Genome-wide QTL and eQTL mapping reveal genes associated with growth rate trait of the Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND: Growth rate is a crucial economic trait for farmed animals, but the genetic regulation of this trait is largely unknown in non-model organisms such as shrimp. RESULTS: In this study, we performed genome-wide phenotypic quantitative trait loci (QTL) and expression quantitative trait loci (eQTL) mapping analyses to identify genes affecting the growth rate of Pacific white shrimp (Litopenaeus vannamei), which is the most commercially-farmed crustacean worldwide. We used RNA-sequencing of 268 individuals in a mapping population, and subsequently validated our findings through gene silencing and shrimp growth experiments. We constructed a high-density genetic linkage map comprising 5533 markers spanning 44 linkage groups, with a total distance of 6205.75 cM and an average marker interval of 1.12 cM. Our analyses identified 11 QTLs significantly correlated with growth rate, and 117,525 eQTLs. By integrating QTL and eQTL data, we identified a gene (metalloreductase STEAP4) highly associated with shrimp growth rate. RNA interference (RNAi) analysis and growth experiments confirmed that STEAP4 was significantly correlated with growth rate in L. vannamei. CONCLUSIONS: Our results indicate that the comprehensive analysis of QTL and eQTL can effectively identify genes involved in complex animal traits. This is important for marker-assisted selection (MAS) of animals. Our work contributes to the development of shrimp breeding and available genetic resources.

How is work-family conflict linked to nurse-assessed patient safety among intensive care unit nurses? A serial multiple mediation analysis.

AIM: The aim of this study was to test whether rumination and negative affectivity mediate the relationship between work-family conflict and nurse-assessed patient safety among intensive care unit nurses. BACKGROUND: Most intensive care unit nurses experience work-family conflicts that jeopardise patient safety. Although prior studies have explored the effect of work-family conflict on patient safety, few have investigated whether work-family conflict is associated with patient safety through rumination and negative affectivity among intensive care unit nurses. DESIGN: Cross-sectional study. METHODS: This study included 209 intensive care unit nurses from five general hospitals. The Work-Family Conflict Scale, the Ruminative Response Scale, the Positive and Negative Affect Schedule-Negative Affectivity, and three items indicating nurses' perception of overall patient safety were used to gather data. Associations between work-family conflict, rumination, negative affectivity, and nurse-assessed patient safety were assessed using correlation and serial multiple mediation analysis. RESULTS: Work-family conflict, rumination, negative affectivity, and nurse-assessed patient safety were significantly correlated (p < 0.01). Work-family conflict can have not only a direct negative impact on the nurse-assessed patient safety (effect = -0.0234; standard error [SE] = 0.0116; 95% confidence interval [CI]: lower limit [LL] = -0.0464, upper limit [UL] = -0.0005) but also an indirect impact on nurse-assessed patient safety through three paths: the independent mediating role of rumination (effect = -0.0118; SE = 0.0063; 95% CI: LL = -0.0251, UL = -0.0006), the independent mediating role of negative affectivity (effect = -0.0055; SE = 0.0039; 95% CI: LL = -0.0153, UL = -0.0001), and the chain-mediating role of rumination and negative affectivity (effect = -0.0078; SE = 0.0031; 95% CI: LL = -0.0152, UL = -0.0027). CONCLUSION: Our findings indicated that work-family conflict could influence nurse-assessed patient safety through increasing rumination and negative affectivity among intensive care unit nurses. Based on the results, interventions aimed at decreasing work-family conflict would be beneficial for intensive care unit nurses' emotional stability and patient safety.

ATP synthase subunit e is a shrimp growth-associated breeding marker.

Penaeus vannamei is one of the most popular aquaculture species in the world. This species is featured with its fast-growing and delicious taste, which drives people develop various strains. During this process identification of trait-associated markers could effectively increase breeding efficiency. Driven by this, we tried to screen fast-growing key regulators via a FACS-based high throughput method, in which 2-NBDG was applied as a fluorescent indicator for direct glucose uptake measurement. Totally six candidate genes were screened out followed by in vitro validation in 293T cells. After that, the correlation between these genes and shrimp growing was further verified in a hybrid lineage. The expression level of two genes including ATP synthase subunit e and inhibitor of apoptosis protein showed some correlation with shrimp growth speed. Furthermore, we tested these two candidate markers in various lineages and confirmed that ATP synthase subunit e could be a shrimp growth-associated breeding marker.

