Integrated transcriptome and microRNA analysis reveals molecular responses to high-temperature stress in the liver of American shad (Alosa sapidissima).

BACKGROUND: Fish reproduction, development and growth are directly affected by temperature, investigating the regulatory mechanisms behind high temperature stress is helpful to construct a finer molecular network. In this study, we systematically analyzed the transcriptome and miRNA information of American shad (Alosa sapidissima) liver tissues at different cultivation temperatures of 24 â„ƒ (Low), 27 â„ƒ (Mid) and 30 â„ƒ (High) based on a high-throughput sequencing platform. RESULTS: The results showed that there were 1594 differentially expressed genes (DEGs) and 660 differentially expressed miRNAs (DEMs) in the LowLi vs. MidLi comparison group, 473 DEGs and 84 DEMs in the MidLi vs. HighLi group, 914 DEGs and 442 DEMs in the LowLi vs. HighLi group. These included some important genes and miRNAs such as calr, hsp90b1, hsp70, ssa-miR-125a-3p, ssa-miR-92b-5p, dre-miR-15a-3p and novel-m1018-5p. The DEGs were mainly enriched in the protein folding, processing and export pathways of the endoplasmic reticulum; the target genes of the DEMs were mainly enriched in the focal adhesion pathway. Furthermore, the association analysis revealed that the key genes were mainly enriched in the metabolic pathway. Interestingly, we found a significant increase in the number of genes and miRNAs involved in the regulation of heat stress during the temperature change from 24 °C to 27 °C. In addition, we examined the tissue expression characteristics of some key genes and miRNAs by qPCR, and found that calr, hsp90b1 and dre-miR-125b-2-3p were significantly highly expressed in the liver at 27 â„ƒ, while novel-m0481-5p, ssa-miR-125a-3p, ssa-miR-92b-5p, dre-miR-15a-3p and novel-m1018-5p had the highest expression in the heart at 30℃. Finally, the quantitative expression trends of 10 randomly selected DEGs and 10 DEMs were consistent with the sequencing data, indicating the reliability of the results. CONCLUSIONS: In summary, this study provides some fundamental data for subsequent in-depth research into the molecular regulatory mechanisms of A. sapidissima response to heat stress, and for the selective breeding of high temperature tolerant varieties.

Proteomics and metabolomics analysis of American shad (Alosa sapidissima) liver responses to heat stress.

The dramatic changes in the global climate pose a major threat to the survival of many organisms, including fish. To date, the regulatory mechanisms behind the physiological responses of fish to temperature changes have been studied, and a comprehensive analysis of the regulatory mechanisms of temperature tolerance will help to propose effective strategies for fish to cope with global warming. In this study, we investigated the expression profiles of proteins and metabolites in liver tissues of American shad (Alosa sapidissima) corresponding to different water temperatures (24 °C, 27 °C and 30 °C) at various times (1-month intervals) under natural culture conditions. Proteomic analysis showed that the expression levels of the heat shock protein family (e.g. HSPE1, HSP70, HSPA5 and HSPA.1) increase significantly with temperature and that many differentially expressed proteins were highly enriched especially in pathways related to the endoplasmic reticulum, oxidative phosphorylation and glycolysis/gluconeogenesis processes. In addition, the results of conjoint metabolomics and proteomics analysis suggested that the contents of several important amino acids and chemical compounds, including L-serine, L-isoleucine, L-cystine, choline and betaine, changed significantly under high-temperature environmental stress, affecting the metabolic levels of starch, amino acid and glucose, which is thought to represent a possible energy conservation method for A. sapidissima to cope with rapid changes in external temperature. In summary, our findings demonstrate that living under high temperatures for a long period of time leads to different physiological defense responses in A. sapidissima, which provides some new ideas for analyzing the molecular regulatory patterns of adaptation to high temperature and also provides a theoretical basis for the subsequent improvement of fish culture in response to global warming.

Molecular characterization and function of hif1a and fih1 in response to acute thermal stress in American shad (Alosa sapidissima).

