Genetic analysis and QTL mapping of seed hardness trait in a soybean (Glycine max) recombinant inbred line (RIL) population.

Seed hardness is a critical quality trait impacting both the suitability of soybeans for consumption and their processing. The primary objective of this study was to explore the genetic foundations underlying seed hardness in soybeans. A 234 recombinant inbred line (RIL) population was evaluated for seed hardness across three years (2015 in Gansu, 2016, and 2017 in Hainan). Notably, the parent varieties, Zhonghuang35 and Jindou21, displayed significant differences in seed hardness. Also, the RIL population exhibited a wide range of genetic variation in seed hardness, with coefficients of variation between 70.53 % and 94.94 %. The frequency distribution of this trait conformed to a relatively normal distribution, making it suitable for QTL analysis. Six QTLs associated with seed hardness were identified with three located on chromosome 2 and three on chromosome 16. The major QTL, qHS-2-1, consistently exhibited the highest percentage of PVE and LOD in Gansu 2015, Hainan 2016, and Hainan 2017, suggesting its central role in determining seed hardness. Further investigation revealed four genes within the qHS-2-1 interval potentially related to seed hardness. GO enrichment analysis provided insights into their functions, including factors such as Glyma.02G307000, a pectin lyase-like superfamily protein, which could influence seed hardness through its role in pectin lyase enzyme activity. Expression analysis of these candidate genes demonstrated significant differences between the two parent varieties, further highlighting their potential role in seed coat hardness. This study offers valuable insights into the genetic mechanisms governing soybean seed coat hardness, providing a foundation for future research and crop improvement efforts.

Identification of two genes potentially related to myogenesis and muscle growth in Fenneropenaeus chinensis: Activin receptor II and Follistatin-like protein.

Activin receptor (ActR) and follistatin-like (FSTL) genes, which are involved in the Myostatin (Mstn) related TGF-ß/Smad signaling pathway, play important roles in regulating the muscle generation, development and growth of muscle in vertebrate. Our previous studies have confirmed that Mstn negatively regulates muscle development and growth in Fenneropenaeus chinensis as that in vertebrate. However, the roles of ActR and FSTL in muscle development and growth in invertebrate remains unclear. In the present study, type II ActR(FcActRII) and FSTL (FcFSTL) genes from F. chinensis were cloned and characterized, and their functions on muscle development and growth were investigated. The full-length cDNAs of FcActRII and FcFSTL were 2366 bp that encoded 572 amino acids and 2474 bp that encoded 717 amino acids, respectively. Sequence analysis revealed that the overall protein sequences of the two genes shared 97% and 96% identities with Penaeus vannamei and 50%-59% and 35%-36% identities with vertebrates, respectively. In the early development stages, muscles firstly appeared in nauplius stage and developed gradually until post larval, and the mRNA expressions of FcActRII increased from gastrula to zoea stage and then decreased from zoea stage to post larval stage while that of FcFSTL was lowest in gastrula stage and increased rapidly in nauplius stage and then expressed stably from nauplius stage to post-larval stage. In the adult shrimp, the two genes were widely distributed in the examined tissues. The FcActRII expression in muscle of L group was significantly lower than that of S group, but the FcFSTL expression showed an opposite result. After down-regulating the expression of FcMstn by RNAi, FcActRII expression was significantly down-regulated while that of FcFSTL was up-regulated. The present study suggested that FcActRII and FcFSTL, regulated by FcMstn, might be involved in myogenesis and muscle growth.

Feed competition reduces heritable variation for body weight in Litopenaeus vannamei.

