Sheng Luan

Transcriptome Analysis of the Hepatopancreas in the Pacific White Shrimp (Litopenaeus vannamei) under Acute Ammonia Stress

In the practical farming of Litopenaeus vannamei, the intensive culture system and environmental pollution usually results in a high concentration of ammonia, which usually brings large detrimental effects to shrimp, such as increasing the susceptibility to pathogens, reducing growth, decreasing osmoregulatory capacity, increasing the molting frequency, and even causing high mortality. However, little information is available on the molecular mechanisms of the detrimental effects of ammonia stress in shrimp. In this study, we performed comparative transcriptome analysis between ammonia-challenged and control groups from the same family of L. vannamei to identify the key genes and pathways response to ammonia stress. The comparative transcriptome analysis identified 136 significantly differentially expressed genes that have high homologies with the known proteins in aquatic species, among which 94 genes are reported potentially related to immune function, and the rest of the genes are involved in apoptosis, growth, molting, and osmoregulation. Fourteen GO terms and 6 KEGG pathways were identified to be significantly changed by ammonia stress. In these GO terms, 13 genes have been studied in aquatic species, and 11 of them were reported potentially involved in immune defense and two genes were related to molting. In the significantly changed KEGG pathways, all the 7 significantly changed genes have been reported in shrimp, and four of them were potentially involved in immune defense and the other three were related to molting, defending toxicity, and osmoregulation, respectively. In addition, majority of the significantly changed genes involved in nitrogen metabolisms that play an important role in reducing ammonia toxicity failed to perform the protection function. The present results have supplied molecular level support for the previous founding of the detrimental effects of ammonia stress in shrimp, which is a prerequisite for better understanding the molecular mechanism of the immunosuppression from ammonia stress.

Identification, cloning and characterization of an extracellular signal-regulated kinase (ERK) from Chinese shrimp, Fenneropenaeus chinensis.

Extracellular signal-regulated kinase (ERK) is a serine/threonine-specific protein kinase, which participates in signaling transduction pathways that control intracellular events, including resumption of meiosis, embryogenesis, cell differentiation, cell proliferation, cell death and response to radiation. Some virus species evolved the ability to hijack the host cell ERK signaling transduction pathway for viral replications and gene expressions. To obtain a better understanding of ERK, we cloned a cDNA encoding ERK from the muscle of Fenneropenaeus chinensis (FcERK). The FcERK contained a 1098 bp open reading frame (ORF) encoding a protein of 365 amino acid residues with a conserved phosphorylation motif TEY in the kinase activation loop. Pair-wise and multiple sequence alignment revealed that ERK is highly conserved across taxa. The FcERK gene expressions in the hepatopancreas and gill were noticeably higher than the expression observed in the muscle. A challenge test was performed to reveal the responses of FcERK in different tissues to white spot syndrome virus (WSSV) infection. Post WSSV challenge, the FcERK expression in the gill significantly increased during the early stage of the viral infection, the FcERK expression in the muscle increased later than that in the gill, and the FcERK expression in the hepatopancreas significantly decreased. The FcERK gene expression profile accorded with the results that the virus primarily infects tissues originating from the ectoderm, with less infection of the tissues originating from the mesoderm, and hardly any infection in the tissues originating from the entoderm. Two single nucleotide polymorphisms (SNPs) were identified in the FcERK gene, involving C/T transition. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps were obtained using a high-resolution melting (HRM) method. In the two groups, the MAFs of both sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distributions of both mutation sites between the two groups were not significantly different. These results lead to a better understanding of the molecular mechanisms of the host-virus interaction and provide useful information for disease control.

Molecular cloning and characterization of a cathepsin B gene from the Chinese shrimp Fenneropenaeus chinensis.

