Genome-wide association analysis reveals the genetic basis of thermal tolerance in dwarf surf clam Mulinia lateralis.

Recently, elevated seawater temperatures have resulted numerous adverse effects, including significant mortality among bivalves. The dwarf surf clam, Mulinia lateralis, is considered a valuable model species for bivalve research due to its rapid growth and short generation time. The successful cultivation in laboratory setting throughout its entire life cycle makes it an ideal candidate for exploring the potential mechanisms underlying bivalve responses to thermal stress. In this study, a total of 600 clams were subjected to a 17-day thermal stress experiment at a temperature of 30 °C which is the semi-lethal temperature for this species. Ninety individuals who perished initially were classified as heat-sensitive populations (HSP), while 89 individuals who survived the experiment were classified as heat-tolerant populations (HTP). Subsequently, 179 individuals were then sequenced, and 21,292 single nucleotide polymorphisms (SNPs) were genotyped for downstream analysis. The heritability estimate for survival status was found to be 0.375 ± 0.127 suggesting a genetic basis for thermal tolerance trait. Furthermore, a genome-wide association study (GWAS) identified three SNPs and 10 candidate genes associated with thermal tolerance trait in M. lateralis. These candidate genes were involved in the ETHR/EHF signaling pathway and played pivotal role in signal sensory, cell adhesion, oxidative stress, DNA damage repair, etc. Additionally, qPCR results indicated that, excluding MGAT4A, ZAN, and RFC1 genes, all others exhibited significantly higher expression in the HTP (p < 0.05), underscoring the critical involvement of the ETHR/EHF signaling pathway in M. lateralis' thermal tolerance. These results unveil the presence of standing genetic variations associated with thermal tolerance in M. lateralis, highlighting the regulatory role of the ETHR/EHF signaling pathway in the bivalve's response to thermal stress, which contribute to comprehension of the genetic basis of thermal tolerance in bivalves.

Dietary antarctic krill improves antioxidant capacity, immunity and reduces lipid accumulation, insights from physiological and transcriptomic analysis of Plectropomus leopardus.

BACKGROUND: Due to its enormous biomass, Antarctic krill (Euphausia superba) plays a crucial role in the Antarctic Ocean ecosystem. In recent years, Antarctic krill has found extensive application in aquaculture, emerging as a sustainable source of aquafeed with ideal nutritional profiles. However, a comprehensive study focused on the detailed effects of dietary Antarctic krill on aquaculture animals, especially farmed marine fishes, is yet to be demonstrated. RESULTS: In this study, a comparative experiment was performed using juvenile P. leopardus, fed with diets supplemented with Antarctic krill (the krill group) or without Antarctic krill (the control group). Histological observation revealed that dietary Antarctic krill could reduce lipid accumulation in the liver while the intestine exhibited no obvious changes. Enzyme activity measurements demonstrated that dietary Antarctic krill had an inhibitory effect on oxidative stress in both the intestine and the liver. By comparative transcriptome analysis, a total of 1,597 and 1,161 differentially expressed genes (DEGs) were identified in the intestine and liver, respectively. Functional analysis of the DEGs showed multiple enriched terms significantly related to cholesterol metabolism, antioxidants, and immunity. Furthermore, the expression profiles of representative DEGs, such as dhcr7, apoa4, sc5d, and scarf1, were validated by qRT-PCR and fluorescence in situ hybridization. Finally, a comparative transcriptome analysis was performed to demonstrate the biased effects of dietary Antarctic krill and astaxanthin on the liver of P. leopardus. CONCLUSIONS: Our study demonstrated that dietary Antarctic krill could reduce lipid accumulation in the liver of P. leopardus, enhance antioxidant capacities in both the intestine and liver, and exhibit molecular-level improvements in lipid metabolism, immunity, and antioxidants. It will contribute to understanding the protective effects of Antarctic krill in P. leopardus and provide insights into aquaculture nutritional strategies.

Therapeutic application of decellularized porcine small intestinal submucosa scaffold in conjunctiva reconstruction.