BACKGROUND: Competition is a common social interaction among shrimp and depending on its intensity, it can affect heritable variation and response to selection. Little is known about the variance of indirect genetic effects (IGE) under competitive and non-competitive conditions in shrimp. In this study, we used extended mixed linear models to estimate genetic parameters for the direct genetic effect (DGE) and IGE on body weight in Litopenaeus vannamei raised under ad libitum (AF, non-competitive environment) and restricted (RF, competitive environment) feeding regimes. RESULTS: Estimates of heritabilities for body weight obtained with a traditional animal model (i.e. without accounting for IGE) were 0.11 ± 0.09 under AF and 0.25 ± 0.11 under RF. With extended animal models that accounted for IGE, the corresponding estimates for body weight were 0.07 ± 0.08 and 0.34 ± 0.11. Thus, heritabilities were higher under the RF regime than under the AF regime, regardless of whether IGE was accounted for or not. The log-likelihood ratio test revealed significant IGE under the RF regime. Although estimates of indirect genetic variance were low (0.0023 ± 0.0013 for AF and 0.0028 ± 0.0012 for RF), they contributed substantially to the total heritable variance: 66.8% for AF and 692.2% for RF. The total heritable variance was smaller under the RF regime (0.7 ± 1.3) than under the AF regime (5.8 ± 2.6) because of the high contribution of the negative covariance between DGE and IGE (- 7.03). Estimates of the correlation between DGE and IGE were 0.32 ± 0.47 under AF and - 0.93 ± 0.15 under RF, those of DGE and IGE for body weight between both regimes were 0.94 ± 0.07 and 0.67 ± 0.20, respectively, and those of IGE for body weight with DGE for survival were - 0.12 ± 0.22 under AF and - 0.58 ± 0.20 under RF. CONCLUSIONS: These results indicate that strong competitive interactions occurred under the RF regime in L. vannamei. Significant reranking and variation in IGE of individuals were observed between the two feeding regimes. Strong competitive interactions reduced the total heritable variation for body weight when food was restricted. These results indicate that the extent of competition among L. vannamei depends on the feeding regime applied and that this competition affects the genetic basis of body weight.

Molecular characterization of myostatin and its inhibitory function on myogenesis and muscle growth in Chinese Shrimp, Fenneropenaeus chinensis.

Myostatin (Mstn) inhibits muscle growth in vertebrates with endoskeleton, but it is still inconclusive that Mstn is a positive or negative regulator in crustacean with exoskeleton, and little information was available for its function on myogenesis. In this study, we identified and characterized the Mstn from Fenneropenaeus chinensis (FcMstn), and investigated its function on myogenesis and muscle growth. Two different cDNA sequences (2628 bp and 2604 bp) encoding for slightly different sizes of proteins were obtained for FcMstn, containing 86 bp of 5' untranslated regions (UTR) and 1258 bp of 3' UTR. The open reading frame of the long sequence and the short sequence contain 1284 bp and 1260 bp cDNA, encoding 427 and 419 amino acid sequence, respectively. Sequence analysis revealed that the overall protein sequence and specific functional sites of FcMstn were highly conserved with those in other crustacean species. In the early development stage, the muscle firstly appeared in nauplius stage and developed gradually until post larval, but the expression of FcMstn at mRNA and protein levels decreased from nauplius stage to post larval stage, indicating that Mstn involved in myogenesis as a negative regulator in shrimp. In the adult shrimp, the expression of FcMstn at mRNA and protein levels in muscle were significantly lower in the larger group than in the smaller group, and the diameter and number of muscle fiber of the muscle were significantly different between the two groups. Moreover, the shrimp with reduced level of FcMstn by RNAi displayed a dramatic faster growth rate compared with the control group. The present study demonstrates that FcMstn involved in myogenesis and muscle growth probably also as a negative regulator in shrimp like in vertebrates.

High accuracy of pooled DNA genotyping by 2b-RAD sequencing in the Pacific white shrimp, Litopenaeus vannamei.

Using pooled DNA genotyping to estimate the proportional contributions from multiple families in a pooled sample is of particular interest for selective breeding in aquaculture. We compared different pooled libraries with separate 2b-RAD sequencing of Litopenaeus vannamei individuals to assess the effect of different population structures (different numbers of individuals and families) on pooled DNA sequencing, the accuracy of parent sequencing of the DNA pools and the effect of SNP numbers on pooled DNA sequencing. We demonstrated that small pooled DNA genotyping of up to 53 individuals by 2b-RAD sequencing could provide a highly accurate assessment of population allele frequencies. The accuracy increased as the number of individuals and families increased. The allele frequencies of the parents from each pool were highly correlated with those of the pools or the corresponding individuals in the pool. We chose 500-28,000 SNPs to test the effect of SNP number on the accuracy of pooled sequencing, and no linear relationship was found between them. When the SNP number was fixed, increasing the number of individuals in the mixed pool resulted in higher accuracy of each pooled genotyping. Our data confirmed that pooled DNA genotyping by 2b-RAD sequencing could achieve higher accuracy than that of individual-based genotyping. The results will provide important information for shrimp breeding programs.

