Penaeus Japonicus Research Articles

RPS27, a sORF-Encoded Polypeptide, Functions Antivirally by Activating the NF-κB Pathway and Interacting With Viral Envelope Proteins in Shrimp.

A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of <100 amino acids in eukaryotes (50 amino acids in prokaryotes). Studies have shown that several sORF-encoded peptides (SEPs) have important physiological functions in different organisms. Many ribosomal proteins belonging to SEPs play important roles in several cellular processes, such as DNA damage repair and apoptosis. Several studies have implicated SEPs in response to infection and innate immunity, but the mechanisms have been unclear for most of them. In this study, we identified a sORF-encoded ribosomal protein S27 (RPS27) in Marsupenaeus japonicus. The expression of MjRPS27 was significantly upregulated in shrimp infected with white spot syndrome virus (WSSV). After knockdown of MjRPS27 by RNA interference, WSSV replication increased significantly. Conversely, after MjRPS27 overexpression, WSSV replication decreased in shrimp and the survival rate of the shrimp increased significantly. These results suggested that MjRPS27 inhibited viral replication. Further study showed that, after MjRPS27 knockdown, the mRNA expression level of MjDorsal, MjRelish, and antimicrobial peptides (AMPs) decreased, and the nuclear translocation of MjDorsal and MjRelish into the nucleus also decreased. These findings indicated that MjRPS27 might activate the NF-κB pathway and regulate the expression of AMPs in shrimp after WSSV challenge, thereby inhibiting viral replication. We also found that MjRPS27 interacted with WSSV's envelope proteins, including VP19, VP24, and VP28, suggesting that MjRPS27 may inhibit WSSV proliferation by preventing virion assembly in shrimp. This study was the first to elucidate the function of the ribosomal protein MjRPS27 in the antiviral immunity of shrimp.

Identification and characterization of a novel mannose-binding C-type lectin (PjLec2) in shrimp Penaeus japonicus

C-type lectins (CTLs) are important molecules involved in the innate immunity of invertebrates. In this investigation, we reported a novel CTL (designated as PjLec2) from the marine shrimp Penaeus japonicus (P. japonicus). The cDNA of PjLec2 is 669 bp in length with an open reading frame (ORF) of 477 bp. It encodes a protein of 159 amino acids (aa) containing a putative 18-aa signal peptide and a carbohydrate recognition domain (CRD). The CRD of PjLec2 possesses an EPN (Glu-Pro-Asn) motif which is putatively responsible for mannose recognition. Expression pattern analysis demonstrated that PjLec2 mRNA is exclusively expressed in hepatopancreas in P. japonicus. At protein level, PjLec2 is detected in hemolymph and hepatopancreas. Recombinant PjLec2 has a mannose binding activity and a strong agglutination activity against Gram-negative bacteria Vibrio alginolyticus (V. alginolyticus) and Aromonas hydrophila (A. hydrophila) in a calcium-dependent manner. In addition, the expression of PjLec2 is significantly upregulated at both mRNA and protein levels after challenging shrimp with A. hydrophila. Our results indicated that PjLec2 may play a fundamental role in the recognition of pathogenic bacteria in shrimp.

Stomach contents of the kuruma shrimp Marsupenaeus japonicus from the western Mediterranean Egyptian coast

The stomach content of 1000 and 1293 specimens from males and females respectively of Marsupenaeus japonicus, inhabiting the western Mediterranean Egyptian coast, were studied monthly from May 2015 to April 2016. The aim of this study was to identify the diet items of M. japonicus based on the analysis of the stomach contents. The stomach analysis was carried out by using the frequency of occurrence (O %) and numerical methods (N %). The kuruma shrimp M. japonocus is omnivorous, but with an important carnivorous component, mainly on crustacean and fish remains. Represented data of this study indicated that M. japonicus has not food preference between sexes. Stomach contents of adult and juvenile specimens were similar. The quantitative analysis and seasonally variations in the feeding intensity was quite high during the summer, autumn and winter.