The objective of this study was to investigate the biological feasibility and surgical applicability of decellularized porcine small intestinal submucosa (DSIS) in conjunctiva reconstruction. A total of 52 Balb/c mice were included in the study. We obtained the DSIS by decellularization, evaluated the physical and biological properties of DSIS in vitro, and further evaluated the effect of surgical transplantation of DSIS scaffold in vivo. The histopathology and ultrastructural analysis results showed that the scaffold retained the integrity of the fibrous morphology while removing cells. Biomechanical analysis showed that the elongation at break of the DSIS (239.00 ± 12.51%) were better than that of natural mouse conjunctiva (170.70 ± 9.41%, P < 0.05). Moreover, in vivo experiments confirmed the excellent biocompatibility of the decellularized scaffolds. In the DSIS group, partial epithelialization occurred at day-3 after operation, and the conjunctival injury healed at day-7, which was significantly faster than that in human amniotic membrane (AM) and sham surgery (SHAM) group (P < 0.05). The number and distribution of goblet cells of transplanted DSIS were significantly better than those of the AM and SHAM groups. Consequently, the DSIS scaffold shows excellent biological characteristics and surgical applicability in the mouse conjunctival defect model, and DSIS is expected to be an alternative scaffold for conjunctival reconstruction.

A unique Ca2+-inhibited C-type lectin in shrimp Fenneropenaeuschinensis.

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca2+-inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight ß-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca2+-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca2+ in invertebrates.

Molecular identification and functional characterization of a C-type lectin gene in Meretrix meretrix.

C-type lectins (CTLs) are a kind of Ca2+-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca2+-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca2+ performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.

Identification of candidate SNPs and genes associated with resistance to nervous necrosis virus in leopard coral grouper (Plectropomus leopardus) using GWAS.

The leopard coral grouper (Plectropomus leopardus), which has become increasingly popular in consumption due to its bright body color and great nutritional, holds a high economic and breeding potential. However, in recent years, the P.leopardus aquaculture industry has been impeded by the nervous necrosis virus (NNV) outbreak, leading to widespread mortality among fry and juvenile grouper. However, the genetic basis of resistance to NNV in P. leopardus remains to be investigated. In the present study, we conducted a genome-wide association analysis (GWAS) on 100 resistant and 100 susceptible samples to discover variants and potential genes linked with NNV resistance. For this study, 157,926 high-quality single nucleotide polymorphisms (SNPs) based on whole genome resequencing were discovered, and eighteen SNPs loci linked to disease resistance were discovered. We annotated six relevant candidate genes, including sik2, herc2, pip5k1c, npr1, mybpc3, and arhgap9, which showed important roles in lipid metabolism, oxidative stress, and neuronal survival. In the brain tissues of resistant and susceptible groups, candidate genes against NNV infection showed significant differential expression. The results indicate that regulating neuronal survival or pathways involved in lipid metabolism may result in increased resistance to NNV. Understanding the molecular mechanisms that lead to NNV resistance will be beneficial for the growth of the P. leopardus breeding sector. Additionally, the identified SNPs could be employed as biomarkers of disease resistance in P. leopardus, which will facilitate the selective breeding of grouper.

SnO2QDs sensitized ZnFe2O4spheres with enhanced acetone sensing performance.

Herein, SnO2QDs (<10 nm) with small size instead of conventional nanoparticles was employed to modify ZnFe2O4to synthesize porous and heterogeneous SnO2/ZnFe2O4(ZFSQ) composites for gas sensing. By an immersion process combined with calcination treatment, the resultant porous ZFSQ composites with different contents of SnO2QDs were obtained, and their sensing properties were investigated. Compared with bare ZnFe2O4and SnO2QDs, porous ZFSQ composites based-sensors showed much improved sensor response to acetone. For contrast, the sensor performance of ZFSQ composites was also compared with that of ZnFe2O4sphere modified by SnO2nanoparticles with different size. The porous ZFSQ composite with 5 wt% SnO2QDs (ZFSQ-5) showed a better acetone sensing response than that of other ZFSQ composites, and it exhibited a high response value of 110-100 ppm of acetone and a low detection limit of 0.3 ppm at 240 °C. In addition to the rich heterojunctions and porous structure, the size effect of SnO2QDs was other indispensable reasons for the improved sensor performance. Finally, the ZFSQ-5 composite sensor was attempted to be applied for acetone sensing in exhaled breath, suggesting its great potential in monitoring acetone.