Integrative phenotypic and gene expression data identify myostatin as a muscle growth inhibitor in Chinese shrimp Fenneropenaeus chinensis.

Growth traits, largely determined by muscle growth, are the most critical economic traits in shrimp breeding. Myostatin (Mstn) is a conserved inhibitor of muscle growth in vertebrates, but until now solid evidence supporting a similar function of Mstn in invertebrates has been lacking. In the present study, we examined the Mstn expression along with growth trait data in a Fenneropenaeus chinensis population, to establish a potential correlation between Mstn and growth. The heritabilities of FcMstn expression, body weight at 190 days of culture, body weight and length at 230 days of culture, and average daily gain were estimated using 773 individuals and a thirteen-generation pedigree. The results showed FcMstn expression was negatively correlated with the growth traits, and the mean FcMstn expression in females was significantly lower than that of males, indicating Mstn negatively regulates muscle growth in shrimp, and its lower expression may underscore the faster growth of females. Low heritabilities were detected for FcMstn expression, suggesting that the expression of Mstn might be heritable in shrimp. These results provide strong support for a growth inhibitory function of Mstn in F. chinensis, and suggest a potential method for selective breeding of this species without substantial experimental resources and labor force.

Screening of the candidate genes related to low-temperature tolerance of Fenneropenaeus chinensis based on high-throughput transcriptome sequencing.

In order to screen the candidate genes of Fenneropenaeus chinensis related to low-temperature tolerance, this research takes juvenile prawns of F. chinensis (P40) in low temperature stress group (4°C) and normal temperature group (18°C) as experimental materials. The results showed that a total of 127,939 Unigenes with average length of 1,190 bp were obtained by assembly, of which 46% were annotated in the Nr database. A total of 1,698 differentially expressed genes were screened by differential gene expression analysis, of which 920 genes showed up-regulated expression and 778 genes showed down-regulated expression. Both GO and KEGG enrichment analysis revealed that differentially expressed genes were enriched in spliceosomes, ribosomes, bile secretion, ABC transport pathways, and cellular nitrogen compound synthesis. A further in-depth analysis obtained 8 genes that may be associated with low-temperature traits of F. chinensis. Five of them displayed up-regulated expression, including ATP-binding cassette protein C, acid ceramidase, glutathione transferase, C-type lectin and heat shock protein HSP70. The remaining three genes, γ-butyl betaine hydroxylase, ß-hexosaminidase A and long chain fatty acid-CoA ligase displayed down-regulated expression. Eight differentially expressed genes were randomly selected and the real time RT-PCR verification showed that their expression levels were consistent with the sequencing results, demonstrating the accuracy of the sequencing results. The results of this study provide basic data for revealing the molecular mechanisms of F. chinensis in response to low temperature stress and the molecular assisted breeding of F. chinensis in low temperature.

Insights into the molecular basis of immunosuppression and increasing pathogen infection severity of ammonia toxicity by transcriptome analysis in pacific white shrimp Litopenaeus vannamei.

The high concentration of ammonia resulting from intensive culture system and environmental pollution could cause disease occurrence in shrimp, but little information is available on its molecular mechanisms. In this study, we performed comparative transcriptome analysis among WSSV-infected shrimp under ammonia stress (LAV), WSSV-infected shrimp under normal water (LV), and normal shrimp under ammonia stress (LA) groups to identify the key genes and pathways involved in immunosuppression and increasing pathogen infection severity caused by ammonia toxicity in Litopenaeus vannamei. Totally, 526 significantly differential expressed genes (DEGs) were identified in LAV group compared to LV and LA groups, among which 270 genes were lost expressed and 67 genes uniquely expressed in the LAV group. According to the public functional reports for the annotated DEGs, they potentially involved in the following functions: (1) accelerating pathogen adhesion, invasion and multiplication; (2) reducing the ability for pathogen defense and immune response; (3) inhibiting positive regulation of apoptotic and antioxidant defense for host homeostasis; (4) inhibiting transcription and protein transport; (5) and increasing protein methylation and ubiquitination, etc. A total of 13 pathways were obtained mainly involving in this process, which mainly led to the following changes: (1) increasing the immunosuppression, anemia, endocrine dysfunction, neurotoxic effect and neuroinvasion, atherosclerosis and thrombogenesis, blood-brain barrier penetration, thyroid disorder, necrosis, inflammation, and circadian disturbance; (2) reducing the ability of vascular remodeling, angiogenesis, cell survival, migration, apoptosis, and lymph transferred to blood stream; (3) leading to cell hypertrophy, cellular shape changes, and mesangial matrix expansion. The present results firstly supplied molecular mechanisms for the ammonia toxicity inhibiting the immune system and increasing pathogen infection severity in shrimp, which is a prerequisite for better understanding the pathogenesis caused by ammonia toxicity.

