Xianhong Meng

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.

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.

Genetic parameter estimation of reproductive traits of Litopenaeus vannamei

In this study, the heritability, repeatability, phenotypic correlation, and genetic correlation of the reproductive and growth traits of L. vannamei were investigated and estimated. Eight traits of 385 shrimps from forty-two families, including the number of eggs (EN), number of nauplii (NN), egg diameter (ED), spawning frequency (SF), spawning success (SS), female body weight (BW) and body length (BL) at insemination, and condition factor (K), were measured,. A total of 519 spawning records including multiple spawning and 91 no spawning records were collected. The genetic parameters were estimated using an animal model, a multinomial logit model (for SF), and a sire-dam and probit model (for SS). Because there were repeated records, permanent environmental effects were included in the models. The heritability estimates for BW, BL, EN, NN, ED, SF, SS, and K were 0.49 ± 0.14, 0.51 ± 0.14, 0.12 ± 0.08, 0, 0.01 ± 0.04, 0.06 ± 0.06, 0.18 ± 0.07, and 0.10 ± 0.06, respectively. The genetic correlation was 0.99 ± 0.01 between BW and BL, 0.90 ± 0.19 between BW and EN, 0.22 ± 0.97 between BW and ED, −0.77 ± 1.14 between EN and ED, and −0.27 ± 0.36 between BW and K. The heritability of EN estimated without a covariate was 0.12 ± 0.08, and the genetic correlation was 0.90 ± 0.19 between BW and EN, indicating that improving BW may be used in selection programs to genetically improve the reproductive output of L. vannamei during the breeding. For EN, the data were also analyzed using body weight as a covariate (EN-2). The heritability of EN-2 was 0.03 ± 0.05, indicating that it is difficult to improve the reproductive output by genetic improvement. Furthermore, excessive pursuit of this selection is often at the expense of growth speed. Therefore, the selection of high-performance spawners using BW and SS may be an important strategy to improve nauplii production.

Heterosis and heritability estimates for the survival of the Pacific white shrimp ( Litopenaeus vannamei ) under the commercial scale ponds

The aim of the present study is to detect the potential of the base population from diallel crosses of eight introduced strains of the Pacific white shrimp (Litopenaeus vannamei) for improving the yield. Heterosis and heritability were estimated for pond survival at commercial farm conditions for the base population that included 207 full-sib families from a nested mating design by artificial insemination. Among all the hybrids, the heterosis ranged from–11.37% (UA1×UA2) to 20.53% (UA3×SIN) with an average of 0.953%. The results showed that more than half of the hybrids (51.85%) have negative heterosis for survival rate, but most of the hybrids with positive heterosis have high estimates. The high proportion of negative heterosis for survival rate reminders us that the survival trait also should be considered in the crossbreeding program to avoid yield decrease. However, high positive heterosis manifested in most of the hybrids for survival indicates the usefulness of these hybrids for improving the survival to obtain higher yield by crossbreeding in this breeding program. The heritability estimate for pond survival was 0.092±0.043 when genetic groups were included in the pedigree, and it was significantly different from zero (P<0.05). The results from this study also indicated that significant improvement for survival is possible through selection in L. vannamei.

iTRAQ-based comparative proteome analysis for molecular mechanism of defense against acute ammonia toxicity in Pacific White shrimp Litopenaeus vannamei

ABSTRACT In the practical farming of Litopenaeus vannamei, the intensive culture system and environmental pollution usually results in a high concentration of ammonia, which brings large detrimental effects to shrimp, such as increasing the susceptibility to pathogens and even causing high mortality. We have revealed that the survival time under acute ammonia stress varied substantially among different families and obtained ammonia‐tolerant (LV_T) and ammonia‐sensitive (LV_S) families. In order to understand the molecular mechanism of defense against ammonia toxicity in shrimp, we performed iTRAQ LC‐MS/MS proteomic analysis between LV_T and LV_S groups of L. vannamei under acute ammonia stress to identify the key proteins and pathways that play an effective role for against ammonia toxicity. By comparative proteome analysis, 202 significantly differentially proteins (DEPs) were identified in LV_T compared to LV_S, and most of the DEPs (60%) were up‐regulated. Excepting for the proteins without function reporting, the meaningful finding is that 77.8% of the DEPs have been reported mainly involving in immune defense and stress tolerant in crustacean species, such as hemocyanin, Rab7, Rab GTPase, Rac1, alpha 2 macroglobulin, Bip, peroxiredoxin, Cu/Zn SOD, glutathione peroxidase, thioredoxin, calreticulin, and Elongation Factor 1‐alpha, etc. These DEPs might potentially play important role in against ammonia toxicity, and it also reflected a relation between ammonia tolerance and pathogen resistance. In addition, a total of 10 significantly changed KEGG pathways were detected, and the network diagram of these KEGG pathways showed that more critical nodes were up‐regulated, which involved in protein synthesis and transport, and against stress stimuli. This study provided important information for understanding the molecular mechanism of defense against ammonia toxicity in shrimp at whole protein level. HIGHLIGHTSThe mechanism of against ammonia toxicity in shrimp analyzed by iTRAQ approach.Ammonia tolerant and sensitive shrimp used for the comparative protein analysis.Most of the significantly different proteins involved in immune and up regulated.Most of the critical nodes in the significantly changed pathways were up regulated.A relation between ammonia tolerance and pathogen resistance was also suggested.