Aquaculture Molecular Breeding Platform (AMBP): a comprehensive web server for genotype imputation and genetic analysis in aquaculture.

It is of vital importance to understand the population structure, dissect the genetic bases of performance traits, and make proper strategies for selection in breeding programs. However, there is no single webserver covering the specific needs in aquaculture. We present Aquaculture Molecular Breeding Platform (AMBP), the first web server for genetic data analysis in aquatic species of farming interest. AMBP integrates the haplotype reference panels of 18 aquaculture species, which greatly improves the accuracy of genotype imputation. It also supports multiple tools to infer genetic structures, dissect the genetic architecture of performance traits, estimate breeding values, and predict optimum contribution. All the tools are coherently linked in a web-interface for users to generate interpretable results and evaluate statistical appropriateness. The webserver supports standard VCF and PLINK (PED, MAP) files, and implements automated pipelines for format transformation and visualization to simplify the process of analysis. As a demonstration, we applied the webserver to Pacific white shrimp and Atlantic salmon datasets. In summary, AMBP constitutes comprehensive resources and analytical tools for exploring genetic data and guiding practical breeding programs. AMBP is available at http://mgb.qnlm.ac.

Development of recombinase amplification assays for the rapid detection of infectious myonecrosis virus.

Infectious myonecrosis virus (IMNV) has affected shrimp farming in many countries, such as northeastern Brazil and southeast Asia, and poses a serious threat to the global shrimp industry. Reverse transcription enzymatic recombinant amplification technology (RT-ERA) is a rapid DNA amplification assay with high specificity in isothermal conditions and has been widely applied to the pathogen's detection. In this study, two novel ERA assays of IMNV, real-time RT-ERA and an RT-ERA combined with lateral flow dipsticks assay (RT-ERA-LFD), were developed and evaluated. The real-time RT-ERA assay could be carried out at 38-42 °C and had the highest end-point fluorescence value and the smallest Ct value at 41 °C. The brightness and width of the detection line were at a maximum at 39 °C and 30 min, and these conditions were selected in RT-ERA-LFD. Both real-time RT-ERA and RT-ERA-LFD produced positive results with IMNV standard plasmids only and showed no cross-reaction with Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis disease (VpAHPND); white spot syndrome virus (WSSV); infectious hypodermal and hematopoietic necrosis virus (IHHNV); or Ecytonucleospora hepatopenaei (EHP). Meanwhile, we compared the sensitivities of nested RT-PCR, real-time RT-PCR, real-time RT-ERA, and RT-ERA-LFD. The sensitivities of real-time RT-ERA and RT-ERA-LFD were both 101 copies/µL. The detection sensitivities of nested RT-PCR and real-time RT-PCR were 100 and 102 copies/µL, respectively. As a result, two ERA assays were determined to be specific, sensitive, and economical methods for the on-site diagnosis of IMNV infection, showing great potential for the control of IMNV infections.

Proteome analysis of outer membrane vesicles from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease.

A specific strain of Vibrio parahaemolyticus (VpAHPND) causes acute hepatopancreatic necrosis disease (AHPND), leading to significant losses in shrimp aquaculture. Outer membrane vesicles (OMVs) are naturally secreted by Gram-negative bacteria, and their significant roles in host-pathogen interactions and pathogenicity have been recognized. In the present study, OMVs were isolated from VpAHPND by differential-ultracentrifugation and used for proteomics analysis. In the Nano-HPLC-MS/MS analysis, totally 645 proteins were determined, including virulence factors, immunogenic proteins, outer membrane protein, bacterial secretory proteins, ribosomal proteins, protease, and iron regulation proteins. Furthermore, GO and KEGG annotations indicated that proteins identified in VpAHPND-OMVs are involved in metabolism, regulation of multiple biological processes, genetic information processes, immunity and more. Meanwhile, toxin proteins PirAvp and PirBvp, associated with VpAHPND pathogenicity, were also identified in the proteome of VpAHPND-OMVs. Our objective is to identify the protein composition of OMVs released by VpAHPND, analyzing the potential for cytotoxicity and immunomodulatory activity of these granule hosts. This study is crucial for understanding the roles played by bacterial-derived vesicles in the disease process, given that these vesicles carry relevant activities inherent to the bacteria that produce them.