Comparative transcriptomic characterization of the eyestalk in Pacific white shrimp (Litopenaeus vannamei) during ovarian maturation.

In crustaceans, some of fundamental regulatory processes related to a range of physiological functions, including ovarian maturation, molting, glucose homeostasis, osmoregulation, etc., occur in the organs of the eyestalk. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites (X-organ/sinus gland, XO/SG) within the eyestalk. As unilateral eyestalk ablation was the most common method used to artificially induce ovarian maturation for farmed Litopenaeus vannamei, to better understand the reproductive regulation mechanism in L. vannamei, we have investigated the transcriptomes of the eyestalk during five ovary developmental stages with or without eyestalk ablation by high-throughput Illumina sequencing technology. The raw reads were assembled and clustered into 127,031 unigenes. Meanwhile, the differentially expressed genes (DEGs) between ovarian development stages were identified. We examined, through DEG enrichment analysis, eyestalk gene expression patterns for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, comparing natural to artificially induced ovarian maturation. We also identified a variety of transcripts that appear to be differentially expressed throughout ovarian maturation. These include transcripts that encode G-protein coupled receptors (GPCRs) and neuropeptides, such as the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and crustacean female sex hormone (CFSH). Furthermore, numerous exoskeleton formation-related genes were found to be down-regulated during ovarian maturation, including cuticle-like proteins, eclosion hormone (EH), and gastrolith-like proteins, of which the latter are the first reported in L. vannamei. Our work is the first reproduction-related investigation of L. vannamei focusing on the eyestalk at the whole transcriptome level. These findings provide novel insight into the function of the eyestalk in reproduction regulation.

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

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.

Transcriptome analysis of 'Huanghai No. 2' Fenneropenaeus chinensis response to WSSV using RNA-seq.

White spot syndrome (WSS) is one of the most damaging phenomena in the culturing of shrimp. To characterize the mechanisms of the molecular responses to WSSV infection in 'Huanghai No. 2'' Fenneropenaeus chinensis, we used next-generation sequencing to observe the transcriptome after oral infection. A total of 108.6 million clean reads were obtained and assembled into 64,103 final unigenes with an average length of 845 bp (N50 = 1534 bp). The assembled unigenes contained 14,263 significant unigenes after BLASTX against the Nr database (E-value cut-off of 10-5). After comparison of digital gene expression data between challenged and control shrimp, a total of 896 DEGs after WSSV infection were identified. Gene pathway analysis indicated that 92, 131 and 142 metabolic pathways were affected at early, peak and late phases respectively. Some pathways were related to the immune response, such as the phagosome, complement and coagulation cascades, the antigen processing and presentation pathway and so on. Many immune-related genes were also identified after pathway analysis. Interestingly, some growth-related genes, such as cathepsin L, myosin regulatory light chain 2 smooth muscle, and alpha-amylase were also differentially expressed after WSSV infection, and the correlation between growth trait and WSSV-resistance trait need further research. The expression patterns of eight DEGs were confirmed by quantitative real-time reverse transcription polymerase chain reaction, and there was good agreement between RNA-seq and qRT-PCR. These data will provide valuable information for characterizing the immune mechanism of the response of shrimp's to WSSV.

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

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.

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

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.

Genetic assessment of residual feed intake as a feed efficiency trait in the Pacific white shrimp Litopenaeus vannamei.