Identification of two novel C-type lectins involved in immune defense against white spot syndrome virus and Vibrio parahaemolyticus from Marsupenaeus japonicus

C-type lectins (CTLs) are vital molecules in crustacean innate immunity with the capacity to recognize and eliminate invaders, such as viruses and bacteria. Here, two novel CTLs were identified from the kuruma shrimp Marsupenaeus japonicus, and their molecular characteristics and immune function were investigated. Sequence analysis revealed that the two CTLs possessed the typical CTL structure and function features. Tissue distribution analysis showed that the two CTLs were most abundantly expressed in the hepatopancreas and weakly expressed in other examined tissues. The transcription of the two CTLs significantly increased in the hepatopancreas of shrimp challenged with both white spot syndrome virus (WSSV) and Vibrio parahaemolyticus, and MjCTL4 was found to be more sensitive to the two pathogens than MjCTL3, being induced at relatively faster and higher increments. GST pull-down assays showed that the two CTLs could directly interact with several WSSV envelope proteins (VP19, VP24, VP26 and VP28). Moreover, the two CTLs displayed obvious binding and antibacterial ability to V. parahaemolyticus, and MjCTL3 exhibited stronger anti-V. parahaemolyticus activity than MjCTL4. These results suggest that the two novel CTLs might function as pattern recognition receptors (PRRs) and antibacterial molecules in M. japonicus innate immunity, and the two CTLs may be alternative agents for the prevention and treatment of diseases caused by WSSV and V. parahaemolyticus.

Physiological and molecular differences in the thermal tolerance of two varieties of kuruma prawn Marsupenaeus japonicus: critical thermal maximum and heat shock protein 70

Temperature is one of the most important variables that influence aquatic organisms. This study aimed to test physiological and molecular differences in the thermal tolerance of two morphologically similar Marsupenaeus japonicus. The physiological parameter was the upper thermal limit, tested via the critical thermal maximum, and the molecular parameter was the relative messenger RNA expression of heat shock proteins (HSP40, HSP60, HSP70 and HSP90), quantified via real-time polymerase chain reaction. Oxygen consumption of shrimp was measured with a respirometer. M. japonicus variety II showed a higher critical thermal maxima and survival rate and lower oxygen consumption rate than variety I, suggesting that it had higher thermal tolerance and acclimatory plasticity. M. japonicus variety I showed significantly higher production of HSP70 and HSP90, suggesting that these shrimp may have experienced more thermal damage. By comparing the differences in the stress response between two morphologically similar M. japonicus, this study provides useful information for the aquaculture of M. japonicus.

Cross talk between heat shock protein 10 and a heat shock factor identified from Marsupenaeus japonicus

Heat shock factors (HSFs) and heat shock proteins (HSPs) are crucial regulators and effectors of the heat shock response (HSR). In this study, the full-length cDNA sequences of MjHSP10 and MjHSF1 were cloned by rapid amplification of cDNA ends (RACE). The deduced MjHSP10 and MjHSF1 amino acid (aa) sequences exhibited conserved structures and the functional features of HSP10 and HSF1, respectively. The tissue distributions and mRNA expression profiles of the two genes in response to heat stress were analyzed by quantitative real-time PCR (qRT-PCR). MjHSP10 and MjHSF1 were ubiquitously expressed in various tissues. Heat stress induced a significant increase in MjHSP10 expression that tend to positively correlate with temperature. Additionally, MjHSF1 transcription was up-regulated less than MjHSP10 transcription under heat stress. MjHSF1 expression in the hepatopancreas was up-regulated under only long-term (48 h) heat stress, and MjHSF1 transcription in the gill increased under only acute (34 °C) heat stress. MjHSF1 knockdown by RNA interference (RNAi) down-regulated MjHSP10 expression. Glutathione-S-transferase (GST) pull-down assays showed an interaction between MjHSP10 and the DNA-binding domain (DBD) of MjHSF1. This study provided new insights into cross talk between HSP10 and HSF1 in Marsupenaeus japonicus.