Cathepsin B is a unique member of the cathepsin superfamily, which acts as both an endopeptidase and peptidyl-dipeptidase. To obtain a better understanding of this enzyme, we cloned a cDNA encoding cathepsin B from the muscle of Fenneropenaeus chinensis (FcCB). FcCB contained a 996-bp open reading frame (ORF) encoding a protein of 331 amino acid residues with a putative signal peptide and a propeptide_C1 at the N-terminal, a glutamine oxyanion hole and active site cysteine, histidine and asparagine residues. A region from residue 79 to 327 conferred the peptidase activity of FcCB. Pair-wise and multiple sequence alignment with 17 other organisms, including ten different vertebrate species, five different invertebrate species and two different plant species, indicated that the signal peptide and the propeptide_C1 at the N-terminal of FcCB were less conserved than the mature protein, except when compared with Penaeus monodon, Litopenaeus vannamei and Marsupenaeus japonicas, all of which belong to the genus Penaeus. The expression of FcCB in the hepatopancreas was higher than that in the gill. The expression of FcCB in the gill was higher than that in the muscle. A challenge test was performed to reveal the responses of FcCB in different tissues to white spot syndrome virus (WSSV) infection, which causes serious economic losses in the shrimp farming industry. The FcCB gene expressions in the ectoderm, mesoderm and entoderm were not the same prior to WSSV infection, but at 6 h after WSSV challenge, the FcCB expression in the gill, hepatopancreas and muscle was up-regulated, suggesting that FcCB might be involved in the immune response to WSSV. Three single nucleotide polymorphisms (SNPs) were identified in the FcCB gene, involving C/T transitions, which are known as mutation hot spots. Notably, the three SNPs constituted a haplotype that can be used as an indicator of the haplotype block. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps, were obtained using a high-resolution melting (HRM) method. Associated factors, including observed heterozygosity (Ho), expected heterozygosity (He), minor allele frequency (MAF) and P-values for the deviation from Hardy-Weinberg equilibrium (HWE), were obtained. For the association analysis with WSSV resistance, the P-values were calculated using Pearsons chi-square test. In the two groups, the MAFs of all sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distribution of the C-984T mutation site between the two groups was not significantly different. These results lead to a better understanding of the molecular mechanisms of the host-virus interaction and provide useful information for solving the WSSV problem.

Genetic parameter estimation for juvenile growth and upper thermal tolerance in turbot (Scophthalmus maximus Linnaeus)

Twenty-six half-sib groups (53 full-sib families) of turbot, Scophthalmus maximus Linnaeus, were obtained by artificial insemination. We measured growth in the offspring (40–50 individuals/family) and subjected them to a thermal tolerance challenge over a period of 34 d. There was no significant difference in daily mortality (range: 0.580%–1.391%) between Days 1–13 during the thermal tolerance challenge. However, daily cumulative mortality increased rapidly between Days 14 and 29, especially on Days 15 and 16 (20.232% and 34.377%, respectively). Mortality was highest on Day 16 (14.145%). We estimated the genetic parameters using the average information restricted maximum likelihood method. We used a likelihood ratio test to evaluate the significance of effects in models with and without identity as an effect, and compared the final log-likelihoods (maximum log L). Lastly, we estimated phenotypic and genetic correlation between the upper thermal tolerance limit (UTT) and body weight (BW). In this study, the positive phenotypic correlation was low between UTT and BW (0.093±0.029). The genetic correlation between UTT and BW was negative (−0.044±0.239). The heritability for upper thermal tolerance was low (0.087±0.032), which is of approximately moderate heritability. The heritability for body weight was high (0.303±0.074). Our results suggest there is significant potential for improvement in the culture of turbot by selective breeding.

The identification of microRNAs involved in the response of Chinese shrimp Fenneropenaeus chinensis to white spot syndrome virus infection