BACKGROUND: Residual feed intake (RFI) was investigated as a measure of feed efficiency in a breeding population of Litopenaeus vannamei. Shrimp from 34 families were housed individually and feed efficiency and growth traits were recorded during two successive growth periods. The objectives of this study were (1) to estimate the heritability of RFI and related traits, including feed efficiency ratio (FER), average daily gain (ADG) and daily feed intake (DFI), (2) to determine the relationships between RFI and other traits, and (3) to evaluate the variation of these traits across two growth periods. RESULTS: Shrimp displayed large inter-individual variation in RFI, FER, ADG and DFI during each growth period. Heritability estimates of all these traits during both periods reached high values (0.577 ± 0.232 to 0.707 ± 0.252). RFI showed weak and no genetic correlations with ADG during the two growth periods between days 1 to 21 (0.135 ± 0.204) and 22 to 42 (-0.018 ± 0.128), respectively, but high positive genetic correlations with DFI (>0.8). Weak and moderate negative genetic correlations were observed between RFI and FER during the two periods (-0.126 ± 0.208 and -0.387 ± 0.183). As evidenced by the high genetic correlations between the two periods for each trait (>0.6), trait performance of the shrimp tended to be consistent across periods. CONCLUSIONS: For the first time, accurate measurement of individual feed efficiency on a large scale was achieved in shrimp. Although the estimated heritability reported here for RFI may be overestimated, it is a heritable trait in L. vannamei that can be improved by genetic improvement. For L. vannamei, the biggest potential advantage in using RFI as a measure of feed efficiency is that it is independent of growth rate, and thus genetic selection on RFI has the potential to improve feed efficiency and reduce feed intake, without compromising growth performance.

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

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.

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.

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

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.

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.

Comparative microarray profile of the hepatopancreas in the response of "Huanghai No. 2" Fenneropenaeus chinensis to white spot syndrome virus.

White spot syndrome virus (WSSV) infects all shrimp species and is the greatest detriment to shrimp culture. To better understand the mechanism of molecular responses to WSSV infection in "Huanghai No. 2" Fenneropenaeus chinensis, a microarray technique was used. Microarray gene expression profiling of 59,137 unigenes identified Differentially Expressed Genes (DEGs) both in live and moribund shrimp at early, peak and late phases. In live shrimp, 1307, 1479 and 1539 DEGs were obtained in the early, peak and late phase, respectively. Meanwhile, 1536, 2181 and 1591 DEGs were obtained in moribund shrimp. Twenty known annotation genes are uniquely expressed in the late phase of live shrimp, including adhesion regulating molecule 1, arginine kinase, BUD31 homolog, and QM. Compared to WSSV-susceptible shrimp, 75 known annotation genes are uniquely expressed in WSSV-resistant shrimp, including arginine kinase, BUD31 homolog, clottable protein 2, caspase 2, cathepsin C, calnexin, HMGBb, Histone 3, and selenoprotein M. The gene expression patterns of the infected shrimp were altered by WSSV infection. To further confirm the expression of differentially expressed genes, real-time RT-PCR was performed to test six randomly selected genes. The data will provide valuable information to understand the immune mechanism of shrimp's response to WSSV.

Isolation and expression analysis of an MAPKK gene from Fenneropenaeus chinensis in response to white spot syndrome virus infection.

Mitogen-activated kinase kinase (MAPKK) is an important gene involved in the host-virus interaction process. To obtain a better understanding of MAPKK in the interaction process between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), we cloned the sequence of an MAPKK cDNA from F. chinensis (FcMAPKK) and investigated the effect of FcMAPKK on WSSV infection. The results showed that the FcMAPKK gene contained a 1227 bp open reading frame (ORF), which encoded a highly conserved protein with a serine/threonine protein kinase catalytic (S_TKc) domain. The deduced amino acid sequence of FcMAPKK shared identities between 11.9 and 92.6% with MAPKKs from vertebrate, invertebrate, plant and fungus species. The FcMAPKK was expressed in all the examined tissues in the normal F. chinensis. FcMAPKK expression level was highest in the hepatopancreas where it was approximately 2.6-fold the expression level in the gill, and lowest in the muscle where it was approximately 0.3-fold the expression level in the hepatopancreas. The FcMAPKK expression levels in the muscle, gill, and hepatopancreas were all changed post WSSV challenge. The FcMAPKK expression was significantly (P < 0.01) up-regulated in the muscle of F. chinensis at 48 h post WSSV infection. The WSSV began to replicate quickly in the normal F. chinensis at 48 h post infection, while the WSSV replication in the U0126-treated F. chinensis could be significantly (P < 0.05) inhibited. The results suggested that FcMAPKK might be involved in the WSSV infection process, and hijacking of FcMAPKK might be required for WSSV replication in F. chinensis.