cDNA cloning and expression analysis of a phosphopyruvate hydratase gene from the chinese shrimp Fenneropenaeus chinensis

Abstract In the present study a cDNA encoding a phosphopyruvate hydratase (enolase) was cloned from the muscle of the Chinese shrimp (Fenneropenaeus chinensis) and named as FcEnolase. The cDNA of FcEnolase encoded a protein of 434 amino acid residues with a molecular mass 47.22 kDa. The residues 342–355 constituted the signature motif “LLLKVNQIGSVTES”. A SNP locus (C96T) in the ORF at 96 bp was identified. The results showed that the FcEnolase was a conserved gene. In the normal F. chinensis, the mRNA level in the muscle was much higher (P < 0.05) than the mRNA level in the gill and hepatopancreas. To verify the mRNA level of FcEnolase in the F. chinensis post WSSV infection, a real‐time RT‐PCR was performed. In the WSSV‐infected F. chinensis, the FcEnolase mRNA level was significantly (P < 0.05) up‐regulated in the muscle at 12 and 24 h post challenge (hpc) to approximately 2.7‐fold and 2.7‐fold the mRNA level in the controls, respectively. The FcEnolase mRNA level in the gill was significantly (P < 0.05) down‐regulated at 6 hpc to approximately 0.3‐fold the mRNA level in the control, followed by a significant (P < 0.05) up‐regulation at 12 hpc to approximately 2.8‐fold the mRNA level in the control. There was no obvious change of FcEnolase mRNA level in the hepatopancreas during the infection process. The expression profile coincided with the fact that WSSV primarily infects the tissues of muscle and gill, but hardly infects hepatopancreas. To verify the protein level of FcEnolase post WSSV infection, a Western blot was performed. The FcEnolase protein level in the muscle at 24 hpc significantly (P < 0.05) increased to approximately 2.1‐fold the level in the control. These results showed the characterization of FcEnolase and suggested that the FcEnolase might be involved in the response of F. chinensis to WSSV infection.

Cloning and characterization of a transcriptional repressor from Fenneropenaeus chinensis (Osbeck, 1765)

In this study, a transcriptional repressor was cloned and characterized from the Chinese shrimp Fenneropenaeus chinensis (Osbeck, 1765) and named FcTR. The results indicate that the full-length cDNA of 1397 bp has an open reading frame encoding a polypeptide of 354 amino acids. The calculated molecular mass of the mature protein was found to be 39.9 kDa and theoretical pI 5.59. The FcTR has four ZnF-C2H2 domains and two putative transcriptional repressor regulating G2/M transition domains (SFP1). A phylogenetic analysis of transcriptional repressor sequences shows that FcTR has a high homology with the invertebrate transcriptional repressors from Megachile rotundata (Fabricius, 1793), Apis mellifera Linnaeus, 1758, Camponotus floridanus (Buckley, 1866), Acromyrmex octospinosus echinatior (Forel, 1899) and Apis florea Fabricius, 1787 (similarity 72%). A qRT-PCR analysis indicated that FcTR was expressed in intestines, hepatopancreas, muscles and gills, and its profile was increased significantly post WSSV (white spot syndrome virus) challenge followed by a decreased trend in all examined tissues. These results indicate that FcTR might be involved in the immune defence response to WSSV in F. chinensis as a negative regulator.

