potential probiotic Bacillus subtilis KM0 on the profile of the gut microbiota and transcription of genes related to intestinal transport of zebrafish (Danio rerio). Zebrafish was exposed by immersion to B. subtilis KM0 for 48 h, and the intestines were collected for metataxonomic analysis and transcription of genes related to transcellular and paracellular transports. Although exposure to B. subtilis changed the intestinal microbiota profile of zebrafish, the diversity indices were not altered. A decrease in the number of genera of potentially pathogenic bacteria (Flavobacterium, Plesiomonas, and Pseudomonas) and downregulation in transcription of transcellular transport genes (cubn and amn) were observed. B. subtilis KM0 strain had the expected probiotic effect, by interfering with the proliferation of potentially pathogenic bacteria and decreasing the transcription of genes codifying for signals involved with a mechanism that can be used for invasion by pathogens. The present study demonstrated that, even with a short-term exposure, a bacterium with probiotic potential such as the KM0 strain of B. subtilis can modify the profile of the host's intestinal microbiota, with an impact on the regulation of intestinal genes related to mechanisms that can be used for invasion by pathogenic bacteria.

Modulation of miR-429 during osmotic stress in the silverside Odontesthes humensis.

Silverside fish inhabit marine coastal waters, coastal lagoons, and estuarine regions in southern South America. Although silversides are not fully adapted to freshwater, they can tolerate a wide range of salinity variations. MicroRNAs (miRNAs) are a class of ∼22 nucleotide noncoding RNAs, which are crucial regulators of gene expression at post-transcriptional level. Current data indicate that miRNAs biogenesis is altered by situations of environmental stress, thereby altering the expression of target mRNAs. Foremost, the silversides were acutely exposed to 30 g.L-1 of salt to reveal in which tissue miR-429 could be differentially expressed. Thus, fish were acclimated to freshwater (0 g.L-1) and to brackish water (10 g.L-1), and then exposed to opposite salinity treatment. Here, we reveal that miR-429, a gill-enriched miRNA, emerges as a prime osmoregulator in silversides. Taken together, our findings suggest that miR-429 is an endogenous regulator of osmotic stress, which may be developed as a biomarker to assist silverside aquaculture.

GH Overexpression Alters Spermatic Cells MicroRNAome Profile in Transgenic Zebrafish.

Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F0104 has earlier been reported to cause increased muscle growth. In addition to this, GH affects a broad range of cellular processes in transgenic fish, such as morphology, physiology, and behavior. Reports show changes such as decreased sperm quality and reduced reproductive performance in transgenic males. It is hypothesized that microRNAs are directly involved in the regulation of fertility potential during spermatogenesis. The primary aim of our study was to verify whether gh overexpression disturbs the sperm miRNA profile and influences the sperm quality in transgenic zebrafish. We report a significant increase in body weight of gh-transgenic males along with associated reduced sperm motility and other kinetic parameters in comparison to the non-transgenic group. MicroRNA transcriptome sequencing of gh-transgenic zebrafish sperms revealed expressions of 186 miRNAs, among which six miRNA were up-regulated (miR-146b, miR-200a-5p, miR-146a, miR-726, miR-184, and miR-738) and sixteen were down-regulated (miR-19d-3p, miR-126a-5p, miR-126b-5p, miR-22a-5p, miR-16c-5p, miR-20a-5p, miR-126b-3p, miR-107a-3p, miR-93, miR-2189, miR-202-5p, miR-221-3p, miR-125a, miR-125b-5p, miR-126a-3p, and miR-30c-5p) in comparison to non-transgenic zebrafish. Some of the dysregulated miRNAs were previously reported to be related to abnormalities in sperm quality and reduced reproduction ability in other species. In this study, an average of 134 differentially expressed miRNAs-targeted genes were predicted using the in silico approach. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that the genes of affected pathways were primarily related to spermatogenesis, sperm motility, and cell apoptosis. Our results suggested that excess GH caused a detrimental effect on sperm microRNAome, consequently reducing the sperm quality and reproductive potential of zebrafish males.

Growth Hormone Overexpression Induces Hyperphagia and Intestinal Morphophysiological Adaptations to Improve Nutrient Uptake in Zebrafish.

The excess of circulating growth hormone (GH) in most transgenic animals implies mandatory growth resulting in higher metabolic demand. Considering that the intestine is the main organ responsible for the digestion, absorption, and direction of dietary nutrients to other tissues, this study aimed to investigate the mechanisms by which gh overexpression modulates the intestine to support higher growth. For this purpose, we designed an 8-weeks feeding trial to evaluate growth parameters, feed intake, and intestinal morphometric indices in the adult gh-transgenic zebrafish (Danio rerio) model. To access the sensitivity of the intestine to the excess of circulating GH, the messenger RNA (mRNA) expression of intestine GH receptors (GHRs) (ghra and ghrb) was analyzed. In addition, the expression of insulin-like growth factor 1a (igf1a) and genes encoding for di and tripeptide transporters (pept1a and pept1b) were assessed. Gh-transgenic zebrafish had better growth performance and higher feed intake compared to non-transgenic sibling controls. Chronic excess of GH upregulates the expression of its cognate receptor (ghrb) and the main growth factor related to trophic effects in the intestine (igf1a). Moreover, transgenic zebrafish showed an increased intestinal absorptive area and higher expression of crucial genes related to the absorption of products from meal protein degradation. These results reinforce the ability of GH to modulate intestinal morphology and the mechanisms of assimilation of nutrients to sustain the energy demand for the continuous growth induced by the excess of circulating GH.