In order to evaluate the function of hypoxia-inducible factor-1 alpha (hif1α) and factor inhibiting hif1α (fih1) in response to thermal stress, we first conducted a functional analysis of A. sapidissima hif1α and fih1, and determined hif1α and fih1 expressions in different tissues in response to thermal stress based on identified housekeeping genes (HKGs). The results showed that hif1α and fih1 were mainly located in the nucleus and cytoplasm. The full length cDNA sequence of hif1α and fih1 was 4073 bp and 2759 bp, respectively. The cDNA sequence of hif1α includes 15 exons encoding 750 amino acid residues, and the full length cDNA sequence of fih1 contains 9 exons encoding 354 amino acid residues. During the acute thermal stress transferring from 16 ± 0.5 °C (control) to 20 ± 0.5 °C, 25 ± 0.5 °C, and 30 ± 0.5 °C for 15 min, it was found that the expression trends of hif1α and fih1 showed an inhibitory regulation in the heart, while they consistently expressed in brain, intestine, muscle, gill, kidney and liver. In conclusion, this is the first study to identify the tissue-specific HKGs in A. sapidissima and found that ef1α and ß-actin are the most suitable HKGs. Hif1α and Fih1 are mainly the nuclear and cytoplasmic proteins, respectively, having high levels in the heart and brain. Alosa sapidissima countered a temperature increase from 16 to 25 ℃ by regulating the expressions of hif1α and fih1, but their physiological regulatory functions were unable to cope with acute thermal stress when the temperature difference was 14 ℃ (from 16 to 30 ℃).

Proximate composition, lipid utilization and validation of a non-lethal method to determine lipid content in migrating American shad Alosa sapidissima.

Lipid content forms the most important energy reserve in anadromous fish and can limit survival, migration and reproductive success. A fat meter was evaluated and compared with a traditional extractive method of measuring available lipid for migrating American shad Alosa sapidissima in the Connecticut River, U.S.A. The fat meter gives rapid (<10 s) and non-lethal lipid measurements, whereas traditional methods require lethal sampling that is both time consuming and expensive. The fat-meter readings had a strong relationship to traditional lipid extractions for 60 fish, 30 whole body (R2 = 0·72) and 30 fillet only (R2 = 0·81). Additional validation showed that fat-meter readings captured the gradual decrease of lipid in individual fish over time, were not affected by removal of gonads or scales and were stable for fish exposed to water or air for 24 h after death. These experiments indicate that the fat meter can be used as a reliable tool for future A. sapidissima energetic studies, allowing for larger sample sizes and non-lethal sampling.

Developmental changes in digestive enzyme activity in American shad, Alosa sapidissima, during early ontogeny.

In order to assess the digestive physiological capacity of the American shad Alosa sapidissima and to establish feeding protocols that match larval nutritional requirements, we investigated the ontogenesis of digestive enzymes (trypsin, amylase, lipase, pepsin, alkaline phosphatase, and leucine aminopeptidase) in larvae, from hatching to 45 days after hatching (DAH). We found that all of the target enzymes were present at hatching, except pepsin, which indicated an initial ability to digest nutrients and precocious digestive system development. Trypsin rapidly increased to a maximum at 14 DAH. Amylase sharply increased until 10 DAH and exhibited a second increase at 33 DAH, which coincided with the introduction of microdiet at 30 DAH, thereby suggesting that the increase was associated with the microdiet carbohydrate content. Lipase increased until 14 DAH, decreased until 27 DAH, and then increased until 45 DAH. Pepsin was first detected at 27 DAH and then sharply increased until 45 DAH, which suggested the formation of a functional stomach. Both alkaline phosphatase and leucine aminopeptidase markedly increased until 18 DAH, which indicated intestinal maturation. According to our results, we conclude that American shad larvae possess the functional digestive system before mouth opening, and the significant increases in lipase, amylase, pepsin, and intestinal enzyme activities between 27 and 33 DAH suggest that larvae can be successfully weaned onto microdiets around this age.