Integrated Analysis of DNA Methylome and Transcriptome Reveals Epigenetic Regulation of Cold Tolerance in Litopenaeus vannamei.

DNA methylation is an important epigenetic modification that has been shown to be associated with responses to non-biological stressors. However, there is currently no research on DNA methylation in response to environmental signals in shrimp. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles and differentially expressed genes between two strains of Litopenaeus vannamei with significantly different cold tolerance through whole genome bisulfite sequencing (WGBS) and transcriptome sequencing. Between Lv-C and Lv-T (constant temperature of 28 °C and low temperatures of 18 °C and 10 °C) under cytosine-guanine (CG) environments, 39,100 differentially methylated regions (DMRs) were identified, corresponding to 9302 DMR-related genes (DMRGs). The DMRs were mainly located in the gene body (exons and introns). Gene Ontology (GO) analysis showed that these DMRGs were significantly enriched in cell parts, catalytic activity, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed significant enrichment of these DMRGs in pathways such as proteasome (ko03050), oxidative phosphorylation (ko00190), mTOR signaling pathway (ko04150), fatty acid metabolism (ko01212), and fatty acid degradation (ko00071). The comprehensive results suggested that L. vannamei mainly regulates gene expression in response to low temperatures through hypermethylation or demethylation of some genes involved in thermogenesis, glycolysis, the autophagy pathway, the peroxisome, and drug metabolism pathways. These results provide important clues for studying DNA methylation patterns and identifying cold tolerance genes in shrimp.

Identification of a quantitative trait loci (QTL) associated with ammonia tolerance in the Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND: Ammonia is one of the most common toxicological environment factors affecting shrimp health. Although ammonia tolerance in shrimp is closely related to successful industrial production, few genetic studies of this trait are available. RESULTS: In this study, we constructed a high-density genetic map of the Pacific white shrimp (Litopenaeus vannamei) using specific length amplified fragment sequencing (SLAF-seq). The constructed genetic map contained 17,338 polymorphic markers spanning 44 linkage groups, with a total distance of 6360.12 centimorgans (cM) and an average distance of 0.37 cM. Using this genetic map, we identified a quantitative trait locus (QTL) that explained 7.41-8.46% of the phenotypic variance in L. vannamei survival time under acute ammonia stress. We then sequenced the transcriptomes of the most ammonia-tolerant and the most ammonia-sensitive individuals from each of four genetically distinct L. vannamei families. We found that 7546 genes were differentially expressed between the ammonia-tolerant and ammonia-sensitive individuals. Using QTL analysis and the transcriptomes, we identified one candidate gene (annotated as an ATP synthase g subunit) associated with ammonia tolerance. CONCLUSIONS: In this study, we constructed a high-density genetic map of L. vannamei and identified a QTL for ammonia tolerance. By combining QTL and transcriptome analyses, we identified a candidate gene associated with ammonia tolerance. Our work provides the basis for future genetic studies focused on molecular marker-assisted selective breeding.

Penaeus stylirostris densovirus proteins CP and NS1 interact with peritrophin of Litopenaeus vannamei.

The Penaeus stylirostris densovirus (PstDNV) is a major virus of shrimps that severely harms the shrimp farming industry. Peritrophin is a peritrophic membrane protein with chitin binding activity. To examine the roles of peritrophin in viral infection, we used yeast two-hybrid to analyze the interaction between the Pacific white shrimp (Litopenaeus vannamei) peritrophin and PstDNV proteins (CP, NS1 and NS2). The yeast two-hybrid results showed that NS1 and peritrophin had an interaction, CP and peritrophin had an interaction as well, and NS2 had no interaction with peritrophin. We validated the interactions with GST pull-down assays. We then conducted RNA interference and qRT-PCR. The results showed that when pre-injection of dsRNA-peritrophin, the quantity of PstDNV in the shrimps injected with viruses was significantly lower than in the control group (P < 0.01), indicating the viral infection was decreased when the peritrophin gene expression was inhibited. The results indicated that peritrophin of L. vannamei participated in the PstDNV infection.