Ultrastructural changes in Marsupenaeus japonicus spermatozoa in the journey from the testis to the female thelycum

Marsupenaeus japonicus is one of the most important penaeid shrimp farmed in China. As a typical closed thelycum penaeid shrimp, the morphological changes in the spermatozoa during the journey from the testis to the female thelycum remain unclear in this species. Herein, an ultrastructural comparison of the spermatozoa in the testis (TE), vas deferens (VD), and the spermatophore in the male terminal ampoule (SA) and the female seminal receptacle (SR) of M. japonicus was conducted. The spermatozoa in the VD, SA and SR shared typical morphological characteristics of the spermatozoa of decapod dendrobranchiates, including a spherical main body and a slender spike. However, no spike was observed in the spermatozoa collected from the TE. The spike of the spermatozoa in the SA and SR was longer than that in the VD, whereas no significant difference in spike length was observed between the spermatozoa from the SA and SR. The basic ultrastructure of the spermatozoa in the VD, SA and SR was similar, except for some differences in the nuclear region. The main body of the spermatozoa comprised the cytoplasmic band, nuclear region, several small and large vesicles, and the basal region of the acrosomal structure. The nuclear materials in the spermatozoa from the TE were more electron-dense than those in the VD, SA and SR, while the nuclear region of the spermatozoa was more electron-lucent in the SR than in the VD and SA. These findings preliminarily reveal the ultrastructural changes in M. japonicus spermatozoa during the journey from the testis to the female thelycum and provided a theoretical foundation for developing effective artificial insemination methods in this species.

Purification, characterization and antibacterial activities of red color-related protein found in the shell of kuruma shrimp, Marsupenaeus japonicus

The well-known red color change plays a significant role in consumer acceptability of crustacean species. In this study, we described the purification of the red color-related protein named MjRCP75 from the shell of Marsupenaeus japonicus. It was a homogeneous monomer with molecular mass of 75 kDa and rich in α-helix conformation. The α-helix content decreased within the increasing of heating temperature and was transformed dominantly to β types. Identification and structural analysis revealed that MjRCP75 belonged to hemocyanin family. The released pigment from heated MjRCP75 showed a λmax at 483 nm in acetone. MjRCP75 showed clearly antibacterial activity against Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus. These findings identify MjRCP75 as the red color-related protein in M. japonicus shell and reveal its involvement in antibacterial activities.

Effect of a peptide complex on the defense mechanism of shrimp, Marsupenaeus japonicus against pathogens and changes in environmental parameters

AbstractThis study examines the effect of a peptide complex extracted from chicken egg yolk on the action of the immune system of kuruma shrimp, Marsupenaeus japonicus, on pathogens as well as changes in environmental parameters. In the design of three independent experiments, we defined the treated shrimp or the control shrimp as shrimp receiving diet with or without peptide complex administration, respectively. In the first experiment, the shrimp were challenged by Vibrio sp. after 1 week of feeding; the results showed that the survival rates of the treated shrimp were 69%. In the other experiments, the shrimp were exposed to low salinity or high temperature to analyze the changes in various immunological parameters. In a low‐salinity test, results showed that the total hemocyte count (THC) of the treated group was significantly higher than that of the control group within 24 hr. In a high‐temperature test, THC and phenoloxidase activity of the shrimp in the treated group displayed significantly higher values than that in the control group during 48 hr. In short, the administration of the peptide complex was significantly effective in the prevention of disease and enhancement of the tolerance of kuruma shrimp to environmental changes.

A Hint of Primitive Mucosal Immunity in Shrimp through Marsupenaeus japonicus Gill C-Type Lectin.