Comparative Transcriptome Analysis of Hepatopancreas Reveals Sexual Dimorphic Response to Methyl Farnesoate Injection in Litopenaeus vannamei.

Sexually dimorphic traits such as growth and body size are often found in various crustaceans. Methyl farnesoate (MF), the main active form of sesquiterpenoid hormone in crustaceans, plays vital roles in the regulation of their molting and reproduction. However, understanding on the sex differences in their hormonal regulation is limited. Here, we carried out a comprehensive investigation on sexual dimorphic responses to MF in the hepatopancreas of the most dominant aquacultural crustacean-the white-leg shrimp (Litopenaeus vannamei). Through comparative transcriptomic analysis of the main MF target tissue (hepatopancreas) from both female and male L. vannamei, two sets of sex-specific and four sets of sex-dose-specific differentially expressed transcripts (DETs) were identified after different doses of MF injection. Functional analysis of DETs showed that the male-specific DETs were mainly related to sugar and lipid metabolism, of which multiple chitinases were significantly up-regulated. In contrast, the female-specific DETs were mainly related to miRNA processing and immune responses. Further co-expression network analysis revealed 8 sex-specific response modules and 55 key regulatory transcripts, of which several key transcripts of genes related to energy metabolism and immune responses were identified, such as arginine kinase, tropomyosin, elongation of very long chain fatty acids protein 6, thioredoxin reductase, cysteine dioxygenase, lysosomal acid lipase, estradiol 17-beta-dehydrogenase 8, and sodium/potassium-transporting ATPase subunit alpha. Altogether, our study demonstrates the sex differences in the hormonal regulatory networks of L. vannamei, providing new insights into the molecular basis of MF regulatory mechanisms and sex dimorphism in prawn aquaculture.

Theoretical Analysis and Expression Profiling of 17ß-Hydroxysteroid Dehydrogenase Genes in Gonadal Development and Steroidogenesis of Leopard Coral Grouper (Plectropomus leopardus).

The differentiation and developmental trajectory of fish gonads, significantly important for fish breeding, culture, and production, has long been a focal point in the fields of fish genetics and developmental biology. However, the mechanism of gonadal differentiation in leopard coral grouper (Plectropomus leopardus) remains unclear. This study investigates the 17ß-Hydroxysteroid Dehydrogenase (Hsd17b) gene family in P. leopardus, with a focus on gene characterization, expression profiling, and functional analysis. The results reveal that the P. leopardus's Hsd17b gene family comprises 11 members, all belonging to the SDR superfamily. The amino acid similarity is only 12.96%, but conserved motifs, such as TGxxxGxG and S-Y-K, are present in these genes. Hsd17b12a and Hsd17b12b are unique homologs in fish, and chromosomal localization has confirmed that they are not derived from different transcripts of the same gene, but rather are two independent genes. The Hsd17b family genes, predominantly expressed in the liver, heart, gills, kidneys, and gonads, are involved in synthesizing or metabolizing sex steroid hormones and neurotransmitters, with their expression patterns during gonadal development categorized into three distinct categories. Notably, Hsd17b4 and Hsd17b12a were highly expressed in the testis and ovary, respectively, suggesting their involvement in the development of reproductive cells in these organs. Fluorescence in situ hybridization (FISH) further indicated specific expression sites for these genes, with Hsd17b4 primarily expressed in germ stem cells and Hsd17b12a in oocytes. This comprehensive study provides foundational insights into the role of the Hsd17b gene family in gonadal development and steroidogenesis in P. leopardus, contributing to the broader understanding of fish reproductive biology and aquaculture breeding.

