Sea cucumbers in a high temperature and low dissolved oxygen world: Roles of miRNAs in the regulation of environmental stresses

Highlights

• MiRNA profiles of sea cucumber under thermal, hypoxic and the combination were constructed.

• The typical DE-miRNAs in response to heat and oxygen deficiency were identified and validated.

• The potential target genes of DE-miRNAs mainly involved in biosynthesis, metabolism, immunity, and etc.

• Potential molecular regulation mechanism of the A. japonicus under global warming were provided.

Abstract

The exacerbation of global warming has driven changes in environmental factors, including water temperature and oxygen concentration. The sea cucumber Apostichopus japonicus, an economically important aquatic animal, is constantly and directly challenged by heat and hypoxia. In this study, 12 small RNA libraries were constructed for this species, and a total of 21, 26 and 22 differentially expressed (DE) miRNAs were clarified in A. japonicus under thermal (26 °C), hypoxic (2 mg/L) and the combined stresses. Comparative miRNA sequencing analysis and real-time PCR were used to identify and validate the representative miRNAs, including Aja-miR-novel-299, Aja-let-7b-3p, Aja-miR-71b-5p, Aja-miR-novel-13218 and Aja-miR-2004 in response to high temperature, and Aja-miR-92b-3p, Aja-miR-210–5p and Aja-miR-novel-26331 in response to oxygen limitation. GO and KEGG pathway analysis revealed that the potential target genes of DE-miRNAs involved in biosynthesis, metabolism, immunity, cell growth and death, translation and signaling transduction. Key DE-miRNAs with potentially targeted genes associated with heat shock and hypoxia response were also determined. These results may help explaining the role of miRNA regulation in stress resistance, as well as the potential molecular regulation mechanism of the echinoderm A. japonicus in the context of global warming.

Introduction

The sea cucumber (Apostichopus japonicus) is one of the most commercially important aquatic species in Asia. However, as a result of global warming, deteriorating water conditions are increasingly challenging the survival of sea cucumbers and their development for the mariculture industry. For example, 174,340 tons of sea cucumbers were harvested in 2018—a reduction of approximately 20.72% compared with 2017 (Ministry of Agriculture, 2018). The main causes of the massive mortality were extremely high temperature and low dissolved oxygen concentration. In China, the mean surface temperature has shown an increase of 0.4–0.6 °C in the last 100 years (IPCC, 2001), and it is predicted to be continue to rise by 1.7 °C in the next 30 years and by 2.2 °C over the next 50 years (Qin, 2003). Based on the measured value of summertime bottom water DO from the 1980s–2010s, a remarkable decline was observed in the central Bohai Sea in China (Zhai, 2019). For example, the DO was at rather high levels of over 190 μmol O₂/L in 1982 (Tang, 1997). But in late summer 2015, the historically lowest measured Bohai Sea DO of 67 μmol O₂/L was observed (Zhai, 2019). Sea cucumbers were greatly impacted by thermal and hypoxic stress at the level of genes, proteins and metabolites (Huo et al., 2019a, 2019b, 2020). Sea cucumbers likely developed specific strategies in response to the environmental stress.

Organisms would like respond with cellular modifications to tolerate the adverse environment, including transcriptional, translational and post-translational modification. The action of non-coding RNA molecules is indispensable in post-transcriptional regulation. MicroRNAs (miRNAs) represent a class of small, non-coding RNA molecules with a length of 18–28 nucleotides (Tanase et al., 2012). Argonautes (Agos) first bind to endogenous miRNA, and guide strands are incorporated into Agos after discarding passenger strands. Finally, mature RNA-induced silencing complex (RISC) is guided to complementary target mRNAs. According to the complementarity, RISC would endonucleolytically cleave the target mRNA or reduced translation and/or stability of target mRNAs (Janas et al., 2012). MiRNAs may play roles in regulating gene expression and restoring homeostasis (Leung and Sharp, 2010), and they were considered as key modulators for the development of abiotic stress tolerance (Noman et al., 2017). In recent years, a multitude of reports have demonstrated that specific miRNAs are involved in the hypoxic response and thermal response, and contribute to the regulation of biologically important genes in low oxygen tension and high temperature, such as hypoxia-inducible factor-1α (HIF-1α) (Liang, 2017), vascular endothelial growth factor (VEGF) (Hua, 2006), heat shock protein 70 (Hsp70) (Yin, 2009), heat shock cognate protein 70 (Scott, 2012), Hsp72 (Beninson, 2014), Hsp60 (Shan, 2010) and etc. In previous studies of sea cucumbers, miRNAs changed in response to skin ulceration syndrome (Sun et al., 2016, 2018), intestine regeneration (Sun et al., 2017), aestivation (Chen et al., 2013), heat shock response (Li and Xu, 2018), salinity stress (Tian et al., 2019) and hypoxia stress (Huo et al., 2017). As shown previously, heat is usually accompanied by hypoxia (Huo et al., 2020). However, little attention has been paid to combined environmental stressors.

Therefore, we conducted experiments to determine the effects of three types of environmental stresses on the miRNA profiles in sea cucumber, including thermal stress, hypoxic stress and a combination of the two. Our main objectives were to identify and characterize the differentially expressed (DE) miRNAs, and to determine the biological processes with which they are mainly involved. Our results confirmed that miRNAs are involved in the regulation mechanisms of aquatic animals exposed to environmental stress. They will also help facilitate understanding on the molecular regulation mechanisms of sea cucumbers in the context of global warming.