Comparisons of Growth and Survival Performance Among Selected Families and Wild Populations of Fenneropenaeus chinensis

In this study, families of selected population for growth (SP_BWT), selected population for white spot syndrome virus (WSSV) resistance (SP_RW), Bohai wild population (WP_BH) and Huanghai wild population (WP_HH) of F. chinensis were constructed through artificial insemination and with the standardized procedure of larvae rearing. Growth and survival performance were studied among four populations after a 70 days common test. The results showed that the maximum least square mean of body weight was 17.50 g in SP_BWT while the minimum was 13.03 g in WP_HH. Compared with WP_BH, body weight of SP_BWT increased by 23.41% (P < 0.01) and that of SP_RW by 12.20% (P > 0.05). Body weights of SP_BWT and SP_RW were significantly higher than that of WP_HH, which increased by 34.31% (P < 0.01) and 22.10% (P < 0.05), respectively. The mean AGR of four populations was 0.19, 0.18, 0.17 and 0.16 g d−1, respectively. Coefficient of variation of body weight among four populations was high, which ranged from 32.67% to 35.25%. Such a range showed that there was the potentiality for further improvement in selected populations. Coefficient of variation of survival rate among four populations was low, varying between 3.20% and 5.90%. The difference of survival was highly significant (P < 0.01) between SP_BWT and WP_BH, and significant (P < 0.05) between SP_RW and WP_BH. However, no significant difference among other populations (P < 0.05) was observed. Different growth performances were also observed among different families in each population. The body weight of 798F family was the highest. The absolute growth rate (AGR) was 0.25 g d−1, 150% higher than that of the lowest one, 0.1 g d−1 in 807F family. Survival rate of families among four populations was different. The highest was 94.74%, and the lowest was 71.88%.

Isolation and characterization of a Raf gene from Chinese shrimp Fenneropenaeus chinensis in response to white spot syndrome virus infection

Abstract Raf is a member in the Ras/Raf/MAPKK/MAPK signaling transduction pathway. To obtain a better understanding of Raf in the interaction between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), the sequence of cDNA of Raf from F. chinensis (FcRaf) was obtained. The FcRaf gene contained a 2421 bp open reading frame (ORF). The FcRaf shared most characteristic of Raf protein, such as the Raf‐like Ras‐binding domain (RBD), phorbol esters/diacylglycerol binding domain (C1 domain), and catalytic domain of the serine/threonine kinases, Raf (STKc_Raf). The sequence of functional domains of Raf protein was relatively conserved. The FcRaf mRNA was detected in the tissues of gill, muscle, and hepatopancreas from normal F. chinensis. The mRNA abundance level of FcRaf in the gill was the highest, which was 2.7‐fold the level in the hepatopancreas. The expression level of FcRaf was significantly (P < 0.05) up‐regulated in the tissues of gill, muscle, and hepatopancreas post WSSV‐infection, which suggested that FcRaf might be involved in the interaction between F. chinensis and WSSV. Two SNP loci were identified in the ORF, one of which was a C‐T mis‐sense mutation, where an Ala was replaced by a Val, and induced the predicted protein secondary structure change. Considering the relatively low MAF (0.07), whether this mis‐sense mutation was a detrimental mutation needs further investigation. HighlightsA cDNA encoding Raf from Fenneropenaeus chinensis (FcRaf) was cloned and sequenced for the first time.The sequence of functional domains of FcRaf protein was relatively conserved.The FcRaf mRNA abundance level in tissues fluctuated post WSSV challenge.Two SNPs were identified in the FcRaf ORF and one of which was a mis‐sense mutation.

Screening white spot syndrome virus (WSSV)-resistant molecular markers from Fenneropenaeus chinensis

White spot syndrome virus (WSSV)-resistant molecular markers were screened from the selectively bred new variety ‘Huanghai No. 2’ of Fenneropenaeus chinensis using unlabeled-probe high-resolution melting (HRM) technique. After the artificial infection with WSSV, the first 96 dead shrimps and the last 96 surviving shrimps were collected, representing WSSV-susceptible and -resistant populations, respectively. The genotypes at well-developed 39 single nucleotide polymorphisms (SNPs) loci were obtained. As revealed in the Chi-square test, 3 SNPs, genotype A/A of contig C364-89AT, genotype A/A of C2635-527CA and genotype C/T of contig C12355-592CT, were positively correlated with disease-resistance traits. Other 2 SNPs, genotype G/G of contig C283-145AG and genotype C/C of contig C12355-592CT, were negatively correlated. Moreover, analysis with BlastX program for disease-resistant SNPs indicated that 3 contigs, Contig283, Contig364 and Contig12355, matched to the functional genes of effector caspase of Penaeus monodon, peptide transporter family 1-like protein, and 40S ribosomal protein S2 of Perca flavescens with high sequence similarity. The results will be helpful to provide theoretical and technical supports for molecular marker-assisted selective breeding of F. chinensis.