The role of regulated necrosis in inflammation and ocular surface diseases.

In recent decades, numerous types of regulated cell death have been identified, including pyroptosis, ferroptosis and necroptosis. Regulated necrosis is characterized by a series of amplified inflammatory responses that result in cell death. Therefore, it has been suggested to play an essential role in the pathogenesis of ocular surface diseases. The cell morphological features and molecular mechanisms of regulated necrosis are discussed in this review. Furthermore, it summarizes the role of ocular surface diseases, such as dry eye, keratitis, and cornea alkali burn, as potential disease prevention and treatment targets.

Molecular characterization and expression analysis of a QM protein gene in Chinese mitten crab Eriocheir sinensis.

QM protein was previously discovered as a tumor suppressor, and numerous studies have shown that QM protein also played important roles in the immune responses. To investigate the potential roles of the QM protein gene in Eriocheir sinensis, the QM protein gene (designated as EsQM) has been cloned from E. sinensis using the rapid amplification of cDNA ends (RACE) technique. The cDNA of EsQM is 781 bp in length, consisting of a 654 bp open reading frame (ORF), encoding 219 amino acids, a 27 bp 5' untranslated region (UTR) and a 94 bp 3' UTR. The EsQM protein has a calculated molecular weight of 25.4 kDa and a theoretical isoelectric point of 10.10. The deduced protein sequence of EsQM contains a Ribosomal_L16 domain, an SH3-binding motif, an N-acylation site, two putative antibiotic binding sites, two putative protein kinase C phosphorylation sites, and two amidation sites. EsQM is extremely conserved and exhibits more than 85% similarities to previously identified arthropod QM protein genes. By real-time quantitative PCR (qPCR) analysis, we found that EsQM mRNA transcripts were detectable in all the examined tissues, with the highest expression in hemocytes. The mRNA expression of EsQM in hemocytes was significantly upregulated after the stimulation of Aeromonas hydrophila or polybrominated diphenyl ether-47 (BDE-47). Moreover, EsQM mRNA expression in hemocytes responded more quickly and lasted longer when stimulated by A.hydrophila than BDE-47. Thus, EsQM can respond to bacterial infection and environmental pollution, and might be involved in the defense mechanism to both biological and non-biological stimulation of arthropods.

Comparative analysis of two cathepsin L genes in Asiatic hard clam (Meretrix meretrix): Similar in sequence features, different in expression profiles.

Cathepsin L is widely found in eukaryotes and prokaryotes, and it plays important roles in innate immunity. In the present study, we cloned two cathepsin L genes (designated as MmCTSL1 and MmCTSL2, respectively) from Asiatic hard clam (Meretrix meretrix). The complete sequence of MmCTSL1 cDNA contained a 5' untranslated region (UTR) of 31 bp, a 3' UTR of 228 bp with a poly (A) tail, and an open reading frame (ORF) of 1005 bp encoding 334 amino acids with predicted molecular weight of 37.5 kDa and theoretical isoelectric point of 5.27, and contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W27 to F87), and a papain family cysteine protease domain (from L118 to T333). The complete sequence of MmCTSL2 cDNA contained a 5' UTR of 50 bp, a 3' UTR of 162 bp with a poly (A) tail, and an ORF of 996 bp encoding a polypeptide of 331 amino acids with predicted molecular weight of 36.8 kDa and theoretical isoelectric point of 7.07. It contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W30 to F89), and a papain family cysteine protease domain (from L115 to T330). Real-time quantitative PCR analysis demonstrated that MmCTSL1 and MmCTSL2 were widely expressed in all the tested tissues, including adductor muscle, foot, gill, hemocytes, hepatopancreas and mantle, with the highest mRNA expression level in hepatopancreas and hemocytes, respectively. After Vibrio splendidus challenge, the mRNA expression levels of MmCTSL1 and MmCTSL2 in hemocytes and hepatopancreas were both significantly up-regulated with different expression profiles. In hemocytes, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks (3.4-fold and 13.0-fold compared with the control, respectively) at 12 h after bacterial challenge, and MmCTSL2 responds earlier than MmCTSL1. In hepatopancreas, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks at 6 h (9.0-fold compared with the control) and 24 h (2.8-fold compared with the control) after bacterial challenge, meaning that MmCTSL1 responds earlier than MmCTSL2. At the same time, whether in hepatopancreas or hemocytes, MmCTSL1 persist for a while after the bacterial challenge peak, while MmCTSL2 would quickly return to the initial level after the bacterial challenge peak. These results indicate that cathepsin L may be involved in the immune process of hard clam against V. splendidus with different potential roles.