Acute exposition to Roundup Transorb® induces systemic oxidative stress and alterations in the expression of newly sequenced genes in silverside fish (Odontesthes humensis).

Roundup Transorb® (RDT) is a glyphosate-based herbicide commonly used in agricultural practices worldwide. This herbicide exerts negative effects on the aquatic ecosystem and affects bioenergetic and detoxification pathways, oxidative stress, and cell damage in marine organisms. These effects might also occur at the transcriptional level; however, the expression of genes associated with oxidative stress has not been studied well. Odontesthes humensis is a native Brazilian aquatic species naturally distributed in the habitats affected by pesticides, including Roundup Transorb® (RDT). This study evaluated the toxic effects of short-term exposure to RDT on O. humensis. Moreover, the genes related to oxidative stress were sequenced and characterized, and their expressions in the gills, hepatopancreas, kidneys, and brain of the fish were quantified by quantitative reverse transcription-polymerase chain reaction. The animals were exposed to two environmentally relevant concentrations of RDT (2.07 and 3.68 mg L-1) for 24 h. Lipid peroxidation, reactive oxygen species (ROS), DNA damage, and apoptosis in erythrocytes were quantified by flow cytometry. The expression of the target genes was modulated in most tissues in the presence of the highest tested concentration of RDT. In erythrocytes, the levels of lipid peroxidation, ROS, and DNA damage were increased in the presence of both the concentrations of RDT, whereas cell apoptosis was increased in the group exposed to 3.68 mg L-1 RDT. In conclusion, acute exposure to RDT caused oxidative stress in the fish, induced negative effects on cells, and modulated the expression of genes related to the enzymatic antioxidant system in O. humensis.

Evaluation of qPCR reference genes in GH-overexpressing transgenic zebrafish (Danio rerio).

Reference genes (RGs) must have a stable expression in tissues in all experimental conditions to normalize real-time quantitative reverse transcription PCR (qRT-PCR) data. F0104 is a highly studied lineage of zebrafish developed to overexpress the growth hormone (GH). It is assumed that the transgenic process may influence the expression levels of commonly used RGs. The objective of the present study was to make a comprehensive analysis of stability of canditade RGs actb1, actb2, b2m, eif2s2, eef1a1, gapdh, rplp2, rpl7, rpl13α, tuba1, and rps18, in gh-transgenic and non-transgenic zebrafish. Liver, brain, intestine and muscle samples from both groups had qRT-PCR results analyzed by dCt, geNorm, NormFinder, BestKeeper, and RefFinder softwares. Consensus analyses among software concluded that rpl13α, rpl7, and eef1a1 are the most stable genes for zebrafish, considering the studied groups and tissues. Gapdh, rps18, and tuba1 suffered variations in stability among different tissues of both groups, and so, they were listed as the genes with lowest stability. Results from an average pairwise variations test indicated that the use of two RGs would generate reliable results for gene expression analysis in the studied tissues. We conclude that genes that are commonly used in mammals for qRT-PCR assays have low stability in both non-transgenic and gh-transgenic zebrafish reinforcing the importance of using species-specific RGs.

Corrigendum: Gene and Blood Analysis Reveal That Transfer from Brackish Water to Freshwater Is More Stressful to the Silverside Odontesthes humensis.

[This corrects the article DOI: 10.3389/fgene.2018.00028.].

Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions.

Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes.

Gene and Blood Analysis Reveal That Transfer from Brackish Water to Freshwater Is More Stressful to the Silverside Odontesthes humensis.

Silversides are fish that inhabit marine coastal waters, coastal lagoons, and estuarine regions in southern South America. The freshwater (FW) silversides have the ability to tolerate salinity variations. Odontesthes humensis have similar habitats and biological characteristics of congeneric O. bonariensis, the most studied silverside species and with great economic importance. Studies revealed that O. bonariensis is not fully adapted to FW, despite inhabiting hyposmotic environments in nature. However, there is little information about stressful environments for cultivation of silverside O. humensis. Thus, the aim of this study was to evaluate the stress and osmoregulation responses triggered by the osmotic transfers on silverside O. humensis. Silversides were acclimated to FW (0 ppt) and to brackish water (BW, 10 ppt) and then they were exposed to opposite salinity treatment. Silverside gills and blood were sampled on pre-transfer (D0) and 1, 7, and 15 days (D1, D7, and D15) after changes in environmental salinity, the expression levels of genes atp1a3a, slc12a2b, kcnh1, and hspa1a were determined by quantitative reverse transcription-PCR for evaluation of osmoregulatory and stress responses. Furthermore, glycemia, hematocrit, and osmolality were also evaluated. The expression of atp1a3a was up- and down-regulated at D1 after the FW-BW and BW-FW transfers, respectively. Slc12a2b was up-regulated after FW-BW transfer. Similarly, kcnh1 and hspa1a were up-regulated at D1 after the BW-FW transfer. O. humensis blood osmolality decreased after the exposure to FW. It remained stable after exposure to BW, indicating an efficient hyposmoregulation. The glycemia had a peak at D1 after BW-FW transfer. No changes were observed in hematocrit. The return to the pre-transfer levels at D7 after the significant increases in responses of almost all evaluated molecular and blood parameters indicated that this period is enough for acclimation to the experimental conditions. In conclusion, our results suggest that BW-FW transfer is more stressful to O. humensis than FW-BW transfer and the physiology of O. humensis is only partially adapted to FW.