Transcriptome analysis reveals fluid shear stress (FSS) and atherosclerosis pathway as a candidate molecular mechanism of short-term low salinity stress tolerance in abalone

Abstract

Background: Transcriptome sequencing is an effective tool to reveal the essential genes and pathways underlying countless biotic and abiotic stress adaptation mechanisms. Although severely challenged by diverse environmental conditions, the Pacific abalone Haliotis discus hannai remains a high-value aquaculture mollusk and a Chinese predominantly cultured abalone species. Salinity is one of such environmental factors whose fluctuation could significantly affect the abalone's cellular and molecular immune responses and result in high mortality and reduced growth rate during prolonged exposure. Meanwhile, hybrids have shown superiority in tolerating diverse environmental stresses over their purebred counterparts and have gained admiration in the Chinese abalone aquaculture industry. The objective of this study was to investigate the molecular and cellular mechanisms of low salinity adaptation in abalone. Therefore, this study used transcriptome analysis of the gill tissues and flow cytometric analysis of hemolymph of H. discus hannai (DD) and interspecific hybrid H. discus hannai ♀ x H. fulgens ♂ (DF) during low salinity exposure. Also, the survival and growth rate of the species under various salinities were assessed.

Results: The transcriptome data revealed that the differentially expressed genes (DEGs) were significantly enriched on the fluid shear stress and atherosclerosis (FSS) pathway. Meanwhile, the expression profiles of some essential genes involved in this pathway suggest that abalone significantly up-regulated calmodulin-4 (CaM-4) and heat-shock protein90 (HSP90), and significantly down-regulated tumor necrosis factor (TNF), bone morphogenetic protein-4 (BMP-4), and nuclear factor kappa B (NF-kB). Also, the hybrid DF showed significantly higher and sustained expression of CaM and HSP90, significantly higher phagocytosis, significantly lower hemocyte mortality, and significantly higher survival at low salinity, suggesting a more active molecular and hemocyte-mediated immune response and a more efficient capacity to tolerate low salinity than DD.

Conclusions: Our study argues that the abalone CaM gene might be necessary to maintain ion equilibrium while HSP90 can offset the adverse changes caused by low salinity, thereby preventing damage to gill epithelial cells (ECs). The data reveal a potential molecular mechanism by which abalone responds to low salinity and confirms that hybridization could be a method for breeding more stress-resilient aquatic species.

Keywords: Abalone; FSS pathway; Immunity; Interspecific hybrid; Low salinity; Transcriptomics.

Fig. 1

Principal Component Analysis Results Map (PCA) for gill tissues of abalone showing separation between the controls (red color), the low salinity-stressed groups at 3 h (blue color) and 24 h (green color), and between the species: Haliotis discus hannai (DD; circles), hybrid H. discus hannai ♀ × H. fulgens ♂ (DF, triangles)

Fig. 2

Volcano plots of the differentially expressed genes (DEGs) of gill tissues of abalone during short-term low salinity exposure: H. discus hannai (DD) and hybrid H. discus hannai ♀ × H. fulgens ♂ (DF). A Comparison between DD and DF at the control, B Comparison between DD and DF after 3 h at low salinity, and C Comparison between DD and DF after 24 h at low salinity

Fig. 3

Comparison of top 20 Kyoto Encyclopedia of Gene and Genome (KEGG) pathways enrichment statistics of gill tissues of abalone during short-term low salinity exposure. The FSS pathway is highlighted with a rectangular red box. H. discus hannai (DD) and hybrid H. discus hannai ♀ × H. fulgens ♂ (DF). A Comparison between control DD (CD) and control DF (CF), B Comparison between low salinity groups of DD after 3 h (D3) and DF after 3 h (F3), and C Comparison between low salinity groups of DD after 24 h (D24) and DF after 24 h (F24). The size of each point represents the number of genes annotated to the KEGG pathway. Different colors from yellow to mauve represent the p-value of the enrichment

Fig. 4

A Heat map showing the expression pattern of some genes involved in the FSS pathway in abalone. B Hypothesized model of the FSS pathway in abalone showing crosstalk between some genes (adapted from Kanehisa and Goto [30]). Hypothetically, up-regulation of all the genes under stable hemolymph flow (on the left) should lead to gill ECs anti-oxidation and anti-inflammation, while up-regulation of all the genes under disturbed hemolymph flow (on the right) should lead to gill ECs inflammation and apoptosis. Oval shapes denote genes that were validated by qRT-PCR: Red color denotes genes that were up-regulated in expression relative to the control. Blue color denotes genes that were down-regulated in expression relative to the control. Grey-colored rectangular boxes denote genes that were not expressed in the current study

Fig. 5

Expression profile of five FSS pathway genes in abalone gills during short-term hyposmotic stress: A The Pacific abalone Haliotis discus hannai (DD), and B Hybrid H. discus hannai ♀ × H. fulgens ♂ (DF), (I) Calmodulin-4, (II) Heat shock protein90, (III) Tumor necrosis factor, (IV) Bone morphogenetic protein-4, and (V) Nuclear factor kappa-B. Sampling times = “0 h”: control group; “3 h”, “12 h”, and “24 h”: exposure times at low salinity. Statistical analysis was performed by One-way ANOVA, followed by Turkey’s HSD test. Different alphabets denote a significant difference between sampling times. Differences were deemed significant at P < 0.05

Fig. 6

Comparison of the gene expression profiles between Haliotis discus hannai (DD) and hybrid H. discus hannai ♀ × H. fulgens ♂ (DF) during short-term low salinity stress: A Calmodulin-4, B Heat shock protein90, C Tumor necrosis factor, D Bone morphogenetic protein-4, and E Nuclear factor kappa-B. Sampling times = “0 h”: control group; “3 h”, “12 h”, and “24 h”: exposure times at low salinity. Statistical analysis was performed by Welch’s t-test at a given sampling time. Differences were deemed significant at P < 0.05. “*”: P = 0.01; “**”: P = 0.004; “***”: P = 0.000

Fig. 7

Flow cytometry analysis of Haliotis discus hannai (DD) and hybrid H. discus hannai ♀ × H. fulgens ♂ (DF) hemocytes during short-term hyposmotic stress: A Total hemocyte count (THC), B Hemocyte mortality, C Reactive oxygen species (ROS), and D Phagocytosis. “0 h”: control group; “3 h”, “12 h”, and “24 h”: exposure times at low salinity. One-way ANOVA, followed by Turkey’s HSD test, was used to analyze the differences between the sampling times for each species. Analysis of the differences between the species at a given sampling time was done by Welch’s t-test. Asterisks = significant difference between DD and DF at the given sampling time: “*”: P = 0.02; “**”: P = 0.002; “***”: P = 0.000. The bar indicates no significant difference between sampling times (P > 0.05) for the individual species

Fig. 8

Survival and growth of Haliotis discus hannai (DD) and hybrid H. discus hannai ♀ × H. fulgens ♂ (DF) cultured under various salinities for 60 days: A Survivorship, B Specific growth rate in shell length (SGR_SL), C Specific growth rate in shell width (SGR_SW), and D Specific growth rate in wet weight (SGR_WT). Analysis of the differences between the species at a given salinity level was done by Welch’s t-test “*”: Significant difference between DD and DF at P < 0.05