The Penaeus stylirostris densovirus capsid interacts with Litopenaeus vannamei troponin I.

The Penaeus stylirostris densovirus (PstDNV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), a very small DNA virus, is a major shrimp pathogen. The PstDNV genome encodes only two nonstructural proteins and one capsid protein. This virus is thus an ideal, simple model for the investigation of virus-host interactions. To explore the role of the PstDNV capsid in viral infections, a yeast two-hybrid (Y2H) cDNA library was constructed based on Pacific white shrimp, Litopenaeus vannamei mRNA. The Y2H library was then screened, using the PstDNV capsid protein as bait. We identified a host protein that interacted strongly with the PstDNV capsid as L. vannamei troponin I (LvTnI). An in vitro co-immunoprecipitation experiment further supported this interaction. In addition, an in vivo neutralization experiment showed that the vaccination with anti-LvTnI significantly reduced PstDNV copies in PstDNV-challenged shrimp, indicating that the interaction between the PstDNV capsid and cellular LvTnI is essential for PstDNV infection. This result has important implications for our understanding of the mechanisms by which PstDNV infects shrimp.

The Penaeus stylirostris densovirus capsid protein interacts with the Litopenaeus vannamei BCCIP protein.

Viral capsid proteins play an important role in the viral infection process. To identify the cellular proteins in shrimp that interact with the Penaeus stylirostris densovirus capsid protein (PstDNV-CP), we constructed a yeast two-hybrid (Y2H) cDNA library of the muscle tissue of Litopenaeus vannamei, and hybridized the bait vector pGBKT7-CP with this library. Cloning and sequencing showed that the shrimp protein interacting with PstDNV-CP was a homolog of BRCA2 and CDKN1A(p21)-interacting protein (BCCIP). We named this protein L. vannamei BCCIP (LvBCCIP). Further analysis showed that LvBCCIP interacted with L. vannamei calmodulin (LvCaM). We validated the interactions between PstDNV-CP and LvBCCIP, and between LvBCCIP and LvCaM, with GST pulldown assays. The gene expression of LvBCCIP increased significantly after PstDNV challenge. In addition, the PstDNV titer of PstDNV-challenged shrimp was significantly reduced after LvBCCIP expression was inhibited using double-stranded RNA (dsRNA) interference. These results indicated that LvBCCIP is critical to PstDNV pathogenesis in L. vannamei. Interestingly, the growth rate of L. vannamei was significantly reduced when LvBCCIP gene expression was silenced, indicating that LvBCCIP may also be associated with growth regulation in L. vannamei. Thus, the interaction between PstDNV-CP and LvBCCIP might explain why PstDNV infection leads to runt-deformity syndrome in shrimp.

A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens.

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US$1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.

The NIa-Pro protein of Turnip mosaic virus improves growth and reproduction of the aphid vector, Myzus persicae (green peach aphid).

Many plant viruses depend on aphids and other phloem-feeding insects for transmission within and among host plants. Thus, viruses may promote their own transmission by manipulating plant physiology to attract aphids and increase aphid reproduction. Consistent with this hypothesis, Myzus persicae (green peach aphids) prefer to settle on Nicotiana benthamiana infected with Turnip mosaic virus (TuMV) and fecundity on virus-infected N. benthamiana and Arabidopsis thaliana (Arabidopsis) is higher than on uninfected controls. TuMV infection suppresses callose deposition, an important plant defense, and increases the amount of free amino acids, the major source of nitrogen for aphids. To investigate the underlying molecular mechanisms of this phenomenon, 10 TuMV genes were over-expressed in plants to determine their effects on aphid reproduction. Production of a single TuMV protein, nuclear inclusion a-protease domain (NIa-Pro), increased M. persicae reproduction on both N. benthamiana and Arabidopsis. Similar to the effects that are observed during TuMV infection, NIa-Pro expression alone increased aphid arrestment, suppressed callose deposition and increased the abundance of free amino acids. Together, these results suggest a function for the TuMV NIa-Pro protein in manipulating the physiology of host plants. By attracting aphid vectors and promoting their reproduction, TuMV may influence plant-aphid interactions to promote its own transmission.