Abstract MicroRNA (miRNA) is a class of small noncoding RNA, which is involved in the post‐transcriptional regulation in all metazoan eukaryotes. MiRNAs might play an important role in the host response to virus infection. However, miRNAs in the aquatic crustacean species were not extensively investigated. To obtain a better understanding of the response of Chinese shrimp Fenneropenaeus chinensis to white spot syndrome virus (WSSV) infection, the sequence and expression profile of miRNAs in the hepatopancreas of WSSV‐infected F. chinensis were obtained by the high‐throughput Illumina HiSeq 2500 deep sequencing technique. A total number of 129 known miRNAs and 44 putative novel miRNAs were identified from the deep sequencing data. The peak size of miRNAs was 22 nt (37.0%). 25 miRNAs were significantly (P < 0.05) differentially expressed post WSSV infection. Six of the differentially expressed miRNAs were randomly selected for further verification by the real‐time RT‐PCR technique. The results showed that there was a consistency between the deep sequencing and real‐time RT‐PCR assay. The target genes of differentially expressed miRNAs were predicted. Each miRNA had 4 target genes on average. The results suggested that some specific miRNAs might be involved in the response of F. chinensis to WSSV infection, and further provided basic information for the investigation of specific miRNAs in F. chinensis. HighlightsA total number of 129 known miRNAs and 44 putative novel miRNAs were identified from the deep sequencing data of the Chinese shrimp Fenneropenaeus chinensis.25 miRNAs were significantly (P < 0.05) differentially expressed post WSSV infection.Differentially expressed miRNAs were verified by the real‐time RT‐PCR technique.

Heritability of body weight and resistance to ammonia in the Pacific white shrimp Litopenaeus vannamei juveniles

Ammonia, toxic to aquaculture organisms, represents a potential problem in aquaculture systems, and the situation is exacerbated in closed and intensive shrimp farming operations, expecially for Litopenaeus vannamei. Assessing the potential for the genetic improvement of resistance to ammonia in L. vannamei requires knowledge of the genetic parameters of this trait. The heritability of resistance to ammonia was estimated using two descriptors in the present study: the survival time (ST) and the survival status at half lethal time (SS50) for each individual under high ammonia challenge. The heritability of ST and SS50 were low (0.154 4±0.044 6 and 0.147 5±0.040 0, respectively), but they were both significantly different from zero (P<0.01). Moreover, these two estimates were basically the same and showed no significant differences from each other (P>0.05), suggesting that ST and SS50 could be used as suitable indicators for resistance to ammonia. There were also positive phenotypic and genetic correlation between resistance to ammonia and body weight, which means that resistance to ammonia can be enhanced by the improvement of husbandry practices that increase the body weight. The results from the present study suggest that the selection for higher body weight does not have any negative consequences for resistance to ammonia. In addition to quantitative genetics, tools from molecular genetics can be applied to selective breeding programs to improve the efficiency of selection for traits with low heritability.

Estimation of genetic parameters and genotype-by-environment interactions related to acute ammonia stress in Pacific white shrimp (Litopenaeus vannamei) juveniles at two different salinity levels.

Regarding the practical farming of Litopenaeus vannamei, the deterioration of water quality from intensive culture systems and environmental pollution is a common but troublesome problem in the cultivation of this species. The toxicities that result from deteriorating water quality, such as that from ammonia stress, have lethal effects on juvenile shrimp and can increase their susceptibility to pathogens. The toxicity of ammonia plays an important role in the frequently high mortality during the early stage on shrimp farms. However, little information is available regarding the genetic parameters of the ammonia tolerance of juveniles in the early stage, but this information is necessary to understand the potential for the genetic improvement of this trait. Considering the euryhalinity of L. vannamei and the fact that low salinity can increase the toxicity of ammonia stress, we estimated the heritability of ammonia tolerance in juveniles in 30‰ (normal) and 5‰ (low) salinity in this study using the survival time (ST) at individual level and the survival status at the half-lethal time (SS50) at the family level. In the normal and low salinity conditions and for the merged data, the heritability estimates of the ST (0.784±0.070, 0.575±0.068, and 0.517±0.058, respectively) and SS50 (0.402±0.061, 0.216±0.050, and 0.264±0.050, respectively) were all significantly greater than zero, which indicates that the ammonia-tolerance of shrimp can be greatly improved. So it might provide an alternative method to reduce mortality, help to enhance resistance to pathogens and reduce the occurrence of infectious diseases. The significant positive genetic correlation between ST and body length suggested that ammonia is more toxic to shrimp in the early stage. The medium-strength genetic correlations of the ST and SS50 between the two environments (0.394±0.097 and 0.377±0.098, respectively) indicate a strong genotype-by-environment (G×E) interaction for ammonia tolerance between the different salinity levels. Therefore, salinity-specific breeding programs for ammonia tolerance in shrimp should be purposefully implemented.