Lectins are found in most living organisms, providing immune surveillance by binding to carbohydrate ligands. In fishes, C-type lectins were isolated from mucus of respiratory organs (skin and gills), where they aid the mucosal immune response in regulating microbiota and suppressing pathogens. In shrimp, however, no mucosal immunity or any form of gill-specific immune defense has been reported, and most identified C-type lectins are associated with hemocyte cellular and humoral responses. Interestingly, our microarray analysis revealed the localization of highly expressed novel biodefense genes in gills, among which is Marsupenaeus japonicus gill C-type lectin (MjGCTL), which we previously reported. Gill mucus collected from M. japonicus displayed similar bacterial agglutination ability as observed with recombinant MjGCTL. This agglutinating ability can be attributed to endogenous MjGCTL (nMjGCTL) detected in gill mucus, which was confirmed with an agglutination assay using purified nMjGCTL from gills. In addition, nMjGCTL also promoted in vivo bacterial phagocytosis by hemocytes. In vivo knockdown of MjGCTL resulted in a compromised immune system, which was manifested by impaired agglutination capacity of gill mucus and downregulation of the gill antimicrobial peptides, crustin and penaeidin. Shrimp immunocompromised by MjCGTL knockdown, apparently lost the ability to respond to attaching and penetrating bacteria. This was evident as increased total bacteria and Vibrio counts in both gills and hemolymph, which were correlated with low survival during a bacterial challenge. These results reveal immune defense by shrimp gills resembling a primitive form of mucosal immunity.

Periphyton and phytoplankton assessment in a shrimp nursery: signature pigments analysis

Understanding microalgae composition is key for an improved aquaculture system management. The primary objective of this research was to estimate microalgae community structure in a Marsupenaeus japonicus nursery. The secondary objective was to analyze the environmental parameters (salinity, pH, dissolved oxygen, total dissolved ammonia, nitrites, nitrates and phosphates) and shrimp density effect on abundance, composition and development of microalgae in a shrimp nursery. Periphyton and phytoplankton composition and abundance were determined using HPLC signature pigment analysis coupled with CHEMTAX software. Environmental parameters were measured in the tanks with probes or in the laboratory following standard procedures of water quality analysis. A nonparametric repeated-measures ANOVA statistical analysis was done to test differences between treatments. Spearman rank correlation analyses were performed on environmental and biological variables with phytoplankton or periphyton groups in order to examine significant relationship. The results showed diatoms were significantly higher than any other groups in both phytoplankton and periphyton communities. Shrimp density effect on periphyton, phytoplankton composition and environmental parameters was minor. Nutrients played a key role on phytoplankton development, but had a minor effect on periphyton, which was more affected by colonization processes and other environmental variables. The analysis of signature pigments allowed to report the presence of previously undetected groups on periphyton, prasinophytes and prymnesiophytes, which are characterized by high nutritional value. This is especially important in nurseries because shrimp grazing on periphyton can increase post-larvae survival.

Effects of dietary Nannochloropsis sp. powder and lipids on the growth performance and fatty acid composition of larval and postlarval kuruma shrimp, Marsupenaeus japonicus

The current study evaluated the effects of fish oil replacement with eicosapentaenoic acid-enriched single-cell microalgae in the diets of larval and postlarval kuruma shrimp. Experimental diets containing different level of Nannochloropsis sp. powder (10, 40, 70 g/kg) and lipids (2, 8, 10, 14, 35, 58 g/kg) were evaluated. The substitution of fish oil with algal powder significantly improved shrimp growth compared to that in the control group, with the highest final body weight recorded at 70 g/kg in larvae and 40 g/kg in postlarvae. Larvae fed algal lipids with a dry weight percentage in the diet of greater than 10 g/kg showed significantly lower performance than those in the control group. The neutral lipids in the tissue of the postlarvae that had been fed algal lipids had an improved fatty acid profile, as the content of highly unsaturated fatty acids increased compared to that in the other groups. The content of docosahexaenoic acid increased in the tissue, indicating the possible occurrence of the metabolism and accumulation of Nannochloropsis sp. essential fatty acids. Algal powder could replace FO up to 140 g/kg in shrimp diets without compromising growth and FA profiles.

MD-2 Homologue Recognizes the White Spot Syndrome Virus Lipid Component and Induces Antiviral Molecule Expression in Shrimp.

