Neurobehavioral disorders induced by environmental zinc in female zebrafish (Danio rerio): Insights from brain and intestine transcriptional and metabolic signatures.

Human activities can cause zinc (Zn) contamination of aquatic environments. Zn is an essential trace metal, but effects of environmentally relevant Zn exposure on the brain-intestine axis in fish are poorly understood. Here, six-month-old female zebrafish (Danio rerio) were exposed to environmentally relevant Zn concentrations for six weeks. Zn significantly accumulated in the brain and intestine, causing anxiety-like behaviors and altered social behaviors. Zn accumulation altered levels of neurotransmitters, including serotonin, glutamate, and γ-aminobutyric acid, in the brain and intestine, and these changes were directly associated with changes in behavior. Zn caused oxidative damage and mitochondrial dysfunction, and impaired NADH dehydrogenase, thereby dysregulating the energy supply in brain. Zn exposure resulted in nucleotide imbalance and dysregulation of DNA replication and the cell cycle, potentially impairing the self-renewal of intestinal cells. Zn also disturbed carbohydrate and peptide metabolism in the intestine. These results indicate that chronic exposure to Zn at environmentally relevant concentrations dysregulates the bidirectional interaction of the brain-intestine axis with respect to neurotransmitters, nutrients, and nucleotide metabolites, thereby causing neurological disorder-like behaviors. Our study highlights the necessity to evaluate the negative impacts of chronic environmentally relevant Zn exposure on the health of humans and aquatic animals.

Changes in aggression and locomotor behaviors in response to zinc is accompanied by brain cell heterogeneity and metabolic and circadian dysregulation of the brain-liver axis.

Zinc is an essential nutrient for life, but over-accumulation can result in toxicity. Anthropogenic activities can increase zinc concentrations in aquatic environments (e.g., to ∼0.46-1.00 mg/L), which are above the safe level of 0.1 mg/L. We investigated the behavior and physiology of zebrafish (Danio rerio) in response to environment-related exposure to zinc chloride at 0.0 (Ctrl), 1.0 (ZnCl2-low) and 1.5 (ZnCl2-high) mg/L for 6 weeks (the zinc conversion ratio of zinc chloride is ∼0.48 and the nominal (measured) values were: Ctrl, 0 (∼0.01); ZnCl2-low, 0.48 (∼0.51); ZnCl2-high, 0.72 (∼0.69) mg/L). Low-zinc exposure resulted in significantly increased locomotion and fast moving behaviors, while high-zinc exposure resulted in significantly increased aggression and freezing frequency. Single cell RNA-seq of neurons, astrocytes, and oligodendrocytes of the brain revealed expression of genes related to ion transport, neuron generation, and immunomodulation that were heterogeneously regulated by zinc exposure. Astrocyte-induced central nervous system inflammation potentially integrated neurotoxicity and behavior. Integrated analyses of brain and hepatic transcriptional signatures showed that genes (and pathways) dysregulated by zinc were associated with sensory functions, circadian rhythm, glucose and lipid metabolism, and amyloid ß-protein clearance. Our results showed that environment-related zinc contamination can be heterogeneously toxic to brain cells and can disturb coordination of brain-liver physiology. This may disrupt neurobehavior and cause a neurodegeneration-like syndrome in adult zebrafish.

Zinc alters behavioral phenotypes, neurotransmitter signatures, and immune homeostasis in male zebrafish (Danio rerio).

Anthropogenic activities discharge zinc into aquatic ecosystems, and the effects of long-term and low-concentration zinc exposure on fish behavior are unclear. We evaluated the behavior and physiology of male zebrafish (Danio rerio) after a 6-week exposure to 1.0 or 1.5 ppm (mg/L) zinc chloride. The exposure caused anxiety-like behaviors and altered the social preferences in both exposure groups. Analysis of transcriptional changes suggested that in the brain, zinc exerted heterogenetic effects on immune and neurotransmitter functions. Exposure to 1.0 ppm zinc chloride resulted in constitutive immune dyshomeostasis, while exposure to 1.5 ppm zinc chloride impaired the neurotransmitter glutamate. In the intestine, zinc dysregulated self-renewal of intestinal cells, a potential loss of defense function. Moreover, exposure to 1.5 ppm zinc chloride suppressed intestinal immune functions and dysregulated tyrosine metabolism. These behavioral alterations suggested that the underlying mechanisms were distinct and concentration-specific. Overall, environmental levels of zinc can alter male zebrafish behaviors by dysregulating neurotransmitter and immunomodulation signatures.

Heat Shock Procedure Affects Cell Division-Associated Genes in Gynogenetic Manipulation.

