Full length articleIdentification and differential expression of hepatopancreas microRNAs in red swamp crayfish fed with emodin diet
Introduction
Red swamp crayfish (Procambarus clarkii) was native to the southeastern United States and had been introduced worldwide. It was the most common freshwater crayfish specie in China now. However, with the development of intensive culture, water pollution, environmental degradation and frequent occurrence of various diseases resulted in economic losses of aquaculture [1]. Like other crustanceans, crayfish lacked an adaptive immune system and depended exclusively on the innate immune system to defend against potential pathogenic bacteria, viruses, and parasites. Therefore, in recent years, appropriate dietary immunoenhancers such as probiotics, functional sugars, were widely used in aquaculture to improve shrimp and crayfish innate immune system.
Emodin (1, 3, 8-trihydroxy-6-methyl-anthraquinone), a medicinal extract from herbs including rhubarb (Rheum officinale Baill), aloe (Aloe barbadensis Miller), senna (Cassia angustifolia), and thunberg (Polygonum multiflorum), had been widely used as a traditional medicine in Eastern Asia, especially in China [2]. Previous reports showed that emodin played a roles in antibacterial and anti-inflammatory [3], antioxidation and free radical scavenging [4], reduction of blood lipid concentration [5], hepatoprotection [6], and immune regulation [7]. In human medical studies, several mechanisms of emodin had been described as possible modes of emodin antitumor action. Firstly, emodin generated a reactive oxygen species, which resulted in cancer cells apoptosis. Secondly, emodin induced primary DNA lesions through alkylation of DNA, which led to the perturbation of the cell cycle. Finally, emodin inhibited specific kinase activities that were required for cancer survival [8].
Now, emodin as immunopotentiator had been studied and applied in fish and crayfish culture. The research indicated that the growth, non-specific immunity and high temperature tolerance of freshwater prawn (Macrobrachium rosenbergii) fed with diet containing emodin were improved significantly [9]. The studies on Wuchang bream (Megalobrama amblycephala) showed that appropriate dietary emodin supplementation (30 mg emodin kg−1 diet) could enhance the growth and immune responses and improve fish resistance to infection by Aeromonas hydrophila [1], [10], [11]. In our previous study, the growth, survival rate of crayfish fed with 75 mg emodin kg−1 diet were significantly increased. Serum lysozyme, ceruloplasmin and alkaline phosphatase activities were significantly improved. Significantly higher levels of hepatopancreas catalase, glutathione and superoxide dismutase activities were also observed [12]. Even so, the detailed mechanism which emodin mediated growth and immune function still remained to be determined.
MicroRNAs (miRNAs) were a large family of 21–22 nucleotide non-coding RNAs, which played very important roles in regulating gene expression by degradation of target mRNAs or by repression of targeted gene translation both in animals and plants [13]. Recently, more and more evidences suggested that miRNAs had diverse biological functions, such as embryo formation, organogenesis, cell death, cell proliferation, lipid metabolism and immune development [14], [15], [16], [17], [18]. Studies on crayfish microRNAs supported that certain miRNAs along with their target genes might be essential in the intricate host–pathogen interaction networks, and should help developing new control strategies for host immune defense against various foreign pathogens's infection in crustaceans [19].
In this paper, we investigated whether miRNAs involved in growth and immunoregulation of crayfish fed with diet containing emodin by using high-throughput Illumina Solexa analysis. This study could contribute to understanding of the miRNAs roles in regulating innate immune response and identification of emodin-associated miRNAs in crayfish.
Section snippets
Experimental animals and feeding trial
All animal experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of China.
Formulation and proximate composition of the basal diet was presented in Table 1. The protein and lipid requirements for this species were set according to Xu et al. (2013) [1]. Fish meal, soybean meal, rapeseed meal and shrimp bran served as protein sources. Soybean oil was used as lipid sources. Wheat flour served as carbohydrate sources. Emodin
Length distribution and small RNAs annotation
Through high throughput Solexa sequencing using Illumina Hiseq2000, the total numbers of raw reads from control and trial small RNA libraries were 13,335,928 and 14,938,951 while high quality reads were 13,216,442 and 14,517,522, respectively (Supplementary File 1). The impurities of raw data including 5′ primer contaminants, no insert tags, oversized insertion, low quality reads, poly A tags and smaller than 18 nt tags were discarded (1.23%). The total of 13,053,344 (98.77%) and 14,517,522
Discussion
The length distribution analysis was helpful to see the compositions of small RNA sample. Normally, the length of small RNA was between 18 nt and 30 nt. miRNA was normally 21 nt or 22 nt, siRNA was 24 nt, and piRNA was 30 nt. The length distribution varied between plants and animals. The peak of plant located in 21 nt or 24 nt while animal located in 22 nt. For fish, the three distinct peaks in the size distribution of Atlantic halibut small RNA consisted of 26–27 nts cluster corresponding to
Conclusions
In summary, we identified 106 mature miRNAs (belong to 68 miRNA gene families) and 5 novel miRNAs from hepatopancreas of crayfish fed with 0 mg and 75 mg emodin kg−1 diets using Solexa sequencing. The expression levels of these miRNAs displayed a large range, and 35 miRNAs showed significantly differential expressions between control and trial libraries. Function annotation of the predicted target genes of 5 novel miRNAs indicated these miRNAs might involve in innate immune response, growth,
Acknowledgment
This research was supported by Fund from the Aquaculture Three Projects of Jiangsu (PJ2010-56) and the Special Fund for Agro-scientific Research in the Public Interest (201003070).