The myeloid differentiation factor 2 (MD-2)-related lipid-recognition (ML) domain is found in multiple proteins, including MD-2, MD-1, Niemann-Pick disease type C2, and mite major allergen proteins. The significance of ML proteins in antibacterial signal transduction and in lipid metabolism has been well studied. However, their function in host-virus interaction remains poorly understood. In the current study, we found that the ML protein family is involved in resistance against white spot syndrome virus in kuruma shrimp, Marsupenaeus japonicus One member, which showed a high similarity to mammalian MD-2/MD-1 and was designated as ML1, participated in the antiviral response by recognizing cholesta-3,5-diene (CD), a lipid component of the white spot syndrome virus envelope. After recognizing CD, ML1 induced the translocation of Rel family NF-κB transcription factor Dorsal into the nucleus, resulting in the expression of Vago, an IFN-like antiviral cytokine in arthropods. Overall, this study revealed the significance of an MD-2 homologue as an immune recognition protein for virus lipids. The identification and characterization of CD-ML1-Dorsal-Vago signaling provided new insights into invertebrate antiviral immunity.

Comparative proteomic investigation of Marsupenaeus japonicus hepatopancreas challenged with Vibrio parahaemolyticus and white spot syndrome virus

This study aimed to use isobaric tags (IBTs) to investigate the immune response of the hepatopancreas of Marsupenaeus japonicas infected with Vibrio parahaemolyticus or white spot syndrome virus (WSSV). Liquid chromatography-tandem mass spectrometry and protein sequencing identified 1005 proteins. Among them, 109 proteins were upregulated and 94 were downregulated after V. parahaemolyticus infection. After WSSV infection, 130 proteins were identified as differentially abundant, including 88 that were upregulated and 42 were downregulated. Fifty-four proteins were identified as differentially abundant after both V. parahaemolyticus and WSSV infection. A number of proteins related to cytoskeletal processes, including actin and myosin, and apoptosis-related proteins were upregulated in shrimp after V. parahaemolyticus and WSSV infection, indicating that phagocytosis and apoptosis may be involved in the response to in V. parahaemolyticus or WSSV infection. Quantitative real-time PCR was carried out to verify the reliability of the proteomic data. These data provide a basis to characterize the immunity-related processes of shrimp in response to infection with WSSV or V. parahaemolyticus.

Transcriptomic analyses of the acute ammonia stress response in the hepatopancreas of the kuruma shrimp (Marsupenaeus japonicus)

The kuruma shrimp Marsupenaeus japonicus is an economically important crustacean. High concentrations of ammonia resulting from intensive culture and environmental pollution affect its aquaculture, but little information is available on the molecular mechanisms. We performed transcriptomic analyses of the hepatopancreas of M. japonicus subjected to ammonia stress. We obtained 44,735,330–56,680,154 clean reads from the control and treatment groups, assembled and clustered a total of 133,486 unigenes with an average of 1178 bp and N50 of 18,769 bp. Comparative transcriptome analyses identified 2294 differentially expressed genes, a large number of which were related to immune function, oxidative stress, apoptosis, molting, and the membrane. In addition, totals of 36 gene ontology terms and 177 Kyoto Encyclopedia of Genes and Genomes pathways were identified. In addition, the genes encoding heat shock protein 90 (HSP90), HSP10, glutamine synthase (involved in the nitrogen pathway), and antioxidant enzymes (catalase and glutathione S-transferase) were significantly upregulated by ammonia stress. Therefore, ammonia probably impaired the immune response, induced oxidative stress, disrupted membranes, and induced apoptosis in M. japonicus. To maintain physiological homeostasis, M. japonicus responded to ammonia stress by upregulating heat shock proteins and antioxidant enzymes, and enhancing the nitrogen metabolism pathway. In conclusion, this is the first comprehensive investigation on the transcriptomic response to ammonia stress in M. japonicus, and the results clarify the response of shrimp to ammonia toxicity. The genes and pathways identified here provide valuable information on the molecular mechanism of the tolerance of shrimp to ammonia stress and will facilitate the molecular-assisted breeding of varieties of M. japonicus with enhanced tolerance to ammonia.