Heat shock procedure is crucial for gynogenetic manipulation leading to diploidization of the maternal genomes; however, the underlying molecular mechanism especially the transcriptomic changes during this procedure has still not been unveiled yet. Here, the artificial gynogenesis of zebrafish (Danio rerio) using inactivated sperm from rare minnow (Gobiocypris rarus) was conducted. We found that artificial gynogenetic manipulation, including pseudo-fertilization and heat shock, decreased hatching rates, whereas heat shock treatment alone had medium hatching rates. The first cleavage changed the expression of genes associated with RNA transcription and protein synthesis. A co-expression network regulated by hub genes GIT1, Sepsecs, and FLNB was significantly correlated with heat shock procedure. The cyclin family and cyclin-dependent kinase-related genes were lowly expressed in embryos from gynogenetic zebrafish, and genes involved in controlling the cell cycle and genomic stability were significantly altered by the gynogenetic treatment. Our results show the effects of artificial gynogenesis on embryos and describe changes in gene expression that suggest drastic changes take place in cell division by heat shock procedure. These findings will contribute to an understanding of the molecular basis for germplasm improving, including the purifying effect and allogynogenetic biological effect by gynogenesis.

[Sequences analysis of jlFABP2 and the correlation between polymorphisms and body weight gain in Cyprinus carpio var. jian].

Two replicate intestine fatty acid binding protein genes (jlFABP2a and jlFABP2b) were cloned from Cyprinus carpio var. jian using PCR. Both ORFs were 399 bp in length sharing 92.2% similarity with each other, and 88.0% and 90.5% with their counterpart in zebrafish, respectively. The gene structure of jlFABP2s was same as other FABPs, which contained four exons and three introns. Sequences and lengths of introns between 2a and 2b. were obviously different Phylogenetic tree displayed that two jlFABP2s corresponded to one zebrafish FABP2 which matches the fact that the chromosome number of common carp was twice of zebrafish. Real time-PCR showed that jlFABP2 genes mainly expressed in intestine and the expression level was very significantly higher than other tissues such as brain, liver, muscle, kidney, and gonad (P<0.01). The expression level of jlFABP2a was significantly (male, P<0.05) or very significantly (females, P<0.01) higher than 2b in intestine; and 2b was expressed slightly higher than 2a in other tissues. It seemed that 2a expressed specifically in intestine, while 2b expressed ubiquitously. Twelve and four SNP loci were found at jlFABP2a and 2b introns through comparison sequences from 8 individuals, respectively. Genotypes of I1-A15G, I1-A99G, I2-C487T, and I3-A27T on jlFABP2a were detected using PCR-RFLP in selection population of C. carpio var. jian. The SNP genotypes and individual weight gain correlation indicated that four SNPs were significantly (P<0.05 or P<0.01) associated with adult weight gain. Diplotype analysis displayed that individuals with genotype AGGGCCXX or AGGGXXAT grew faster than other individuals by 15%. The individuals with these two genotypes only occupied 9% in total selection populations, indicating the presence of large selection space. The 4 SNPs detected in this experiment can be used in C. carpio var. Jian growth selection breeding plan.

Two Δ6-desaturase-like genes in common carp (Cyprinus carpio var. Jian): structure characterization, mRNA expression, temperature and nutritional regulation.

Δ6-Desaturase is the rate-limiting enzyme involved in highly unsaturated fatty acid (HUFA) biosynthesis. There is very little information on the evolution and functional characterization of Δ6Fad-a and Δ6Fad-b in common carp (Cyprinus carpio var. Jian). In the present study, the genomic sequences and structures of two putative Δ6-desaturase-like genes in common carp genome were obtained. We investigated the mRNA expression patterns of Δ6Fad-a and Δ6Fad-b in tissue, hatching carp embryos, larvae by temperature shock and juveniles under nutritional regulation. Our results showed that the two Δ6Fad genes had identical coding exon structures, being comprised of 12 coding exons, and with introns of distinct size and sequence composition. They were not allelic variants of a single gene. Both Δ6Fad genes were highly expressed in liver, intestine (pyloric caeca) and brain. The Δ6Fad-a and Δ6Fad-b mRNAs showed an increase in expression from newly hatched to 25 days after hatching. The expression levels of Δ6Fad-a were obviously regulated by temperature, whereas Δ6Fad-b was not affected by temperature. The regulation of Δ6Fad-a and Δ6Fad-b in response to dietary fatty acid composition was determined in liver, brain and intestine (pyloric caeca) of common carp fed with diets: diet1with fish oil (FO) rich in n-3 HUFA, diet2 with corn oil (CO, 18:2n-6) and diet3 with linseed oil (LO, 18:3n-3). The differential expression of Δ6Fad-a and Δ6Fad-b genes in liver, brain and intestine in common carps was fed with different oil sources, respectively. Further work is in progress to determine the mechanism of differential expression of the Δ6Fad-a and Δ6Fad-b genes in different tissues and the roles of transcription factors in regulating HUFA synthesis.

Identification of housekeeping genes suitable for gene expression analysis in Jian carp (Cyprinus carpio var. jian).