References (32)
- et al.
The influence of various feeding patterns of emodin on growth, non-specific immune responses, and disease resistance to Aeromonas hydrophila in juvenile Wuchang bream (Megalobrama amblycephala)
Fish Shellfish Immunol
(2014) - et al.
Hepatoprotective effects of emodin from Ventilago leiocarpa
J Ethnopharmacol
(1996) - et al.
Effects of anthraquin extract from Rheum officinale Bail on the growth performance and physicological responses of Macrobrachium rosenbergii under high temperature stress
Fish Shellfish Immunol
(2010) - et al.
Effects of anthraquinone extract from Rheum officinale Bail on the physiological responses and HSP70 gene expression of Megalobrama amblycephala under Aeromonas hydrophila infection
Fish Shellfish Immunol
(2012) - et al.
Effects of dietary emodin supplementation on growth performance, non-specific immune responses, and disease resistance to Aeromonas hydrophila in juvenile Wuchang bream (Megalobrama amblycephala)
Isr J Aquac Bamidgeh
(2014) MicroRNAs: genomics, biogenesis, mechanism, and function
Cell
(2004)- et al.
Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans
Cell
(1993) - et al.
The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA
Cell
(1997) - et al.
The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism
Curr Biol
(2003) - et al.
RAS is regulated by the let-7 microRNA family
Cell
(2005)
Transcriptome-wide identification and characterization of the Procambarus clarkii microRNAs potentially related to immunity against Spiroplasma eriocheiris infection
Fish Shellfish Immunol
Epigenetic regulation of miR-184 by MBD1 governs neural stem cell proliferation and differentiation
Cell Stem
Isolation and identification of novel microRNAs from Marsupenaeus japonicus
Fish Shellfish Immunol
Identification and comparative analysis of the Eriocheir sinensis microRNA transcriptome response to Spiroplasma eriocheiris infection using a deep sequencing approach
Fish Shellfish Immunol
MicroRNA control in the immune system: basic principles
Cell
Effect of different dietary protein and lipid levels on growth performance, body composition of juvenile red swamp crayfish (Procambarus clarkii)
Aquac Int
Cited by (19)
The nuclear factor interleukin 3-regulated (NFIL3) transcription factor involved in innate immunity by activating NF-κB pathway in mud crab Scylla paramamosain
2019, Developmental and Comparative ImmunologyHigh-throughput sequencing analysis of microRNAs in gills of red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus
2018, Fish and Shellfish ImmunologyCitation Excerpt :At the last step, the small RNA library's quality was measured with the Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). Subsequently, two qualified small RNA libraries (WG and NG) were sequenced using an Illumina HiSeq 2000 platform [18]. To confirm the results, sequencing work was repeated three times.
Integrated analysis of mRNA and miRNA expression profiles in Ptychobarbus dipogon and Schizothorax oconnori, insight into genetic mechanisms of high altitude adaptation in the schizothoracine fishes
2017, Gene ReportsCitation Excerpt :Such gene regulatory mechanisms mainly rely on base pairing of the miRNA seed sequence (6–8 nucleotides at the 5′ end of the miRNA) to complementary target mRNA regions (usually located in the 3′ untranslated regions), leading to mRNA degradation or translational repression (Bartel, 2009; Akat et al., 2014). In recent years, several studies have identified and quantified abundant miRNAs in fish species using the NGS technologies (Chi et al., 2011; Zhu et al., 2012; Yi et al., 2013; Xu et al., 2014, 2015), and have also identified some miRNAs associated with growth (Yi et al., 2013), nutrition metabolism (Xu et al., 2014) and immunity (Xu et al., 2015). However, the target genes of these miRNAs, which may directly affect phenotypic traits, have not been analyzed in these studies.
Identification and characterization of pro-interleukin-16 from mud crab Scylla paramamosain: The first evidence of proinflammatory cytokine in crab species
2017, Fish and Shellfish ImmunologyCitation Excerpt :Notably, a relatively high transcripts level of Sp-pro-IL-16 was examined in hepatopancreas and intestine. Beyond being the primary organ responsible of absorption and storage of ingested materials, the hepatopancreas is also the important immune organs in crustacean [33]. Meanwhile, IL-16 was considerably increased at both the mRNA and protein levels in pathologic intestinal tissues compared with healthy control individuals, suggesting IL-16 had important roles in intestinal inflammatory response [34,35].
Identification and characterization of lymph organ microRNAs in red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus
2017, Fish and Shellfish ImmunologyCitation Excerpt :Next, a pair of adaptors was ligated to the 5′ and 3′ ends of the small RNA using T4 RNA ligase. Then, a reverse transcription reaction was carried out using the adaptor-ligated small RNAs, and the cDNA was used as a template to amplify to produce sequencing libraries, using PCR with Illumina's small RNA primer set [22]. The quality of the two small RNA libraries was tested with the Agilent Bioanalyzer 2100 system (Agilent Technologies, USA).