Genetic variability of MHC class II DQB exon 2 alleles in yak (Bos grunniens).

The major histocompatibility class (MHC) DQ molecules are dimeric glycoproteins revealing antigen presentation to CD(4+) T cells. In the present study, the exon 2 of the MHC class II DQB gene from 32 yaks (Bos grunniens) was cloned, sequenced and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 25 DQB exon 2 alleles among 32 yaks, all alleles are found to belong to DQB1 loci. These alleles exhibited a high degree of nucleotide and amino acid polymorphisms with most amino acid variations occurring at positions forming the peptide-binding sites. The DQB loci were analyzed for patterns of synonymous (d S) and non-synonymous (d N) substitution. The yak was observed to be under strong positive selection in the DQB exon 2 peptide-binding sites (d N = 0.15, P < 0.001). It appears that this variability among yaks confers the ability to mount immune responses to a wide variety of peptides or pathogens.

Intermodal travel planning and decision support integrated with transportation and energy systems.

The fast urbanization in China makes it all the more important to find sustainable solutions that are both comprehensive and energy-efficient. Because of its important role in lowering logistical expenses and pollutant emissions, intermodal transport is generally seen as an effective method of coordinating transportation operations, helping to address the growing economic and environmental issues. Considering the characteristics of a growing city, this article lays out a multi-criteria method for selecting which new initiatives for China's public transportation system should be prioritized. "Electric municipality bus," "light rail system," and "modernization to the current fleet and optimization" are the three enhancement initiatives that are outlined. Using transportation-related economic, social, and environmental sub-criteria, this research applies TOPSIS, an analytic hierarchy method and fuzzy approach for order preference by resemblance to ideal circumstance application, to prioritize transportation projects. The study aims to improve city life in Chongqing, China, by identifying the most environmentally friendly development projects. Applying the analytical hierarchy method (AHP), the relative importance of several sustainability criteria was established for use in making strategic decisions. The alternative projects for the given city have been ranked using the fuzzy TOPSIS approach. However, the investigated results show the supportive response of hybrid to environmental sustainability and vice versa for non-hybrid vehicles. However, the energy consumption in public transport remains a leading hurdle in sustainability across the three modes of transport: taxis, rail transit and buses. Finally, computer trains in public transport also surprisingly deal with environmental sustainability to keep the current & forthcoming generation from ecological harm. However, theoretical and empirical policy suggestions have been proposed to become clean & green shortly.

The potential of glycinin basic peptide derived from soybean as a promising candidate for the natural food additive and preservative: A review.

Glycinin basic peptide (GBP) is the basic polypeptide of soybean glycinin that is isolated using cheap and readily available raw materials (soybean meals). GBP can bear high-temperature processing and has good functional properties, such as emulsification and adhesion properties et al. GBP exhibits broad-spectrum antimicrobial activities against Gram-positive and Gram-negative bacteria as well as fungi. Beyond that, GBP shows enormous application potential to improve the quality and extend the shelf life of food products. This review will systematically provide information on the purification, physicochemical and functional properties of GBP. Moreover, the antimicrobial activities and multi-target antimicrobial mechanism of GBP as well as the applications of GBP in different food products are also reviewed and discussed in detail. This review aims to offer valuable insights for the applications of GBP in the food industry as a promising natural food additive and preservative.

Biochemical indicators, cell apoptosis, and metabolomic analyses of the low-temperature stress response and cold tolerance mechanisms in Litopenaeus vannamei.

The cold tolerance of Litopenaeus vannamei is important for breeding in specific areas. To explore the cold tolerance mechanism of L. vannamei, this study analyzed biochemical indicators, cell apoptosis, and metabolomic responses in cold-tolerant (Lv-T) and common (Lv-C) L. vannamei under low-temperature stress (18 °C and 10 °C). TUNEL analysis showed a significant increase in apoptosis of hepatopancreatic duct cells in L. vannamei under low-temperature stress. Biochemical analysis showed that Lv-T had significantly increased levels of superoxide dismutase (SOD) and triglycerides (TG), while alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH-L), and uric acid (UA) levels were significantly decreased compared to Lv-C (p < 0.05). Metabolomic analysis displayed significant increases in metabolites such as LysoPC (P-16:0), 11beta-Hydroxy-3,20-dioxopregn-4-en-21-oic acid, and Pirbuterol, while metabolites such as 4-Hydroxystachydrine, Oxolan-3-one, and 3-Methyldioxyindole were significantly decreased in Lv-T compared to Lv-C. The differentially regulated metabolites were mainly enriched in pathways such as Protein digestion and absorption, Central carbon metabolism in cancer and ABC transporters. Our study indicate that low temperature induces damage to the hepatopancreatic duct of shrimp, thereby affecting its metabolic function. The cold resistance mechanism of Lv-T L. vannamei may be due to the enhancement of antioxidant enzymes and lipid metabolism.