Bioaccumulation and physiological responses in juvenile Marsupenaeus japonicus exposed to cadmium

The heavy metal cadmium readily accumulates in organisms, causing damage. In this study, juvenile marine shrimp Marsupenaeus japonicus were exposed to cadmium (Cd2+; 5, 50 and 500 μg L–1). Cd accumulation and antioxidant-related indices were determined, and damage to biomolecules was assessed, after 24, 48 and 96 h. Cd bioaccumulation in M. japonicus increased with exposure time and concentration, which reached the highest value at 96 h. The data showed that 5, 50 and 500 μg L–1 Cd increased glutathione (GSH) content and the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST) in a Cd-dose-dependent manner, but 5 and 50 μg L–1 Cd had no effect on caspase-3 activity. The expression levels of SOD, GST, heat shock protein 70 (HSP70), metallothionein (MT), p53 and caspase-3 genes were rapidly increased after 50 and 500 μg L–1 Cd exposure, and remained at a significantly higher level than in the control after 96 h of exposure. After exposure to 5, 50 and 500 μg L–1 Cd, F-value (the ratio between double-stranded DNA and total DNA) remained high at 24 h, however, as the exposure time increased, the F-value decreased in a dose-dependent manner. An increase in malondialdehyde content was also observed following exposure to 50 and 500 μg L–1 Cd. Our data suggest that Cd induces oxidative stress, molecular damage and apoptosis in juvenile M. japonicus in a concentration- and time-dependent manner.

Shrimp miRNA suppresses the stemness of human cancer stem cellsviathe PIN1 pathway

A tumor can be regarded as a cell mass with a biologic hierarchy that is orchestrated by cancer stem cells. The specific features of cancer stem cells may result from a series of gene expression regulatory mechanisms. As important regulatory factors of gene expression, microRNAs (miRNAs) have critical roles in the self-renewal, pluripotency, differentiation, and tumorigenicity of cancer stem cells. However, the effects of animal miRNAs on cancer stem cells have not been investigated extensively. Here, we report that miRNA of a shrimp, Marsupenaeus japonicus (mja-miR-35-3p), which possesses antiviral activity in shrimp, could suppress the proliferation of melanoma and breast cancer stem cells, but not cancer nonstem cells, by arresting the cell cycle in the G1 phase and inducing apoptosis. Shrimp mja-miR-35-3p (named mja-miR-35) targets the human peptidylprolyl cis/trans isomerase, never in mitosis gene a-interacting 1 (PIN1) gene, which is up-regulated in cancer stem cells. The results indicated that PIN1 silencing caused cell cycle arrest in the G1 phase, induced apoptosis, and decreased the sphere-forming capacity of cancer stem cells, but not cancer nonstem cells, showing that PIN1 had beneficial effects against the stemness of cancer stem cells. The in vivo data revealed that shrimp mja-miR-35 suppressed the stemness of cancer stem cells in mice by inhibiting the PIN1 antiapoptotic-cell cycle pathway. These findings demonstrated that the miRNAs of aquatic animals might be an important source for discovering human antitumor drugs.-Zhang, S., Zhang, X. Shrimp miRNA suppresses the stemness of human cancer stem cells via the PIN1 pathway.

Evolutionary adaptation analysis of immune defense and hypoxia tolerance in two closely related Marsupenaeus species based on comparative transcriptomics

