Elsevier

Fish & Shellfish Immunology

Volume 74, March 2018, Pages 152-161
Fish & Shellfish Immunology

Full length article
Differential expression of microRNAs in hemocytes from white shrimp Litopenaeus vannamei under copper stress

https://doi.org/10.1016/j.fsi.2017.12.053Get rights and content

Highlights

  • miRNA deep sequencing was investigated in Cu exposed shrimp Litopenaeus vannamei.

  • 4524 known miRNAs and 73 novel miRNAs were significantly (P < 0.05) differentially expressed after Cu exposure.

  • Pearson's correlation coefficient (R) confirmed the consistency and accuracy between miRNA deep sequencing and qPCR.

  • Provided valuable information to take an insight into molecular mechanism of L. vannamei against environmental stress.

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that regulate diverse cellular processes, including organismal stress response, through posttranscriptional repression of gene transcripts. They are known to have antiviral functions in aquatic crustacean species, but little is known about the role of miRNAs against environmental stress caused by Cu, a common chemical contaminant in aquatic environment. We performed small RNA sequencing to characterize the differentially expressed microRNAs in Cu exposed shrimp. A total of 4524 known miRNAs and 73 novel miRNAs were significantly (P < .05) differentially expressed after Cu exposure. The peak size of miRNAs was 22 nt. Among them, 218 miRNAs were conserved across 115 species. The validation of 12 miRNAs by stem-loop quantitative RT-PCR were found to be coherent with the expression profile of deep sequencing data as evaluated with Pearson's correlation coefficient (r = 0.707). Target genes of these differentially expressed miRNAs related to immune defense, apoptosis, and xenobiotics metabolism also showed significant changes in expression under Cu stress. The present study provides the first characterization of L. vannamei miRNAs and some target genes expression in response to Cu stress, and the findings support the hypothesis that certain miRNAs along with their target genes might be essential in the intricate adaptive response regulation networks. Our current study will provide valuable information to take an insight into molecular mechanism of L. vannamei against environmental stress.

Introduction

Litopenaeus vannamei is the most important farmed shrimp species and is economically valuable. With the development of industrialization and intensive aquaculture, aquatic pollution has become a severe and growing problem. Among various environmental pollutants, heavy metals have been of great concern because of their inherent toxicity, non-degradability and persistence [1]. In aquaculture practices, copper sulfate is commonly used to eradicate filamentous algae and phytoplankton in shrimp farms. The excess application of copper sulfate in pond management may deteriorate the aquatic environment due to Cu accumulation in the pond sediments [2,3].

In crustaceans, hemocytes in the circulating hemolymph have vital roles in immune responses against pathogens, such as phagocytosis, coagulation, encapsulation, nodulation, production of antimicrobial peptides (AMPs) and proPO activity [4,5]. Consequently, toxic effects on hemocytes potentially affect the survival of these animals [6]. Cu has been reported to decrease the total hemocyte count (THC), respiratory burst activity and phenoloxidase activity of hemocytes in L. vannamei [7]. In our previous study, exposure of L. vannamei to Cu induced hemocyte apoptosis and the expression of antioxidant biomarker genes, apoptosis-related genes and a specific biomarker gene of heavy metal pollution [8]. However, the molecular mechanism of Cu stress in shrimp remains largely unclear.

Biological systems use a variety of mechanisms to maintain their functions in the face of environmental and genetic perturbations [9]. MicroRNAs (miRNAs), a class of small noncoding RNAs with 20–24 nt in length, can regulate gene expression at the post-transcriptional level [10] and play crucial regulatory roles in a large variety of biological processes, such as development, cell differentiation, apoptosis, oncogenesis, immune and stress response in various organisms [[11], [12], [13], [14]]. In animals, miRNAs regulate gene expression through imperfect sequence-specific binding to the 3′-untranslated regions (3′UTR) of the target mRNAs and usually cause translational repression [15]. A growing number of miRNAs were discovered and studied in variety of organisms since the first miRNA lin-4 has been discovered in Caenorhabditis elegans. To date, over 28 thousand miRNAs have been discovered across 223 species (miRBase, release 21.0; June 2014).

Initially, miRNAs were cloned and identified by a traditional method in M. japonicus [16]. However, PCR cloning followed by traditional sequencing remains very labor- and cost-intensive with a limited dynamic range to detect and define relative miRNA expression [17]. Recently, next-generation sequencing (NGS) technologies, including the Illumina Genome Analyzer (GA), Applied Biosystems SOLiD System, and 454 Life Sciences (Roche) FLX instruments, have emerged as well-established approaches for miRNA profiling, which can rapidly produce millions of sequence reads simultaneously with lower cost than Sanger sequencing and allows for the identification of low abundance miRNAs and genome-wide discovery of novel tissue-specific miRNAs with high specificity and sensitivity [[18], [19], [20]]. Deep-sequencing technologies have facilitated a sharp rise in the rate of novel microRNA discovery [21]. In recent years, miRNA studies have been performed using NGS technologies in crustaceans with limited genomic information [[22], [23], [24]].

Although hundreds of miRNAs have been identified, only a small number of shrimp miRNAs have been discovered and functionally identified in Marsupenaeus japonicus [22], Penaeus monodon [25] and L. vannamei [23] that responded to Vibrio alginolyticus or white spot syndrome virus infection. The investigation of miRNAs that responds to copper stress has not yet been performed. The objective of this study was to identify and characterize the differentially expressed miRNAs in shrimp L. vannamei in response to copper stress. The results will extend the knowledge of crustacean miRNA regulation and provide information for further research on shrimp response against environmental stress.

Section snippets

Animals

The experimental shrimp L. vannamei (4.94 ± 0.50 g) were obtained from a commercial farm in Zhanjiang (Guangdong, China), and acclimated for 2 weeks prior to experiment in cycling-filtered plastic tanks with aerated seawater at 26 ± 2 °C and a salinity of 5‰. During the acclimation period, shrimp were fed twice daily with shrimp diet (40% protein, 5.0% fat, 5.0% fiber and 16% ash, supplied by a commercial diet, China) until 24 h before the experimental treatments began. Only shrimp apparently

Illumina sequencing of miRNAs

To identify miRNAs involved in the response of L. vannamei to Cu exposure, two separate libraries of small RNA were constructed, with the mixed pools of hemocytes from shrimp after exposure for 0 h and 3 h, respectively. The two libraries were subjected to Illumina sequencing, and a total of 28,879,125 (0 h) and 24,495,187 (3 h) raw sequences were obtained, respectively. After removal of low quality reads, adapter sequences, poly A sequences, and sequences smaller than 18 nt, a total of

Discussion

Living animals are constantly faced with various environmental stress that challenge normal life. In recent years, many studies in comparative biochemistry field are now on the verge of dissecting the role of miRNAs in adaptation to environmental stress [37]. It has been suggested that miRNAs serve as key players in a robust adaptive response against stress in animals through their capacity to fine-tune gene expression [38]. The role of miRNAs in environmental stress response has been studied

Acknowledgements

This research was supported by the National Natural Science Foundation of China (31600321), Guangdong Provincial Natural Science Foundation (2015A030310438), Program for Scientific Research Start-Up Funds of Guangdong Ocean University (201710566003), Special Program for Outstanding Young Teachers of Guangdong Ocean University (HDYQ2015003). Guangdong Ocean University Student's Platform for Innovation and Entrepreneurship Training.

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