Genome-wide analysis of ATP-binding cassette (ABC) transporter in Penaeus vannamei and identification of two ABC genes involved in immune defense against Vibrio parahaemolyticus by affecting NF-κB signaling pathway.

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5, PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates.

Workplace violence against nursing interns and patient safety: The multiple mediation effect of professional identity and professional burnout.

BACKGROUND: Most nursing interns have suffered some form of workplace violence in clinical settings, which has been linked to the jeopardizing of patient safety. Although previous research studies have examined the effect of workplace violence on patient safety, few studies have examined whether workplace violence is associated with patient safety through professional identity and professional burnout among nursing interns. AIMS: To test whether professional identity and professional burnout play mediating roles in the relationship of workplace violence and patient safety among nursing interns. DESIGN: Cross-sectional study. METHODS: The study included 466 nursing interns from three tertiary grade A hospitals. The Workplace Violence Scale, the Professional Identity Scale, the Maslach Burnout Inventory-General Survey, and the Patient Safety Behaviour Scale were used to gather data. Associations among workplace violence, professional identity, professional burnout, and patient safety were assessed by correlation and the serial-multiple mediation analysis. RESULTS: Workplace violence, professional identity, professional burnout and patient safety were significantly correlated. Workplace violence can have a direct positive impact on patient safety of nursing interns, but also an indirect impact on patient safety through three paths: the independent mediating role of professional identity, the independent mediating role of professional burnout, and the chain mediating role of professional identity and professional burnout. CONCLUSIONS: Our findings suggest that workplace violence can affect patient safety through decreasing professional identity and increasing professional burnout among nursing interns. Interventions aimed at decreasing workplace violence among nursing interns would be beneficial for professional attitude and patient safety.

Negative association between workplace violence and patient safety behaviour in male, but not female, nursing interns: A cross-section study.

AIM: The aim of the study was to compare the associations between workplace violence and patient safety behaviour between male and female nursing interns. DESIGN: A cross-sectional survey. METHODS: A cross-sectional survey was carried out at three general hospitals in Shandong Province in China to collect data from 466 nursing interns. We evaluated the associations between workplace violence and patient safety behaviours in men and women using multiple linear regressions. RESULTS: Sex moderated the association between workplace violence and patient safety behaviour (B = 1.046, [SE = 0.477]; p = 0.029). Among male nursing interns, there was a significant association between workplace violence and patient safety (B = -1.353, 95% CI [-2.556, -0.151]; p = 0.028). In male nursing interns, verbal violence and sexual violence were significantly negatively associated with patient safety (B = -1.569, SE = 0.492, p = 0.002; B = -45.663, SE = 5.554, p < 0.001). No significant association was found in female nursing interns. PATIENT OR PUBLIC CONTRIBUTION: This study did not have a patient or public contribution.

Mediating role of psychological resilience in the relationship between self-efficacy and professional identity among nurses.