MolluscDB: an integrated functional and evolutionary genomics database for the hyper-diverse animal phylum Mollusca.

Mollusca represents the second largest animal phylum but remains poorly explored from a genomic perspective. While the recent increase in genomic resources holds great promise for a deep understanding of molluscan biology and evolution, access and utilization of these resources still pose a challenge. Here, we present the first comprehensive molluscan genomics database, MolluscDB (http://mgbase.qnlm.ac), which compiles and integrates current molluscan genomic/transcriptomic resources and provides convenient tools for multi-level integrative and comparative genomic analyses. MolluscDB enables a systematic view of genomic information from various aspects, such as genome assembly statistics, genome phylogenies, fossil records, gene information, expression profiles, gene families, transcription factors, transposable elements and mitogenome organization information. Moreover, MolluscDB offers valuable customized datasets or resources, such as gene coexpression networks across various developmental stages and adult tissues/organs, core gene repertoires inferred for major molluscan lineages, and macrosynteny analysis for chromosomal evolution. MolluscDB presents an integrative and comprehensive genomics platform that will allow the molluscan community to cope with ever-growing genomic resources and will expedite new scientific discoveries for understanding molluscan biology and evolution.

Development of a real-time enzymatic recombinase amplification assay for rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp Penaeus vannamei.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is classified as a reportable crustacean disease by the World Organisation for Animal Health (WOAH), which causes poor growth in Penaeus vannamei. According to genome sequence alignment analysis, enzymatic recombinase amplification (ERA) primers and probe were designed based on the ORF1 region of IHHNV, and a real-time ERA assay for IHHNV detection (IHHNV-ERA) was established. The experimental results show that IHHNV-F2/IHHNV-R2 and IHHNV-Probe can effectively amplify the target gene, and the sensitivity is 1.4 × 101 copies/µL within 14.97 ± 0.19 min, while the qPCR using primers 309F/309R could reach the detection limit of 1.4 × 101 copies/µL within 21.76 ± 0.63 min, and the sensitivity results of one-step PCR could be as low as 1.4 copies/µL with expense of time and false positives. The IHHNV-ERA system can effectively amplify the target gene at 42 ℃ within 20 min, and has no cross-reaction with white spot syndrome virus (WSSV), Ecytonucleospora hepatopenaei (EHP), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), and healthy shrimp genomic DNA. Test results of practical samples showed that the detection rate of IHHNV-ERA (18/20) was better than the industrial standard qPCR assay (17/20). Compared with the existing technology, the useful results of this detection assay are: (1) get rid of the dependence on the thermal cycle instrument in the PCR process; (2) the experimental procedure is simple, time-consuming and fast; (3) the detection sensitivity is high. This study provides an ERA based detection assay for IHHNV, which can be used not only for the rapid detection of IHHNV infection, but also for the field screening of pathogens. This assay can also be applied to clinical inspection, customs detection, enterprise quality inspection and other fields, and has obvious practical application value.

An isothermal enzymatic recombinase amplification (ERA) assay for rapid and accurate detection of Enterocytozoon hepatopenaei infection in shrimp.