Genome-wide mining, characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats (SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class (44.08%), followed by mononucleotides (29.67%), trinucleotides (18.96%), tetranucleotides (5.66%), hexanucleotides (1.07%), and pentanucleotides (0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci (90.0%) were successfully amplified with specific products and 24 (80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17 (with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.

Comparative transcriptome analysis of the Pacific White Shrimp ( Litopenaeus vannamei ) muscle reveals the molecular basis of residual feed intake

Feed efficiency is an economically important trait in genetic improvement programs of L. vannamei. Residual feed intake (RFI), an ideal measure of feed efficiency, is the difference between observed feed intake and expected feed requirement predicted from maintenance and production. Exploring the molecular basis of RFI is essential to facilitate the genetic breeding of feed efficiency in L. vannamei. However, few studies have been reported in this aspect. In this study, we sequenced muscle transcriptomes of a high-efficiency group, a low-efficiency group and a control group originating from two families, and compared the gene expression patterns between each extreme group and the control group. A total of 383 differentially expressed genes were identified, most of which were involved in cell proliferation, growth and signaling, glucose homeostasis, energy and nutrients metabolism. Functional enrichment analysis of these genes revealed 13 significantly enriched biological pathways, including signaling pathways such as PI3K-Akt signaling pathway, AMPK signaling pathway and mTOR signaling pathway, as well as some important pathways such as ubiquitin mediated proteolysis, cell cycle, pentose phosphate pathway and glycolysis/gluconeogenesis. These genes and pathways provide initial insight into the molecular mechanisms driving the feed efficiency in L. vannamei.

Identification of SNP markers associated with tolerance to ammonia toxicity by selective genotyping from de novo assembled transcriptome in Litopenaeus vannamei

ABSTRACT The high concentration of ammonia from deteriorated aquaculture environments and the intensive culture system could increase the susceptibility to pathogens and even cause high mortality in Litopenaeus vannamei. In addition, we have revealed that the ammonia‐tolerant shrimp also have high disease resistance in L. vannamei. In the present study, in order to identify SNP markers associated with tolerance to ammonia toxicity, we developed and characterized SNPs from our previous transcriptome sequencing data of ammonia‐stressed and control groups, and a marker‐trait association analysis was performed for marker‐assisted selection (MAS) to increase production in L. vannamei. A total of 318,919 SNPs were identified from the transcriptome sequences, and 25,772 SNPs were found from the 1826 ammonia‐responsive genes with functional annotation. We selected 49 SNPs from 26 ammonia‐responsive genes that had strong homologies to known genes in the shrimp and probably involved in immune function as candidate markers for genotyping, among which 39 SNPs were polymorphic for further marker‐trait association analysis with the ammonia‐tolerant (AT) and ammonia‐sensitive (AS) groups. Finally, 12 out of the 49 SNP markers were identified to be associated with ammonia tolerance, containing 10 loci with significantly different allele frequencies and 10 loci with significantly different genotyping frequencies between the AT and AS groups. Among the associated markers, the G allele of TSP‐1 (the first locus from the thrombospondin gene), the A allele of TSP‐3, and the C allele of XBP1–5 (the fifth locus from X‐box binding protein 1) only presented in the AT groups, but they were absent from the AS groups, which would be the preference of the MAS for the ammonia‐tolerant shrimp. In addition, when the 12 associated SNP markers were used for analysis, the genetic diversity of the AT groups were significantly higher than that of the AS groups, but when the 39 loci were used there was no difference. This is the first report for the markers associated with ammonia tolerance in this species, indirectly with disease resistance, which provided important potential for genetic selection to increase survival rate and production in shrimp farming. HIGHLIGHTS318919 SNPs identified from 42149 unigenes of the transcriptome sequences.25772 SNPs identified from 1826 ammonia‐responsive genes with functional annotation.Ammonia‐tolerant associated SNP markers were identified with selective genotyping.12 SNP markers were detected to be associated with tolerance to ammonia toxicity.The markers could be used for marker‐assisted selection to increase production.