High-temperature stress response: Insights into the molecular regulation of American shad (Alosa sapidissima) using a multi-omics approach.

High temperature is an important abiotic stressor that limits the survival and growth of aquatic organisms. American shad (Alosa sapidissima), a migratory fish suitable for culturing at low temperatures, is known for its delicious taste and thus has high economic value. Studies concerning changes in A. sapidissima under high temperature are limited, especially at the gene expression and protein levels. High-temperature stress significantly reduced the survival rates and increased vacuolar degeneration and inflammatory infiltration in the gills and liver. High temperature increased the activities of SOD, CAT, and cortisol, with a trend of initial increase followed by decreases in MDA, ALP, and LDH, and irregular changes in T-AOC and Na-K-ATPase. Comprehensive analysis of the transcriptome, proteome, and metabolome of gills from fish treated with different culture temperatures (24, 27, and 30 °C) revealed that differentially expressed genes, proteins, and metabolites were highly enriched in pathways involved in protein digestion and absorption, protein processing in endoplasmic reticulum, metabolic pathways, and purine metabolism. Gene expression and protein profiles indicated that genes coding for antioxidants (i.e., cat and alpl) and members of the heat shock protein (i.e., HSP70, HSP90AA1, and HSP5) were significantly upregulated. Additionally, a conjoint analysis revealed that several key enzymes, including nucleoside diphosphate kinase 2, adenosine deaminase, and ectonucleoside triphosphate diphosphohydrolase 5/6 were altered, thereby affecting the metabolism of guanosine, guanine, and inosine. An interaction network further confirmed that levels of the essential amino acids DL-arginine and L-histidine were significantly reduced, and corticosterone levels were significantly increased, suggesting that A. sapidissima may be more dependent on amino acids for energy in vivo. Overall, this work suggests that living in a high-temperature environment leads to differential defense responses in fishes. The results provide novel perspectives for studying the molecular basis of adaptation to climate change in A. sapidissima and for genetic selection.

First report of de novo assembly and annotation from brain and blood transcriptome of an anadromous shad, Alosa sapidissima.

OBJECTIVES: American shad (Alosa sapidissima) is an important migratory fish under Alosinae and has long been valued for its economic, nutritional and cultural attributes. Overfishing and barriers across the passage made it vulnerable to sustain. To protect this valuable species, aquaculture action plans have been taken though there are no published genetic resources prevailing yet. Here, we reported the first de novo assembled and annotated transcriptome of A. sapidissima using blood and brain tissues. DATA DESCRIPTION: We generated 160,481 and 129,040 non-redundant transcripts from brain and blood tissues. The entire work strategy involved RNA extraction, library preparation, sequencing, de novo assembly, filtering, annotation and validation. Both coding and non-coding transcripts were annotated against Swissprot and Pfam datasets. Nearly, 83% coding transcripts were functionally assigned. Protein clustering with clupeiform and non-clupeiform taxa revealed ~ 82% coding transcripts retained the orthologue relationship which improved confidence over annotation procedure. This study will serve as a useful resource in future for the research community to elucidate molecular mechanisms for several key traits like migration which is fascinating in clupeiform shads.

The next-generation sequencing reveals the complete mitochondrial genome of Alosa sapidissima (Perciformes: Clupeidae) with phylogenetic consideration.

The complete mitochondrial genome of the Alosa sapidissima is presented in this study. The mitochondrial genome is 16,741 bp long and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region. The gene order and composition of Epinephelus awoara mitochondrial genome was similar to that of most other vertebrates. The nucleotide compositions of the light strand in descending order is 28.79% of G, 28.39% of T, 24.94% of A, and 17.88% of C. With the exception of the NADH dehydrogenase subunit 6 (ND6) and 8 tRNA genes, all other mitochondrial genes are encoded on the heavy strand. The phylogenetic analysis by maximum-likelihood (ML) method showed that the A. sapidissima has the closer relationship to between the A. alosa and A. pseudoharengus in the phylogenetic relationship.