Enterocytozoon hepatopenaei (EHP) is a kind of microsporidian parasite belonging to fungi, and poses a serious threat to prawn farmers. Due to the lack of effective treatments for EHP, the establishment of a rapid and sensitive detection method would be beneficial to the control and prevention of this prawn parasitic disease. In this study, an isothermal enzymatic recombinase amplification (EHP-ERA) assay that could diagnose EHP within 20 min at 42 °C was developed and evaluated. The determined final concentrations of primers and probe in the reaction system were 400 nM and 120 nM, respectively. EHP-ERA was carried out within 13 min (24.31 ± 0.37 Ct) with a detection limit of 10 copies/µL. The results of specificity test showed that EHP-ERA had no cross-reactivity with white spot syndrome virus (WSSV), Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (VpAHPND), and infectious hypodermal and hematopoietic necrosis virus (IHHNV) and specific pathogen free (SPF) shrimp. Using 32 clinical samples, the practical diagnostic results of EHP-ERA was consistent with nested PCR and real-time PCR (qPCR) under the premise of less time-consuming and simpler operation. In summary, we established a simple, rapid, and effective ERA assay for the detection of EHP, which had great potential to be widely used in both lab and practical usage.

Development of a high-throughput SNP array for sea cucumber (Apostichopus japonicus) and its application in genomic selection with MCP regularized deep neural networks.

High-throughput single nucleotide polymorphism (SNP) genotyping assays are powerful tools for genetic studies and genomic breeding applications for many species. Though large numbers of SNPs have been identified in sea cucumber (Apostichopus japonicus), but, as yet, no high-throughput genotyping platform is available for this species. In this study, we designed and developed a high-throughput 24 K SNP genotyping array named HaishenSNP24K for A. japonicus, based on the multi-objective-local optimization (MOLO) algorithm and HD-Marker genotyping method. The SNP array exhibited a relatively high genotyping call rate (> 96%), genotyping accuracy (>95%) and exhibited highly polymorphic in sea cucumber populations. In addition, we also assessed its application in genomic selection (GS). Deep neural networks (DNN) that can capture the complicated interactions of genes have been proposed as a promising tool in GS for SNP-based genomic prediction of complex traits in animal breeding. To overcome the problem of over-fitting when using the HaishenSNP24K array as high-dimensional DNN input, we developed minmax concave penalty (MCP) regularization for sparse deep neural networks (DNN-MCP) that finds an optimal sparse structure of a DNN by minimizing the square error subject to the non-convex penalty MCP on the parameters (weights and biases). Compared to two linear models, namely RR-GBLUP and Bayes B, and the nonlinear model DNN, DNN-MCP has greatly improved the genomic prediction ability for three quantitative traits (e.g., wet weight, dry weight and survival time) in the sea cucumber population. To the best of our knowledge, this is the first work to develop a high-throughput SNP array for A. japonicus and a new model DNN-MCP for genomic prediction of complex traits in GS. The present results provide evidence that supports the HaishenSNP24K array with DNN-MCP will be valuable for genetic studies and molecular breeding in A. japonicus.

Characterization and Preliminary Application of a Novel Lytic Vibrio parahaemolyticus Bacteriophage vB_VpaP_SJSY21.

Litopenaeus vannamei is one of the most economically significant aquatic species globally. However, the emergence of acute hepatopancreatic necrosis disease (AHPND) in recent years has resulted in substantial losses within the L. vannamei farming industry. Phage therapy holds promise as an effective strategy for preventing and controlling bacterial infections like AHPND, thereby promoting the healthy and sustainable growth of the shrimp aquaculture sector. In this study, a novel and unique Vibrio parahaemolyticus bacteriophage, named vB_VpaP_SJSY21, was successfully isolated from sewage samples. Using transmission electron microscopy, it was observed that phage SJSY21 has an elongated shell. Notably, phage SJSY21 exhibited high infection efficiency, with an optimal multiplicity of infection (MOI) of only 0.01 and a remarkably short latent period of 10 min, resulting in a lysis quantity of 508. Furthermore, phage SJSY21 demonstrated notable heat resistance and the capacity to withstand high temperatures during preservation, thus holding potential for application in phage therapy. Whole-genome sequencing and analysis confirmed that phage SJSY21 has a genome size of 110,776 bp, classifying it as a new member of the short-tailed bacteriophage family. Additionally, cultivation experiments indicated that phage SJSY21 has the potential to enhance the survival of L. vannamei in culture systems, thereby offering innovative prospects for the application of phage therapy in aquaculture.