Kuruma shrimp, a major farmed shrimp species in the world, includes two cryptic or sibling species, Form I (Marsupenaeus japonicus) and Form II (Marsupenaeus pulchricaudatus). Due to the lack of genomic resources, little is known about the molecular mechanisms associated with immune defense and hypoxia tolerance. Here, we sequenced the transcriptomes of two closely related Marsupenaeus species and compared genomic divergence. This study obtained 77049 and 84561 unigenes with N50 values of 1281bp and 1244bp for M. japonicus and M. pulchricaudatus, respectively, and 5036 pairs of putative orthologs were identified between two Marsupenaeus species. Estimation of Ka/Ks ratios indicated that 165 orthologous genes may be under positive selection (Ka/Ks > 0.5), including 49 pairs with a Ka/Ks ratio >1. According to the peak of synonymous rates, the divergence time between M. japonicus and M. pulchricaudatus was about 0.26–0.69 Mya. These positively selected orthologous genes related to the immune process mainly comprised single VWC domain protein, legumain, ras-related C3 botulinum, caspase, C-type lectin and were enriched in functions related to immune (Toll-like receptor and PI3K-Akt signaling) and hypoxia signaling (HIF-1 signaling and VEGF signaling). In this study, dozens of caspase-like unigenes were screened from two Marsupenaeus transcriptomes. Among these, the PjCaspase orthologous gene was subjected to positive selection (Ka/Ks = 1.22), which had different secondary and three-dimensional structure prediction. Based on the single copy caspase gene, eight populations of Marsupenaeus species were divided into two phylogeographic lineages from the East and South China. We characterized the transcriptomes of the two Marsupenaeus species and obtained several key orthologs associated with immune defense and hypoxia tolerance, which provides new insights into the immunity and genetic divergence of the two varieties. Moreover, this study will facilitate further comparative genomic studies of the two varieties.

Identification of ovarian miRNAs in banana shrimp (Fenneropenaeus merguiensis) during ovarian development between non-vitellogenic and vitellogenic shrimp

Banana shrimp (Fenneropenaeus merguiensis) is one of the popular shrimp for the consumer while the shrimp farming is not well practice due to its narrow tolerant to the environmental culture that leads to the overfishing crisis. To increase in the population number, the ovarian controlling mechanism need to be identified where the small RNAs particularly microRNAs that could regulate the expression of ovarian genes involved in ovarian development and maturation. Identification of ovarian miRNAs in banana shrimp was performed using the Illumina Hiseq 4000 system. Total 22,785,700 and 23,154,313 clean tag were counted from non-vitellogenic and vitellogenic shrimp respectively with the amount of 58 novel miRNAs. Five known miRNAs including miR-6489, miR-6491, miR-6492, miR-6493 and miR-6494, which are belonging to kuruma shrimp (Marsupenaeus japonicus) were classified together with their target prediction using RNAhybrid tool. Then, the expression of these miRNAs were quantified by RT-qPCR and found higher expression in the vitellogenic stage of all miRNAs than those in the non-vitellogenic stage. Moreover, their predicted targets were confirmed for the binding activity by luciferase assay and intramuscular injection of miRNAs into the shrimp. This data provides more understanding of the roles of some miRNAs in the regulation of the closely related shrimp ovarian gene expression.

Differentially expressed genes of the shrimp Marsupenaeus japonicus in response to infection by white spot syndrome virus or Vibrio alginolyticus

This is the first study to use a next-generation high-throughput DNA sequencing technique, the Illumina Hiseq2000 method, to analyze the transcriptome from hemocytes of Marsupenaeus japonicus. A total of 80,929,652 Illumina reads, including 79,525,942 high quality reads were obtained in this study. From these, 40,231 unigenes with a mean length of 1557 bp were assembled using Trinity de novo software and 28,746 cDNA were matched in the NCBI database. Then we compared the transcriptome changes after white spot syndrome virus (WSSV) or Vibrio alginolyticus infection. A total of 19,872 putative proteins were classified functionally into 25 molecular families in the cluster of orthologous groups. KEGG pathway analysis identified that the metabolic pathway possessed more unigenes (1358 unigenes), followed by biosynthesis of secondary metabolites, Huntington's disease and RNA transport. Important immune functions like apoptosis, phagocytosis, and lysosomes were in response to WSSV and V. alginolyticus early infection. Only 26 transcripts were significantly up-regulated or down-regulated after WSSV infection showed compared with V. alginolyticus. Crustin-like protein, endonuclease-reverse transcriptase, putative nuclease HARBI1-like, diphthamide biosynthesis protein 7 and hormone receptor 3 were involved in the immune response to WSSV or V. alginolyticus infection. These transcriptome datasets accelerate our understanding of the innate immune mechanisms in M. japonicus and other crustaceans.