Jian carp (Cyprinus carpio var. jian) is an important economic fish species cultured in China. In this report, we performed a systematic analysis to identify an appropriate housekeeping (HK) gene for the study of gene expression in Jian carp. For this purpose, partial DNA sequences of four potential candidate genes (elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate (GAPDH), beta-actin (ACTB), and 18S ribosomal RNA (18S rRNA) were isolated, and their expression levels were studied using RNA extracted from nine tissues (forebrain, hypothalamus, liver, fore-intestine, hind-intestine, ovary, muscle, heart, kidney) in juvenile and adult Jian carp. Gene expression levels were quantified by quantitative real time RT-PCR (qRT-PCR), and expression stability was evaluated by comparing the coefficients of variation (CV) of the Ct values. The results showed that EF-1α was the most suitable HK gene in all tissues of juvenile and adult Jian carp. However, at distinct juvenile and adult developmental stages, there was not a single optimal gene for normalization of expression levels in all tissues. EF-1α was the most stable gene only in forebrain, hypothalamus, liver, heart, and kidney. These results provide data that can be expected to aid gene expression analysis in Jian carp research, but underline the importance of identifying the optimal HK gene for each new experimental paradigm.

Influence of dietary fatty acids on muscle fatty acid composition and expression levels of Δ6 desaturase-like and Elovl5-like elongase in common carp (Cyprinus carpio var. Jian).

The effects of dietary fatty acids on muscle fatty acid composition and liver expression levels of Δ6 desaturase-like and Elovl5-like elongase were investigated in common carp (Cyprinus carpio var. Jian). Two Δ6 desaturase-like cDNAs (Fad6-a and Fad6-b) and two Elovl5-like elongase (Elovl5-a and Elovl5-b) cDNAs were cloned. Juvenile carp were fed three semi-purified diets (D1-3) for 6 weeks with different lipid sources: D1, fish oil with high highly unsaturated fatty acids (HUFAs); D2, corn oil with high linoleic acid (LA), but no HUFAs; and D3, linseed oil with high α-linolenic acid (LNA), but no HUFAs. Comparing muscle fatty acids among fish fed D1-3, the content of LA and arachidonic acid (AA) in common carp fed D2 and the content of LNA, EPA and DHA in common carp fed D3 were higher than initial levels (P<0.05), respectively. The liver transcript levels of Fad6-a and Elovl5-a in fish fed D2 and D3 were higher than initial levels (P<0.05), but Fad6-b and Elovl5-b levels were seldom affected by the diets. The dietary fatty acids affect the muscle fatty acid composition and the liver Fad6-a and Elovl5-a gene expression levels in common carp, and further studies should be undertaken.

Molecular and expression analysis of apolipoprotein E gene in the Chinese sturgeon, Acipenser sinensis.

Apolipoproteins are carrier proteins that bind to lipids to form lipoprotein particles and have been shown to play an important role in lipid metabolism. In this study, a full-length cDNA for apolipoprotein E, named AsapoE, was cloned from the Chinese sturgeon (Acipenser sinensis). This cDNA sequence is 1289 bp in length, and codes for a polypeptide of 274 amino acid residues, which is 45% and 42% identical to that of the rainbow trout and zebrafish, respectively, and 39%, 30%, and 29% identical to frog, mouse, and human respectively. The predicted AsApoE protein has a conserved amphipathic α-helix region with the potential to bind to lipids. RT-PCR analysis reveals that AsapoE is expressed in all tissues examined with a preferential expression in the kidney and liver. During the embryo development stage, AsapoE mRNA is low but still detectable at gastrula stage embryos; then AsapoE mRNAs reach a higher level in muscle contraction stage embryos, this relatively stable expression persists during the following embryogenic stages and declines 1 day after hatching. These results will serve as a basis for comparative studies on vertebrate apoE genes.

Cloning, structure, and expression pattern of the P-450 aromatase gene in rice field eel (Monopterus albus).

We report the cloning, tissue expression, and structural analysis of the aromatase gene in the rice field eel (Monopterus albus). The ovary-derived cDNA (1,802 bp) has a 49 bp 5'-untranslated region (UTR), a 202 bp 3'-UTR, and a 1,551 bp open-reading frame, which encodes a protein of 517 amino acid residues with a predicted molecular weight of 58.2 kDa. The amino acid sequence alignment suggests that the rice field eel ovarian P-450 aromatase shares 63-80% identity with that of other fish species, reduced to 59-60% with brain-derived aromatases of other fishes and to 50% with human placenta aromatases. Between the 5' and 3' untranslated terminal regions, the rice field eel CYP19 gene contained seven introns at the same sites as in medaka and human but lacked an intron between the I-helix and the aromatase-specific conserved region. All introns conformed to the GT/AG rule. Sequence analysis of the 1,065 bp upstream of the translation start site revealed that the transcription initiation site was 51 bp upstream from the translation start site. This region had one estrogen receptor recognition half site (nt -62), five copies of an SRY/iSRY binding motif, a C/EBP (CCAAT enhancer binding protein) binding site (nt -751), chicken ovalbumin upstream promoter-transcription factor (nt -986) and GATA-2 (nt -186, -249) recognition sequences, but no binding sequence for steroidogenic factor-1 and the cAMP response element binding protein activating transcription factor family. In females, levels of relative expression were, in descending order, hypothalamus, pituitary, forebrain, ovary, and liver. In males, P450arom was detected only in the pituitary and the liver, with half the expression found in females. In fry, the P450arom expression level increased during development and was significantly higher in the brain than in the gonad.