To explore the mediating role of resilience in the relationship between general self-efficacy and professional identity of nurses during the COVID-19 pandemic. A cross-sectional design was employed. A total of 982 nurses from four Grade III, class A hospitals in Shandong Province were investigated using general information questionnaire, nurses' professional identity rating scale, general self-efficacy scale (GSES), and Connor-Davidson flexibility scale (CD-RISC). SPSS22.0 and Amos21.0 were used for data analysis and structural equation modeling. p % counseling The nurses had a score of （27.038±5.933） for general self-efficacy score, 38.290±6.234 for psychological resilience, and （114.99±16.209） for professional identity. A positive correlation between general self-efficacy, professional identity, and psychological resilience (<0.01) was found. The SEM analysis shows that psychological resilience plays a mediating role between general self-efficacy and professional identity. The ratio of the effect is 75.155. During the COVID-19 pandemic, the levels of general self-efficacy and professional identity of nurses was medium, while psychological resilience was high. Nurses' general self-efficacy can affect their professional identity through psychological resilience. During the pandemic, the psychological status of nurses should not be ignored. Nursing managers should fully utilize of group and cognitive therapy based on mindfulness to improve nurses' psychological resilience and general self-efficacy, and to promote nurses' professional identity, so as to ensure the lower turnover rate.

Fucosyltransferase 2 is involved in immune-related functions in Penaeus vannamei by modulating antimicrobial peptides' expression.

In mammals fucosyltransferase 2 (FUT2) plays an important regulatory role in inflammation, bacterial or viral infection, and tumor metastasis. However, the specific role of FUT2 in invertebrate immunity has not been reported. Here, the FUT2 homolog of Penaeus vannamei (designated as PvFUT2) was cloned and found to have a full-length cDNA of 1104 bp with an open reading frame (ORF) encoding 316 amino acids. PvFUT2 is constitutively expressed in all shrimp tissues tested with the highest found in intestines. Moreover, PvFUT2 was induced in the main immune organs (hemocytes and hepatopancreas) of shrimp by Gram-positive (Vibrio parahaemolyticus), Gram-negative (Streptococcus iniae) bacteria and virus (White Spot Syndrome Virus), indicating the involvement of PvFUT2 in shrimp antimicrobial response. Intriguingly, PvFUT2 knockdown with or without pathogen challenge reduced the expression of Pvß-catenin and antimicrobial peptides genes, particularly anti lipopolysaccharide factor and lysozyme. Further analysis revealed that the knockdown of PvFUT2 increased Vibrio abundance in hemolymph and resulted in an increase in shrimp cumulative mortality rate. Thus, during pathogen challenge, the expression of PvFUT2 is induced to regulate ß-catenin and subsequently antimicrobial peptides expression to augment shrimp antimicrobial immune response.

Cloning, Identification, and Functional Analysis of the Foxl2 Gene in Procambarus clarkii.

Procambarus clarkii is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 (Foxl2) gene has been found to be involved in the reproductive development of many crustaceans. To understand the role of the Foxl2 gene in the gonad development of P. clarkii, we designed CDS-specific primers for the P. clarkii Foxl2 (PcFoxl2) gene and cloned its CDS sequence using RT-PCR. The nucleotide and protein sequence information was then analyzed through bioinformatics analysis. The expression and subcellular localization of PcFoxl2 in various tissues were detected using qRT-PCR and in situ hybridization. The effects of PcFoxl2 knockdown on gonad development were investigated using RNA interference. The results showed that the CDS length of the PcFoxl2 gene was 1614 bp and encoded 537 amino acids. Protein sequence comparison and phylogenetic analysis showed that PcFoxl2 was the closest relative to Crayfish. qRT-PCR analysis indicated that the expression level of PcFoxl2 in the testis was significantly higher (>40 fold) than that in the ovary (p < 0.01). The in situ hybridization results showed that PcFoxl2 was expressed in both the cytoplasm and the nucleus of egg cells, and that the expression was strongest in egg cells at the early stage of yolk synthesis, while weak in the secondary oocytes. The positive signal was strongest in the spermatocyte nucleolus, while only a trace signal was observed in the cytoplasm. After interfering with the PcFoxl2 gene using dsRNA, the expression of PcFoxl2 in the RNA interference group was significantly lower than that in the control group, and this interference effect lasted for one week. Moreover, the gonad index of the experimental group was significantly lower than that of the control group (p < 0.05) after 10 days of P. clarkii cultivation following PcFoxl2 knockdown. The expression levels of the nanos and S3a genes, which are related to gonad development, decreased significantly after PcFoxl2 gene interference. The results suggest that the Foxl2 gene is involved in the growth and development of gonads, particularly in the development of testis, and is related to the early development of oocytes. This study provides a theoretical basis for the artificial breeding of P. clarkii.